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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.)

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.)

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.)

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.)

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]

Silibinin is a natural polyphenolic flavonoid, isolated from the seeds of the milk thistle of Silybum marianum (L.) Gaertn. Silibinin has been widely used clinically as a traditional medicine for liver diseases. This study investigated the protective role of silibinin in ethanol- or acetaldehyde-induced apoptosis in human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells, focusing on elucidation of the underlying mechanism in vitro. The toxicity of ethanol or acetaldehyde was evaluated by MTT assay. Apoptosis-related proteins, mitochondrial fission-associated proteins and mitochondrial fusion-associated proteins were analyzed by western blotting and immunofluorescence microscopy. Present experimental results demonstrated that silibinin improved cell viability, reduced the enzyme activities of AST/ALT and ALDH/ADH, inhibited apoptosis and recovered mitochondrial function in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. Silibinin reduced the expression of mitochondrial fission-associated proteins, dynamin-related protein 1 (DRP1), but increased mitochondrial fusion-associated proteins, optic atrophy 1 (OPA1) and mitofusin 1 (MFN1). Accordingly, inhibition of DRP1 activity with its pharmacological inhibitor or siDRP1 efficiently attenuated ethanol- or acetaldehyde-induced apoptosis, whereas activation of DRP1 by using staurosporine (STS) further increased apoptosis in ethanol- or acetaldehyde-treated HepG2 or HL7702 cells. The results show that silibinin protects cells against ethanol- or acetaldehyde-induced mitochondrial fission that results in apoptosis. • Ethanol or acetaldehyde induces apoptosis in HepG2 and HL7702 cells. • Silibinin reduces ethanol- or acetaldehyde-induced apoptosis. • Ethanol or acetaldehyde aggravates mitochondrial fission resulting in apoptosis. • Silibinin restores ethanol- or acetaldehyde-caused mitochondrial disorders. [ABSTRACT FROM AUTHOR]

Silibinin, one of the flavonoids isolated from milk thistle seeds of Silybum marianum , has hepatoprotective properties against toxins in clinical. However, the detailed mechanisms have remained unclear. This study investigates the underlying mechanism of silibinin in the protection against ethanol- or acetaldehyde-induced damage of neonatal mouse primary hepatocytes in vitro. The results show that ethanol inhibited proliferation of hepatocytes in a time (12, 24, 36 h) and dose-dependent (0–800 mM) manner. However, silibinin did not show protective effect on ethanol (500 mM)-induced suppression of hepatocyte proliferation. Acetaldehyde, the toxic metabolite of ethanol, appearing immediately in individuals after drink also inhibited the proliferation of hepatocytes in a dose-dependent (0–12 mM) manner. Surprisingly, silibinin significantly increased the cell viability and reduced the leakage of alanine amino transferase (ALT) and aspartate amino transferase (AST) in acetaldehyde-treated hepatocytes, suggesting that silibinin protected cell injury caused by acetaldehyde treatment. The apoptosis-inducing effect of acetaldehyde was demonstrated by the increased number of cells in sub-G1 phase as well as caspase-3 activation. Further study shows that acetaldehyde induced autophagy in the hepatocytes. The autophagy inhibitors, 3-Methyladenine (3-MA) and chloroquine (CQ), further decreased the viability of cells treated with acetaldehyde, suggesting that autophagy plays a protective role against apoptosis. Consistently, silibinin (20 μM) significantly reduced the activation of caspase 3 or apoptosis and increased the conversion of LC3-I to LC3-II or autophagy. Taken together, it is concluded that silibinin does not repress the ethanol- induced hepatocyte injury, whereas silibinin reduces acetaldehyde-caused hepatocyte injury through down-regulation of apoptosis and up-regulation of autophagy. • Acetaldehyde (ACH) induced apoptosis in liver cells; • AcH induced autophagy in liver cells; • Silibinin protects AcH-induced death, but not ethanol-induced death of hepatocytes. [ABSTRACT FROM AUTHOR]