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Collagen I is a major component of extracellular matrix in human skin, and is also widely used in a variety of skin-care products. In this study, we investigated the modulatory roles of collagen I on human immortalized keratinocytes HaCaT, especially when cells were irradiated with UVB. Interestingly, the cells grown on plates coated by molecular collagen I, but not fibrillar collagen I, acquired certain resistance against UVB damages, as shown by increased survival and reduced apoptosis. The accumulation of dysfunctional mitochondria in UVB-treated cells was attenuated by molecular collagen I-coating. Interestingly, molecular collagen I rescued the loss of mitochondrial biogenesis in cells treated with UVB. Loss of PINK1/parkin-mediated mitophagy was dominant for the accumulation of dysfunctional mitochondria after UVB irradiation. Of note, cells cultured on molecular collagen I-precoated plates exhibited reserved mitophagy after UVB irradiation, as reflected by the enhanced protein level of PINK1/parkin, increased mitochondrial ubiquitin and the co-localization of lysosomes and mitochondria. Moreover, in UVB-treated cells, inhibiting mitophagy by Cyclosporin A, or by silencing PINK1 or parkin, disturbed the resolution of mitochondrial stress and reduced the protective effect of molecular collagen I, indicating that mitophagy is pivotal for the protection of collagen I against UVB damage in keratinocytes HaCaT. Collectively, this study reveals an unexpected protective role of collagen I, which facilitates mitophagy to rescue cells under UVB irradiation, providing a new direction for clinical application of collagen products., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Excessive physical exercise (overtraining, OT) charactered by long-term and excessive training results in the damage of multiple vital tissues including hippocampus which plays a critical role in learning and memory. A combination of dasatinib (D) plus quercetin (Q) (D+Q) belongs to senolytic drugs which selectively kill senescent cells in vitro and vivo. In this study, the rats that suffered a five-week excessive swimming training were subjected to the oral administration of D+Q. D+Q alleviated the decline in exercise performance of OT rats during the swimming training, and prevented learning and memory deficits in Morris water maze, Y-maze and novel object recognition tests after excessive swimming training. Analytical results by SA-β-gal staining and western blotting showed that D+Q significantly reduced senescent cells with repressed expression of senescence-related proteins, p53 and p21, in hippocampus. Nissl and immunohistochemical staining showed that D+Q significantly attenuated neuronal loss caused by apoptosis. Interestingly, we observed elevated level of cleaved caspase 3, an apoptosis executor protein, in p21 positive hippocampus cells by D+Q treatment in immunofluorescent staining, suggesting that senescent cells were induced to apoptosis in D+Q-treated rats. The positive control drug, silibinin, showed similar protective effect against OT, but did not induce the apoptosis of senescent cells, suggesting a difference in the protective mechanisms. These results indicated that D+Q alleviates overtraining-induced deficits in learning and memory through elimination of senescent cells and reduction of apoptotic cell number., Competing Interests: Declarations of interest None., (Copyright © 2022. Published by Elsevier B.V.)

Exposure to ultraviolet B (UVB) from sunlight causes DNA damage, serious cellular inflammation and aging, and even cell death in the skin, commonly known as sunburn, leading to cutaneous tissue disorders. DNA damage can be sensed as a danger-associated molecular pattern (DAMP) by the innate immune system. It has not been studied, however, whether cGAS-STING activation is involved in the apoptosis induced by UVB irradiation or by cisplatin treatment. Here we report the findings that within hours of DNA damages keratinocytes show an innate immune response, which involves the activation of cGAS-STING; a cytosolic DNA receptor, cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase), cyclic GMP-AMP (cGAMP) synthase, and DNA sensing adaptor, STING (protein stimulator of interferon genes). Either UVB irradiation or cisplatin treatment can cause DNA damages, releasing fragmented DNA from nucleus and/or mitochondria. Roles of cGAS-STING were examined in the HaCaT cells with DNA damages caused by UVB irradiation or cisplatin treatment. Silencing STING by siRNA rescued HaCaT cells from UVB or cisplatin-induced apoptosis. NF-κB, one of the major downstream components of STING pathway, which usually regulates the classical STING apoptotic pathway, was translocated to nucleus in the HaCaT cells irradiated with UVB. This translocation was attenuated by STING silencing. Treatment with BAY, an inhibitor of NF-κB pathway, blocked UVB-induced apoptosis. cGAS-STING-mediated production of IFNβ was induced by nuclear translocation of interferon regulatory factor 3 (IRF3). UVB irradiation inceased the nuclear translocation of IRF3, accompanied by enhanced expression level of IFNβ mRNA. The nuclear translocation of IRF3 and expression of IFNβ mRNA were attenuated by STING silencing. Treatment with MRT67307, an inhibitor of TBK1-IRF3-IFNβ pathway, blocked UVB-induced apoptosis. Therefore, we conclude that NF-κB pathway and IFNβ pathway residing in the downstream of STING are resposible for apoptosis of UVB-irradiated or cisplatin-treated HaCaT cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Glioma alone accounts for 30% of various kinds of primary brain tumors and is the highest cause of mortality associated with intracranial malignant cancers. In the present study, Suzuki-coupling products of remimazolan were synthesized and investigated for anti-neoplastic property against glioma cells. RFMSP treatment for 48 hr suppressed viabilities of U-118MG and U87MG cells in dose dependent manner. Exposure of primary astrocytes to RFMSP at 2-20 μM concentration range minimally affected viabilities. RFMSP treatment at 5 μM doses raised apoptotic cell count to 53.8 ± 2.3% and 48.2 ± 1.8%, respectively in U-118MG and U87MG cells. Treatment of the cells with RFMSP induced nuclear condensation and subsequent fragmentation. In RFMSP treated U-118MG and U87MG cells, NF-κB p65 expression was markedly suppressed compared to the control cells. Additionally, RFMSP treatment decreased the ratio of nuclear to total NF-κB p65 level in both the cell lines. Treatment of U-118MG and U87MG cells with 5 μM RFMSP for 48 hr caused a marked down-regulation in survivin and XIAP levels. Treatment with RFMSP promoted Bax expression and suppressed Bcl-2 level. The caspase-9 and -3 activation was markedly induced by RFMSP treatment in U-118MG and U87MG cells compared to the control cells. In summary, the RFMSP synthesized by Suzuki-coupling of RFMSP inhibited glioma cell survival via DNA damage mediated apoptosis. The anti-glioma potential of RFMSP involved down-regulation of NF-κB expression, targeted survivin & XIAP levels and induced caspase activation in glioma cells. Therefore, RFMSP may be studied further as therapeutic agent for the treatment of glioma., (© 2020 International Union of Biochemistry and Molecular Biology.)

Elucidating virus-cell interactions is fundamental to understanding viral replication and identifying targets for therapeutic control of viral infection. The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate pathogenesis during many viral infections, but its role during coronavirus infection is undetermined. Infectious bronchitis virus is the representative strain of Gammacoronavirus, which causes acute and highly contagious diseases in the poultry farm. In this study, we investigated the role of ERK1/2 signaling pathway in IBV infection. We found that IBV infection activated ERK1/2 signaling and the up-regulation of phosphatase DUSP6 formed a negative regulation loop. Pharmacological inhibition of MEK1/2-ERK1/2 signaling suppressed the expression of DUSP6, promoted cell death, and restricted virus replication. In contrast, suppression of DUSP6 by chemical inhibitor or siRNA increased the phosphorylation of ERK1/2, protected cells from apoptosis, and facilitated IBV replication. Overexpression of DUSP6 decreased the level of phospho-ERK1/2, promoted apoptosis, while dominant negative mutant DUSP6-DN lost the regulation function on ERK1/2 signaling and apoptosis. In conclusion, these data suggest that MEK-ERK1/2 signaling pathway facilitates IBV infection, probably by promoting cell survival; meanwhile, induction of DUSP6 forms a negative regulation loop to restrict ERK1/2 signaling, correlated with increased apoptosis and reduced viral load. Consequently, components of the ERK pathway, such as MEK1/2 and DUSP6, represent excellent targets for the development of antiviral drugs.

We reported previously that higher doses (150-250 μM) of silibinin enhanced fission and inhibited fusion of mitochondria, accompanying apoptosis of double-positive breast cancer cell line MCF-7 cells and triple-negative breast cancer cell line MDA-MB-231 cells. We report here three important questions yet unclarified in the previous study; 1) Whether enhanced fission of mitochondria by the treatment of silibinin leads to mitophagy, 2) Whether mitophagy positively contributes to apoptosis and 3) Whether estrogen receptor-positive (ER + ) MCF-7 cells and estrogen receptor-negative (ER - ) MDA-MB-231 cells are affected in a different way by silibinin treatment, since silibinin often works through ERs signaling pathway. Mitophagy driven by Pink1/Parkin signaling, plays an important role in eliminating damaged mitochondria. Indeed, increased expression of Pink1 and the recruitment of Parkin and LC3-II to mitochondria by the treatment with silibinin account for silibinin induction of mitophagy. In this study, the effects of mitochondrial division inhibitor 1 (mdivi-1) and small interfering RNA targeting dynamin-related protein 1 (DRP1) were examined to reveal the effect of mitochondrial fission on mitophagy. As expected, mdivi-1 or siRNA targeting DRP1 reversed silibinin-induced mitochondrial fission due to down-regulation in the expression of DRP1. Inhibition of mitochondrial fission by mdivi-1 prevented induction of mitophagy as well as autophagy in both MCF-7 and MDA-MB-231 cells, indicating that silibinin-induced mitochondrial fission leads to mitophagy. Inhibition of mitochondrial fission efficiently prevented silibinin-induced apoptosis in MCF-7 and MDA-MB-231 cells in our previous work, and the second point of the present study, inhibition of mitophagy by Pink1 or Parkin knockdown increased silibinin-induced apoptosis of these cells, respectively, suggesting that the mitophagy induced by silibinin treatment serves as a cytoprotective effect, resulting in reduction of apoptosis of cancer cells in both cells. In the third point, we studied whether estrogen receptors (ERs) played a role in silibinin-induced mitophagy and apoptosis in MCF-7 and MDA-MB-231 cells. ERα and ERβ are not involved in silibinin-induced mitophagic process in MCF-7 and MDA-MB-231 cells. These findings demonstrated that silibinin induced mitochondria fission leads to mitophagy, which attenuates silibinin-induced apoptosis not through ERs-Pink1 or -Parkin pathway in MCF-7 and MDA-MB-231., (Copyright © 2020. Published by Elsevier Inc.)

Hydrogen sulfide (H 2 S) has been recognized as the third endogenous signaling gasotransmitter following nitric oxide (NO) and carbon monoxide (CO), and exhibits antiproliferative activity against several cancer cells. In order to stably and controllably release H 2 S, H 2 S donating compound (ADT-OH) was used in the present study and 18H 2 S releasing natural ent-kaurane diterpenoid oridonin derivatives were designed and synthesized. Most derivatives showed more potent antiproliferative activities than oridonin against HepG2 and K562 cell lines, while they were lack of sensitivity to HCT-116 and B16 cells. In particular, 12b showed the most potent antiproliferative activities against HepG2, HCT-116 and K562 cells with IC 50 values of 2.57, 5.81 and 0.95 μM, respectively. Through cell cycle analysis, 12b caused cell cycle arrest at S phase in K562 cells and G1 phase in HepG2 cells. In Hoechst 33258 staining assay, cell shrinkage and fragmentation of cell nuclei indicated apoptotic morphological changes. Considering the decline of mitochondrial membrane potential and changes in the levels of apoptosis-related proteins, 12b was shown to induce apoptosis through extrinsic and intrinsic apoptosis pathways., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Alzheimer's disease (AD) is a neurodegenerative disease, mainly characterized by cognitive dysfunction and memory impairment. Due to its pathological similarities to type 2 diabetes mellitus (T2DM), such as β-amyloid deposition, oxidative stress, inflammation, disordered glucose metabolism, impaired signaling pathways of insulin and insulin-like growth factor-1 (IGF-1), we speculate that AD is another form of brain diabetes. Clarifying the relationship between T2DM and AD is important for us to better understand the exact pathological mechanisms of AD. Silibinin, a polyphenolic flavonoid extracted from the seeds of Silybum marianum, exerts hepatoprotective, anti- diabetic and neuroprotective effects. Streptozotocin (STZ), which is used to disrupt the insulin signal transduction pathway, could well mimic the sporadic AD models by intracerebroventricular (ICV) injection. Therefore, we selected ICV injection of STZ (ICV-STZ) to investigate the neuroprotective effects of silibinin in rats and to make a foundation for further exploring the relationship between AD and T2DM. ICV-STZ obviously caused memory damage, sharply reduced the number of nissl bodies and destroyed morphological structure of hippocampal neuronal cells, while silibinin attenuated the damages. Moreover, silibinin significantly decreased STZ-induced tau hyperphosphorylation (ser404) in hippocampus and cerebral cortex, markedly inhibited apoptosis of neurons induced by STZ, and up-regulated insulin signal transduction pathway. Silibinin exerts neuroprotective effect in STZ-treated rats, indicating the potential of silibinin for the treatment of AD patients with T2DM in future., (Copyright © 2019 Elsevier Inc. All rights reserved.)

High levels of circulating free fatty acids often trigger pancreatic β cell dysfunction during the development of type 2 diabetes. Silibinin, the main component of Silybum marianum fruit extract (silymarin), is reported to have anti-diabetic effect. This study is designed to determine the protective effect of silibinin on palmitic acid-induced damage in a rat pancreatic β-cell line, INS-1 cells. Our results demonstrate that silibinin improves cell viability, enhances insulin synthesis and secretion, and resumes normal mitochondrial function in palmitic acid-treated INS-1 cells. An accumulating body of evidence has shown that the estrogen receptors are key molecules involved in glucose and lipid metabolism. Our results suggest that silibinin upregulates ERα signaling pathway from the finding that ERα-specific inhibitors abolish the anti-lipotoxic effect of silibinin. In conclusion, these findings suggest that silibinin protects INS-1 cells against apoptosis and mitochondrial damage through upregulation of ERα pathway.

Mitochondria are dynamically regulated by fission and fusion processes. Silibinin induces apoptosis of MCF-7 and MDA-MB-231 human breast cancer cells. However, whether or not mitochondria dysfunction is involved in the apoptosis induction with silibinin of both types of the cells remains unknown. We here report that silibinin decreases the mitochondrial mass in terms of MitoTracker Green staining in both breast cancer cells. Silibinin induces morphological changes of mitochondria from oval to truncated or fragmented shapes accordingly. Condensed crests are observed in mitochondria by transmission electron microscopy. Silibinin causes mitochondrial membrane potential reduced. The expression of mitochondrial fission-associated proteins including dynamin-related protein 1 (DRP1) is up-regulated, whereas expression of the mitochondrial fusion-associated proteins, optic atrophy 1 and mitofusin 1, is down-regulated. In addition, silibinin treatment down-regulates ATP content as well as the levels of mitochondrial biogenesis-regulators including mitochondrial transcription factor A, peroxisome proliferator-activated receptor gamma coactivator 1 and nuclear respiratory factor 2. Moreover, treatments with DRP1 inhibitor, mdivi-1, or with DRP1-targetted siRNA efficiently prevent silibinin-induced apoptosis in the breast cancer cells, whereas inhibition of DRP1 phosphorylation with staurosporine increases apoptosis furthermore. Taken together, we conclude that silibinin impairs mitochondrial dynamics and biogenesis, leading to apoptosis of MCF-7 and MDA-MB-123 cells., (Copyright © 2019. Published by Elsevier Inc.)

Excessive physical exercise (overtraining; OT) increases oxidative stress and induces damage in multiple organs including the brain, especially the hippocampus that plays an important role in learning and memory. Silibinin, a natural flavonoid derived from milk thistle of Silybum marianum, has been reported to exert neuroprotective effect. In this study, rats were subjected to overtraining exercise, and the protective effects of silibinin were investigated in these models. Morris water maze and novel object recognition tests showed that silibinin significantly attenuated memory defects in overtrained rats. At the same time, the results of Nissl, TUNEL and SA-β-gal staining showed that silibinin reversed neuronal loss caused by apoptosis, and delayed cell senescence of the hippocampus in the overtrained rats, respectively. In addition, silibinin decreased malondialdehyde (MDA) levels which is associated with reactive oxygen species (ROS) generation. Silibinin prevented impairment of learning and memory caused by excessive physical exercise in rats, accompanied by reduced apoptosis and senescence in hippocampus cells.

The objective was to investigate the mechanism of the protective effect of silibinin on amylin/Aβ 1-42 -induced INS-1 cell apoptosis, with special reference to the roles of glucagon-like peptide-1 receptor (GLP-1R) and protein kinase A (PKA). The effects of silibinin on apoptosis, insulin secretion, GLP-1R, and PKA expression in the INS-1 cells treated with amylin/Aβ 1-42  were examined. INS-1 cells exposed to amylin showed increased TUNEL-positive ratio, reduced expression of GLP-1R and PKA. GLP-1R antagonists or PKA inhibitor enhanced the expression of apoptosis-associated proteins and TUNEL-positive ratio. Silibinin exerted antiapoptotic effect on and upregulation of GLP-1R and PKA. However, Aβ 1-42 -induced INS-1 cell apoptosis, GLP-1R, and PKA expressions were not changed. Our results indicate that down-regulation of GLP-1R and PKA contributes to INS-1 cell apoptosis induced with amylin. Silibinin protects INS-1 cells from amylin-induced apoptosis through activation of GLP-1R/PKA signaling. Silibinin's inhibition of the toxic effects of Aβ 1-42 is independent of GLP-1R/PKA pathway.

Toll-like receptor 3 (TLR3), a member of pattern recognition receptors, is reported to initiate skin inflammation by recognizing double-strand RNA (dsRNA) released from UVB-irradiated cells. Recently, we have discovered the NF-κB pathway activated by TLR3 is involved in apoptosis of UVB-Poly I:C-treated HaCaT cells. The real culprit for apoptosis has not been precisely identified since the system of NF-κB pathway is complex. In this study, we silenced main transcriptional factors in NF-κB family, RelA, RelB and c-Rel, but to our surprise the results show that none of them participate in apoptosis induction in UVB-Poly I:C-treated HaCaT cells. Therefore, we moved to investigate the apoptosis-associated molecules in the upstream of NF-κB pathway. We firstly checked the expression of IκBα, an NF-κB inhibitor. UVB (4.8 mJ/cm 2 ) and Poly I:C (0.3 μg/mL) co-treatment decreased IκBα expression level in a time-dependent manner. Silencing IκBα with siRNA further enhanced UVB-Poly I:C-induced cell death. We then investigated IκB kinase (IKK) complex that contributes to the degradation of IκBα. IKK is composed of IKKα, IKKβ and NEMO. Treatment with IKK-16, an IKKα/β inhibitor, significantly diminished UVB-Poly I:C-induced IκBα degradation and thus apoptosis. Silencing either IKKα or NEMO but not IKKβ with corresponding siRNA inhibited apoptosis. Tumor repressor p73, a homologue of p53, is reported to mediate IKKα-induced apoptosis in DNA damage response. Silencing p73 reduced cell apoptosis in UVB-Poly I:C-treated HaCaT cells. In summary, UVB and Poly I:C co-treatment activates IKKα and NEMO, which diminishes anti-apoptotic IκBα, resulting in enhancement of apoptosis through p73. The findings partially clarify the possible molecular mechanism of pro-apoptotic NF-κB pathway activated by TLR3 in the fate of UVB-irradiated epidermis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ultraviolet B (UVB) irradiation exerts multiple effects on skin cells, inducing apoptosis, senescence and carcinogenesis. Toll-like receptor 3, a member of pattern recognition receptors, is reported to initiate inflammation by recognizing double-strand RNA (dsRNA) released from UVB-irradiated cells. It has not been studied, however, whether apoptosis induction in UVB irradiation is attributed to TLR3 activation. Here, we report on the pro-apoptotic role of TLR3 in UVB-irradiated epidermal cells. Poly I:C, an analogue of dsRNA that activates TLR3, was used in combination with sub-lethal UVB (4.8 mJ/cm 2 ) irradiation for investigating the effects of TLR3 activation on human immortalized keratinocyte HaCaT cells. Although sub-lethal dose of either Poly I:C or UVB alone did not induce cell death, UVB-Poly I:C co-treatment synergistically induced cell death by activation of caspase-3 and cleavages of ICAD and PARP, with apoptotic features when stained with Annexin V/PI or Hoechst 33342. Treatment with pan-caspase inhibitor, Z-VAD, attenuated UVB-Poly I:C-induced cell death. Silencing TLR3 by siRNA rescued HaCaT cells from UVB-Poly I:C-induced apoptosis. NF-κB, a major downstream component of TLR3 pathway, that usually negatively regulates the classical TLR3 apoptotic pathway, was analyzed by western blotting and immunofluorescence confocal microscopy. The results indicate to our surprise that NF-κB is translocated to nucleus in the cells co-treated with UVB-Poly I:C. The nuclear translocation of NF-κB is attenuated by TLR3 silencing. Treatment with BAY, an inhibitor of NF-κB pathway, blocked UVB-Poly I:C-induced apoptosis. Therefore, we conclude that NF-κB pathway plays a cytotoxic role in UVB-Poly I:C-treated HaCaT cells, mediating TLR3-related apoptosis., (Copyright © 2018. Published by Elsevier Ltd.)

Background: Although inhibition of SGK1 has been shown to delay cancer progression, the underlying mechanisms have not yet been elucidated., Methods: We investigated the cellular responses to GSK650394 treatment and SGK1 silencing (or overexpression) in human prostate cancer (PCa) cell lines and PC3 xenografts by flow cytometry, western blotting, immunofluorescence, transmission electron microscopy and immunohistochemistry., Results: In the present study, we demonstrated that SGK1 inhibition, mediated by either GSK650394 or SGK1 shRNA, induced G2/M arrest, apoptosis and autophagy. Furthermore, 3MA-mediated autophagy inhibition attenuated SGK1 inhibition-induced apoptosis, suggesting that induction of autophagy precedes apoptosis. Moreover, ectopic expression of SGK1 significantly attenuated the GSK650394-induced effects. Suppression of mTOR and Foxo3a phosphorylation is critical for blockade of SGK1-induced autophagy and apoptosis, at least partially via pFoxo3a (S253)-LC3 and pFoxo3a (S253)-p27 interactions. Dual inhibition of mTOR and SGK1 enhances autophagy activation and leads to synergistic cytocidal effects in PCa cells., Conclusions: In summary, our findings show that SGK1 inhibition exhibits significant antitumour effects against PCa in vitro and in vivo. This study uncovered a novel mechanism of SGK1 inhibition in PCa, which is mediated, at least in part, by inducing autophagy-dependent apoptosis via the mTOR-Foxo3a pathway.

We previously reported that silibinin induced a loss of cell viability in breast cancer (MCF-7) cells by ERα down-regulation. But whether this cytotoxicity depends on another estrogen receptor, ERβ, has yet to be elucidated. Therefore, we sought to explore the effects of ERβ modulation on cell viability by using an ERβ-selective agonist (Diarylprepionitrile, DPN) and an antagonist (PHTPP). Our data demonstrated that ERβ served as a growth suppressor in MCF-7 cells, and the incubation of silibinin, elevated ERβ expression, resulting in the tumor growth inhibition. The cytotoxic effect of silibinin was diminished by PHTPP and enhanced by DPN. Silencing of ERβ by siRNA confirmed these results. Apoptotic cascades, including the sequential activation of caspase-9 and -6, and finally the cleavage of caspase substrates, PARP and ICAD, caused by treatment with silibinin, were all repressed by PHTPP pre-treatment but exacerbated by DPN. Unlike ERα, ERβ did not involve autophagic process in the regulation, since neither autophagic inhibitor (3-MA) nor the inducer (rapamycin) affected the cell survival rates regardless ERβ activity. Taken together, silibinin induced apoptosis through mitochondrial pathway by up-regulating ERβ pathways in MCF-7 cells without the involvement of autophagy., (Copyright © 2016. Published by Elsevier Inc.)

In this study, the effects of mixed corn peptides and synthetic pentapeptide (QLLPF) on hepatocyte apoptosis induced by ethanol were investigated in vivo. QLLPF, was previously characterized from corn protein hydrolysis, which had been shown to exert good facilitating alcohol metabolism activity. Mice were pre-treated with the mixed corn peptides and the pentapeptide for 1 week and then treated with ethanol. After treatment of three weeks, the biochemical indices and the key ethanol metabolizing enzymes, the serum TNF-α, liver TGF-β1 concentrations and the protein expressions related to apoptosis were determined. We found that the Bcl-2, Bax and cytochrome c expressions in the intrinsic pathway and the Fas, FasL and NF-κB expressions in the extrinsic pathway together with higher TNF-α and TGF-β1 concentrations were reversed compared with the model group by both the mixed corn peptides and the pentapeptide. The activation of caspase3 was also suppressed. Additionally, apoptosis was further confirmed with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the TUNEL assay demonstrated peptides suppressed hepatocyte apoptosis. Our results suggest that apoptosis induced by ethanol is alleviated in response to the treatment of corn peptides, potentially due to reversing the related protein expression.

The estrogen receptor alpha (ERα) has been proven to be one of the most important therapeutic targets in breast cancer over the last 30 years. Previous studies pointed out that a natural flavonoid, silibinin, induced apoptosis in human breast cancer MCF-7 cells. In the present study we report that exposure of MCF-7 cells to silibinin led to cell death through the down-regulation of ERα expression. Silibinin-induced apoptosis of MCF-7 cells through up-regulation of caspase 6 due to ERα signalling repression was further boosted by ERα antagonist. Moreover, up-regulation of autophagy induced by silibinin accounted for apoptotic exacerbation, being further enhanced by ERα inhibition. Upon ERα activation, series of downstream signalling pathways can be activated. We found that silibinin reduced the expressions of Akt/mTOR and extracellular-signal-related kinase (ERK), which respectively accounted for the induction of autophagy and apoptosis. These effects were further augmented by co-treatment with ERα inhibitor. We conclude that the treatment with silibinin of ERα-positive MCF-7 cells down-regulates the expression of ERα, and subsequently mTOR and ERK signaling pathways, ERα downstream, finally resulting in induction of autophagy and apoptosis., (Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the effects of interleukin-18 (IL-18) expression on regulating the viability and apoptosis of tongue squamous cell carcinoma (TSCC) cells in vitro and examine the underlying molecular events. Human IL-18 cDNA was cloned into the vector pcDNA3.1 (+) and transfected into CRL-1623™ cells. Quantitative reverse transcription-PCR (RT-qPCR), western blot analysis, immunofluorescence, cell viability MTT assay, flow cytometric Annexin V/propidium iodide (PI), Giemsa staining, and caspase-3 activity assay were performed. The data showed that overexpression of IL-18 protein reduced TSCC cell viability by inducing apoptosis. Compared with cells transfected with the control vector, IL-18 expression activated caspase-3, -7, and -9 by inducing their cleavage and increased the expression of interferon (IFN)-γ and cytochrome c mRNA, but reduced cyclin D1 and A1 expression in TSCC cells. IL-18 expression upregulated the expression and phosphorylation of glycogen synthase kinase (GSK)-3β protein in CRL1623 cells, whereas the selective GSK-3β inhibitor kenpaullone antagonized the effects of IL-18 protein on TSCC cells in vitro. The results indicated that IL-18 played an important role in the inhibition of TSCC cell growth and may be further investigated as a novel therapeutic target against TSCC.

Cucurbitacin B (CuB) is a tetracyclic triterpene that is contained in extracts from cucurbitaceous plants and has been demonstrated to have anticancer and anti‑inflammatory activities. The purpose of the present study was to determine whether CuB exhibits anticancer effects on SH‑SY5Y human neuroblastoma cells and to analyze the underlying molecular mechanism. The results demonstrated that CuB not only induced cell cycle arrest at the G2/M phase, but also induced apoptosis as characterized by positive Annexin V staining, downregulation of phospho‑Janus kinase 2 (p‑JAK2), phospho‑signal transducer and activator of transcription 3 (p‑STAT3), phospho‑extracellular signal‑regulated kinases and the activation of c‑Jun N‑terminal kinase and p38 mitogen activated protein kinase (MAPK). CuB also altered the expression of gene products that mediated cell proliferation (Cyclin B1 and cyclin‑dependent kinase 1), cell survival (B‑cell lymphoma 2, Bcl2‑associated X protein) and increased the expression of p53 and p21. These results provide the evidence that JAK2/STAT3 and MAPKs have crucial roles in CuB‑induced growth inhibition and apoptosis in SH‑SY5Y human neuroblastoma cells.

Interleukin-10 (IL-10) may have therapeutic potential in various inflammatory diseases, including atherosclerosis, as it can inhibit oxLDL-induced foam cell formation and apoptosis in macrophages. This study investigated the effect of IL-10 on mitogen-activated protein kinase (MAPK) activation, and apoptosis induced by oxidized low-density lipoprotein (oxLDL) in cultured human umbilical vein endothelial cells (HUVEC). The results demonstrated that IL-10 significantly blocked the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) and apoptosis induced by oxLDL. The inhibitory effect of IL-10 on oxLDL-induced apoptosis was partially dependent on reduced p38, but not JNK, phosphorylation. This study also discovered a linkage between IL-10 and p38 MAPK signaling in oxLDL-induced endothelial cell apoptosis. Interestingly, this study found that lectin-like oxidized LDL receptor-1 (LOX-1) was the only scavenger receptor, on the surface of HUVEC, that was upregulated by oxLDL and the increase in LOX-1 was not suppressed by IL-10. This study confirmed that IL-10 significantly upregulated the expression of suppressor of cytokine signaling-3 (SOCS3), whereas SOCS3 knockdown by siRNA effectively blocked the inhibitory effect of IL-10 on p38 MAPK-dependent apoptosis induced by oxLDL. These results showed for the first time, that IL-10 modulated oxLDL-induced apoptosis by upregulating SOCS3, which then interrupted p38 MAPK activation in endothelial cells. These findings support the essential role of p38 MAPK in the interplay of oxLDL and IL-10 in endothelial apoptosis., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Ultraviolet B (UVB) from sunlight is a major cause of cutaneous lesion. Silibinin, a traditional hepatic protectant, elicits protective effects against UVB-induced cellular damage. In A431 cells, the insulin-like growth factor-1 receptor (IGF-1R) was markedly up-regulated by UVB irradiation. The activation of the IGF-1R signalling pathways contributed to apoptosis of the cells rather than rescuing the cells from death. Up-regulated IGF-1R stimulated downstream mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinases (JNK) and extracellular signal-regulated protein kinases 1/2 (ERK1/2). The subsequent activation of caspase-8 and caspase-3 led to apoptosis. The activation of IGF-1R signalling pathways is the cause of A431 cell death. The pharmacological inhibitors and the small interfering RNA (siRNA) targeting IGF-1R suppressed the downstream activation of JNK/ERK-caspases to help the survival of the UVB-irradiated A431 cells. Indeed, silibinin treatment suppressed the IGF-1R-JNK/ERK pathways and thus protected the cells from UVB-induced apoptosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Skin inflammation induced by ultraviolet B (UVB) radiation is characterized by migration and chemotaxis of inflammatory cells, epidermic thickening and erythema. Apoptosis and autophagy of epidermal and dermal cells are involved in its development through the adjustment of balance between cell survival and death. In this study, the role of balance between cell survival and apoptosis in dermis and epidermis in UVB-induced skin inflammation and the effect of autophagy on the balance were elucidated, and the protective mechanism of silibinin was investigated through the examination of the influence of autophagy activation or inhibition on erythema, migration, and chemotaxis of inflammatory cells as well as apoptosis adjustments. In UVB-irradiated controls, dermal apoptosis was retarded and the survival of inflammatory cells was promoted through the up-regulation of dermal autophagic level; epidermal apoptosis was increased through the down-regulation of epidermal autophagic level, causing migration and chemotaxis of neutrophils and mast cells as well as skin erythema. In silibinin-treated group (50 mg/kg/day for 4 days), dermal apoptosis was increased through inhibiting dermal autophagy; improper adjustment of epidermal apoptosis was attenuated through promoting epidermal autophagy, presenting dual effects on the balance between autophagy and apoptosis of epidermal and dermal cells and the protection.

This document is a correction notice for an article titled "Circ-ZEB1 promotes PIK3CA expression by silencing miR-199a-3p and affects the proliferation and apoptosis of hepatocellular carcinoma" published in the journal Molecular Cancer. The correction addresses two errors in the figures of the original article, specifically a typo in the subscript of the X-axis in Fig. 1B and an incorrect image in Fig. 3C. The authors provide the correct figures and state that these errors do not affect the overall results and conclusions of the study. The corrected figures show the expression of circ-ZEB1 and PIK3CA in hepatocellular carcinoma tissues and cells, as well as the effects of circ-ZEB1 downregulation on cell proliferation. The correction notice is authored by Weiwei Liu, Lu zheng, Rongguiyi Zhang, Ping Hou, Jiakun Wang, Linquan Wu, and Jing Li. [Extracted from the article]

NLRP3 inflammasome plays a critical spatiotemporal role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). This study reports a mechanistic insight into noncanonical NLRP3 inflammasome activation in microglia for the effector stage of EAE. Microglia-specific deficiency of ASC (apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment [CARD] domain) attenuated T cell expansion and neutrophil recruitment during EAE pathogenesis. Mechanistically, TLR stimulation led to IRAKM-caspase-8-ASC complex formation, resulting in the activation of caspase-8 and IL-1[beta] release in microglia. Noncanonical inflammasome-derived IL- 1[beta] produced by microglia in the CNS helped to expand the microglia population in an autocrine manner and amplified the production of inflammatory cytokines/chemokines. Furthermore, active caspase-8 was markedly increased in the microglia in the brain tissue from patients with multiple sclerosis. Taken together, our study suggests that microglia-derived IL-1[beta] via noncanonical caspase-8-dependent inflammasome is necessary for microglia to exert their pathogenic role during CNS inflammation., IntroductionNumerous studies indicate that the inflammatory process in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is initiated by T cells that are reactive against myelin (1-6). Importantly, a growing [...]

IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide and it is characterized by mesangial IgA deposits. Proteinuria is a common clinical feature of IgAN, which has a critical connection to podocyte injury and has been used as a clinical prognostic factor for IgAN. Evidence has shown that TNF-α released from mesangial cells may lead to podocyte apoptosis. Forty male BALB/c mouse were randomly divided into the control group and IgAN group. A mice model of IgAN was developed by oral administration of bovine serum albumin (BSA) combined with Staphylococcus Enterotoxin B (SEB) tail vein injection. Urinary protein concentrations, renal function, renal morphological, IgA deposition, apoptosis situation, and the mRNA and protein expression of nephrin, podocin, TNF-α, TNFR1, caspase-8 and caspase-3, were detected after 12 weeks. BSA and SEB can successfully establish an IgAN mouse model, and the main pathological changes are the IgA immune complex deposition in the mesangial area. The gene and protein expression levels of nephrin and podocin were found to be downregulated, and death receptor pathway-related indicators were upregulated, and they were involved in TNF-α-activated podocyte injury and apoptosis in IgAN mice. TNF-α may play an important role in the pathogenesis of podocyte apoptosis in IgAN, and its effects may be mediated through the apoptotic death receptor pathway. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) predominantly consists of squamous cells and is the tumor with the highest incidence of the head and neck. Carnosic acid (CA), a natural monomer drug obtained from rosemary and salvia, shows various pharmacological effects, including of tumor development. This study aimed to assess for an effect of CA on the development of OSCC and the underlying mechanisms. In CAL27 and SCC9 cells, CA inhibited cell proliferation and migration, increased intracellular levels of reactive oxygen species (ROS) and Ca2+, decreased the mitochondrial membrane potential (MMP), and promoted apoptosis. In CAL27- and SCC9-xenotransplanted BALB/c nude mice, CA inhibited the tumor growth without affecting the body weight and tissue morphology. CA upregulated Bax, Bad, cleaved Caspase-3 and -9 levels, and the cleaved PARP1/PARP1 ratio but downregulated Bcl-2 in CA-treated OSCC cells and OSCC cells-xenotransplanted BALB/c nude mice. These results indicate that CA suppresses OSCC at least via the mitochondrial apoptotic pathway and offers this natural compound as a potential therapeutic against OSCC. [ABSTRACT FROM AUTHOR]

This study aimed to elucidate the role of ribophorin II (RPN2) in nasopharyngeal cancer (NPC) cell survival and death. RPN2 expression was upregulated in 22 human NPC specimens and 5–8F and CNE1 cells compared with that in adjacent normal tissues and normal nasopharyngeal NP69 cells. CCK-8 and colony formation assays indicated that the silencing of RPN2 hindered the proliferation and growth of 5–8F and CNE1 cells. RPN2 expression was upregulated in 22 human NPC specimens as well as in 5–8F and CNE1 cells compared with that in adjacent normal tissues and NP69 cells. CCK-8 and colony formation assays indicated that the silencing of RPN2 reduced the proliferation and growth of 5–8F and CNE1 cells. Annexin V/PI flow cytometry and Bcl-2/Bax analysis showed that RPN2 silencing led to increased apoptosis. Moreover, JAK1 was found to interact with RPN2, and total JAK1, STAT3, and phosphorylated STAT3 levels were dramatically decreased in cells with RPN2 silencing. Furthermore, the nuclear localization of STAT3 was blocked by the silencing of RPN2. The administration of the STAT3 activator colivelin could offset the inhibitory effect of RPN2 silencing on the survival and apoptosis of NPC cells. RPN2 is upregulated in NPC tissues or cells, and RPN2 silencing repressed NPC cell proliferation and elicited apoptosis. RPN2 overexpression is possibly associated with JAK1/STAT3 silencing and activation. Finally, RPN2 represents a promising target for NPC treatment. [ABSTRACT FROM AUTHOR]

A series of furoxan derivatives of chromone were prepared. The antiproliferative activities were tested against five cancer cell lines HepG2, MCF-7, HCT-116, B16, and K562, and two normal human cell lines L-02 and PBMCs. Among them, compound 15a exhibited the most potent antiproliferative activity. It was also found 15a produced more than 8 µM of NO at the peak time of 45 min by Griess assay. Generally, antiproliferative activity is positively related to NO release to some extent. Further in-depth studies on apoptosis-related mechanisms showed that 15a caused S-phase cell cycle arrest in a concentration-dependent manner and induced apoptosis significantly through mitochondria-related pathways. Human apoptosis protein array assay also demonstrated 15a increased the expression levels of pro-apoptotic Bax, Bad, HtrA2 and Trail R2/DR5. The expression of catalase and cell cycle blocker claspin were similarly up-regulated. In balance, 15a induced K562 cells death through both endogenous and exogenous pathways. [ABSTRACT FROM AUTHOR]

Benzo[b]fluoranthene (BbF) is one of the main pollutants of polycyclic aromatic hydrocarbons (PAHs), which are generated from organic materials combustion and diesel exhaust. It has been reported that after maternal exposure, BbF crosses the placental barrier, leading to offspring defects. However, the effect of BbF on the female reproductive system, especially on oocyte maturation has not been studied. To elucidate the effect and precise mechanism of BbF on oocyte maturation, nuclear, and cytoplasm maturation were evaluated after exposing mouse oocytes to different concentrations of BbF. Results showed that BbF exposure shows no effect on the meiotic progression, but it caused defects on nuclear maturation via impairment on chromosome alignment. In addition, the treatment of BbF displayed the defects on the cytoplasmic maturation by leading to the mitochondrial dysfunction, DNA damage accumulation, early apoptosis and the loss of H3K4me3. To investigate the mechanism, we found that BbF impaired the oocyte maturation via the AMPK pathway. BbF exposure caused the phosphorylation of AMPK, which cause the DNA damage accumulation and apoptotic incidence. Taken together, our results demonstrated that BbF exposure impaired the mouse oocyte maturation due to mitochondrial dysfunction and early apoptosis. [ABSTRACT FROM AUTHOR]

Amylin and amyloid-β (Aβ) were found to induce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat pancreatic β-cell line, INS-1 cells, leading to cell death. In this study, we report on reciprocal relationship between the expression of estrogen receptors (ERs) α and β (ERα and ERβ) and generation of ROS/RNS in amylin/Aβ1-42 -treated INS-1 cells. That is, pharmacological activation of ERs in INS-1 cells significantly decreases ROS/RNS generation, but blockage of ERs increases ROS/RNS generation. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with phytoestrogen activities, also known as silybin. Treatment with silibinin down-regulated ROS/RNS production induced by treatment with amylin/Aβ1-42 in the cells. Silencing ERs expression with siRNAs targeting ERs showed that the protective effect of silibinin was markedly weakened, indicating that silibinin protection is largely attributed to activation of ERs' signaling. The binding of silibinin to ERs implies that the protective effect of silibinin on amylin/Aβ1-42 -treated INS-1 cells owes to down-regulation of ROS/RNS through the activation of ERs phosphorylation. Amylin and Aβ1-42 cotreatment enhanced furthermore ROS/RNS generation and cytotoxicity through further down-regulation of ERs phosphorylation, and this was reversed by silibinin. Silibinin also protects INS-1 cells from amylin and Aβ1-42 cotreatment. These results indicate that protective effect of silibinin is mediated by enhancement of ERs phosphorylation that depresses ROS/RNS generation in amylin/Aβ1-42 -treated INS-1 cells. [ABSTRACT FROM AUTHOR]

Ultraviolet B ( UVB) is a major cause of skin inflammation, leading to skin damage. Our previous in vivo study revealed that a natural flavonoid silibinin had marked anti-inflammatory effect on UVB-exposed murine skin. UVB exposure caused reduced autophagy in epidermis while it promoted autophagy in dermis. Nevertheless, silibinin inhibited the inflammatory flux in the skin epidermis as well as dermis through the modulation of autophagy. In order to elucidate the underlying protective mechanisms of silibinin for UVB damage on skin, separate studies on epidermis and dermis are helpful. Derived from the normal tissue of the mouse, L929 cells are capable of representing some characteristics of dermal cells. UVB irradiation caused L929 cell apoptosis in a time- and dose-dependent manner. Ataxia-telangiectasia-mutated ( ATM) protein and p53 were activated to cause cell apoptosis, accompanying upregulation of the autophagic flux. The pharmacological inhibition of ATM, p53 and autophagy or the transfection with autophagy-associated protein-targeted small interfering RNAs showed that the UVB-activated ATM-p53 axis and autophagy formed a positive feedback loop, which synergistically promoted cell apoptosis. Silibinin treatment simultaneously repressed the activation of ATM-p53 and autophagy and thereby protected UVB-irradiated L929 cells from apoptotic death. [ABSTRACT FROM AUTHOR]

Highlights: [•] Silibinin-induced autophagy protects A431 cells from apoptosis induced by UVB. [•] Activation of the IGF-1R-Akt axis by UVB results in the reduction of autophagy. [•] Silencing of the autophagy-related molecules reverses silibinin’s protection. [Copyright &y& Elsevier]

Apoptosis in murine dermal cells is retarded by ultraviolet B (UVB) irradiation-induced autophagic intervention while simultaneously epidermal cells commit apoptosis, during which inflammatory cytokines released from the lost epidermal cells promote immune responses of dermal inflammatory cells, forming morphological symptoms of acute cutaneous diseases. Autophagy is involved in prevention or provocation of apoptosis of dermal or epidermal cells of UVB-irradiated mice via modulation of intracellular metabolism, intervening the balance between cell death and survival in dermis and epidermis. p53 expressed in immune system affects autophagy function through activating or inactivating genes encoding apoptotic factors and inflammatory cytokines. Silibinin protects dermal and epidermal cells of UVB irradiated skin against abnormally autophagy-mediated apoptosis adjustments. In this study, how UVB irradiation intervenes autophagy in dermal and epidermal cells as well as how silibinin protects UVB irradiated skin through physiological recovering of autophagy function in dermis and epidermis are focused and elucidated preliminarily. Silibinin treatment (50 mg/kg/day for 4 days) reversed dermal and epidermal autophagy levels from UVB irradiation-induced improper autophagy intervention, repaired the balance between cell survival and death in dermis and epidermis, and protected skin against damage through mediation of p53 activation in dermal and epidermal cells. [ABSTRACT FROM PUBLISHER]

Our previous studies showed that silibinin promoted activation of caspases to induce apoptosis in human breast cancer MCF-7 cells by down-regulating the protein expression level of estrogen receptor (ER) α and up-regulating ERβ. Recently, it has been reported that silibinin-induced apoptosis also involved nuclear translocation of apoptosis-inducing factor (AIF). Here we report that silibinin induces nuclear translocation of AIF through the down-regulation of ERα and up-regulation of ERβ in a concentration dependent manner in MCF-7 cells. AIF knockdown with siRNA significantly reverses silibinin-induced apoptosis. The nuclear translocation of AIF is enhanced by treatment with MPP, an ERα antagonist, and blocked with PPT, an ERα agonist. In contrast to ERα activity, the nuclear AIF is increased with an ERβ agonist, DPN and blocked with an ERβ antagonist, PHTPP. Autophagy, negatively regulated by ERα, positively controls AIF-mediated apoptosis, as evidenced by the preventive effect of autophagy inhibitor 3-MA and siRNA targeting LC3, on the nuclear translocation of AIF and cell death induced by silibinin co-treatment with or without MPP. In sum we conclude that AIF in nuclei is involved in silibinin-induced apoptosis, and the nuclear translocation of AIF is increased by down-regulated ERα pathway and/or up-regulated ERβ pathway in MCF-7 cells, accompanying up-regulation of autophagy. Image 1 • Silibinin induces apoptosis-inducing factor-dependent apoptosis in MCF-7 cells. • Estrogen receptor α reduced by silibinin inhibits AIF nuclear translocation. • Estrogen receptor β increased by silibinin promotes AIF nuclear translocation. • Autophagy under the negative control of estrogen receptor α increases nuclear AIF. [ABSTRACT FROM AUTHOR]

Ultraviolet B (UVB) irradiation causes skin inflammation and apoptosis. Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in skin damages, little is known about the roles of mitochondrial dynamics in these processes. UVB irradiation increases abnormal mitochondrial content but decreases mitochondrial volume in immortalized human keratinocyte HaCaT cells. UVB irradiation resulted in marked upregulation of mitochondrial fission protein dynamin-related protein 1 (DRP1) and downregulation of mitochondrial outer membrane fusion proteins 1 and 2 (MFN1 and MFN2) in HaCaT cells. Mitochondrial dynamics was discovered to be crucial for NLRP3 inflammasome and cGAS-STING pathway activation, as well as the induction of apoptosis. Inhibition of mitochondrial fission by treatments with a DRP1 inhibitor, mdivi-1, or with DRP1-targeted siRNA, efficiently prevented UVB-induced NLRP3/cGAS-STING mediated pro-inflammatory pathways or apoptosis in the HaCaT cells, whereas inhibition of mitochondrial fusion with MFN1and 2 siRNA increased these pro-inflammatory pathways or apoptosis. The enhanced mitochondrial fission and reduced fusion caused the up-regulation of reactive oxygen species (ROS). Application of an antioxidant, N -acetyl- l -cysteine (NAC), which scavenges excessive ROS, attenuated inflammatory responses through suppressing NLRP3 inflammasome and cGAS-STING pathway activation, and rescued cells from apoptosis caused by UVB-irradiation. Together, our findings revealed the regulation of NLRP3/cGAS-STING inflammatory pathways and apoptosis by mitochondrial fission/fusion dynamics in UVB-irradiated HaCaT cells, providing a new strategy for the therapy of UVB skin injury. [Display omitted] • UVB perturbs mitochondrial fission/fusion dynamics in HaCaT cells. • Disturbed mitochondrial dynamics control the amount of mitochondria. • Disturbed mitochondrial dynamics regulate NLRP3/cGAS-STING mediated pro-inflammatory pathways and apoptosis. • ROS are responsible for pro-inflammatory pathways and apoptosis initiated by the dysregulated mitochondrial dynamics. [ABSTRACT FROM AUTHOR]

Ultraviolet B (UVB) irradiation causes skin damages. In this study, we focus on the involvement of mitochondrial disorders in UVB injury. Surprisingly, UVB irradiation increases the amounts of mitochondria in human immortalized keratinocytes HaCaT. However, further analysis shows that ATP levels decreased by UVB treatment in accordance with the collapse of mitochondrial membrane potential (MMP), suggesting an accumulation of dysfunctional mitochondria in UVB-irradiated HaCaT cells. Mitophagy, mainly mediated by PINK1 and parkin, is critical for the elimination of damaged mitochondria. Western blot results show that the levels of both PINK1 and parkin are decreased in UVB-irradiated cells, indicating the impairment of mitophagy. Silencing the expression of PINK1 or parkin by transfection of siRNA shows essentially the same damage to the cells as UVB irradiation does, including increased mitochondrial amount, decreased MMP and ATP production, and enhanced apoptosis, evidencing that repression of PINK1/parkin-mediated mitophagy plays a primary cause of UVB-caused cells damages. We previously found that HaCaT cells exposed to UVB showed activation of the cGAS-STING pathway and apoptosis. Here, silencing PINK1 or parkin also increases the protein levels of cGAS and STING, facilitates nuclear accumulation of NF-κB, and promotes the transcription of IFNβ, suggesting for the activation of STING pathway. Mitophagy impairment either by UVB-irradiation or by PINK1/parkin silencing initiates caspase-3-mediated apoptosis, as shown by the activation of caspase-3 and cleavage of PARP, as well as the increase of Hoechst-positive stained cells and Annexin V-positive cells. Further studies find that Bax-mediated permeabilization of mitochondrial membrane is critical for cell apoptosis, as well as the cytosolic leakage of mtDNA in UVB-treated cells, which results in cGAS-STING activation, and these processes are negatively-regulated by PINK1/parkin-mediated mitophagy. This study reveals the involvement of dysfunctional mitochondria due to impaired mitophagy in the damaging effect of UVB irradiation on HaCaT cells. Restoring the mitophagy has the potential to be developed as a new strategy to protect skin from UVB damages. [Display omitted] • UVB decreases mitophagy and accumulates dysfunctional mitochondria in HaCaT cells. • UVB reduces PINK1/parkin expression that is involved in mitophagy of damaged mitochondria. • Mitophagy arrest caused by UVB leads to the activation of cGAS-STING/apoptosis pathway. • Mitophagy arrest caused by UVB increases mitochondrial membrane permeability. • Mitophagy arrest caused by UVB results in the mtDNA leakage into cytosol. [ABSTRACT FROM AUTHOR]

Abstract In our previous study, we demonstrated that oridonin enhances phagocytosis of apoptotic bodies by macrophage-like cells by inducing autophagy. However, the direct sensor of autophagy and the key event controlling phagocytosis remains unknown. Herein, we showed that Toll-like receptor 4 (TLR4), known to mediate immune responses, was activated by oridonin. Activated TLR4 contributes to phagocytosis of apoptotic cells by RAW264.7 macrophages. Indeed, inhibition or small interfering RNA (siRNA) silencing of TLR4 significantly attenuated oridonin-induced phagocytosis. Inhibition of TLR4 also decreased the level of autophagy and its associated proteins, Beclin-1 and light chain 3 (LC3), suggesting that activated TLR4 is involved in activation of autophagy. LPS-induced activation of TLR4 promoted phagocytosis and autophagy progression. Activation of TLR4 accompany increase in activities of lysosome acid phosphatase and cathepsin B as well as in up-regulation of lysosomal-associated membrane protein (LAMP 1 and 2) levels. Furthermore, TLR4 in association with translocation to cytoplasm leads to macrophage motility or migration through increased plasticity of skeleton and/or membrane structure. These results suggest that oridonin-induced phagocytosis of apoptotic bodies by macrophages is TLR4 signal pathway-mediated, via activation of the autophagy-lysosome pathway as well as increase of cell migration. Graphical abstract Unlabelled Image Highlights • Oridonin might possess binding sites with TLR4 and activate TLR4 signal pathways. • Activation of TLR4 promotes oridonin-induced phagocytosis of apoptotic cells by Raw264.7 macrophages. • Activated TLR4 enhances phagocytosis of apoptotic bodies by mediating autophagy and lysosome pathway. • Activated TLR4 translocates into cytoplasm, promoting formation of phagolysosomes that may accompany increased cell motility or migration. [ABSTRACT FROM AUTHOR]

Abstract: We reported previously that phagocytosis of apoptotic cells by U937 cells was enhanced by the treatment with oridonin that showed high activity to induce the generation of reactive oxygen species (ROS) in many cells. ROS, important signaling molecules, are involved in the immune defenses, cell repair and proliferation. In this study, oridonin caused modest amount of ROS generation in U937 cells, with hydrogen peroxide (H2O2) and hydroxyl free radical (e major types. Meanwhile, H2O2 and ositive regulators involved in oridonin-enhanced engulfment of apoptotic cells through down-regulating mitochondrial membrane potential (MMP) and inducing autophagy. The ROS-mediated phagocytosis was independent of cellular adenosine triphosphate (ATP) levels. H2O2 and tion also activated phosphatidylinositol 3-kinases-Akt (PI3K-Akt) and phospholipase C γ-protein kinase C(PLC γ)-Ras-Raf-ERK signaling pathways, which were essential for oridonin-induced engulfment of apoptotic cells. Phagocytosis, the loss of MMP, autophagy and the activated signaling pathways were all suppressed by ROS scavenger N-acetyl-l-cysteine (NAC), H2O2 scavenger catalase or ger glutathione (GSH). However, superoxide anion (O2 and its scavenger superoxide dismutase (SOD) did not significantly affect these oridonin-induced biological processes. [Copyright &y& Elsevier]