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Background/aims: Diabetic retinopathy is the most common eye disease that causes blindness in the working population. Neurodegeneration is the early sign of diabetic retinopathy, but no drug has been approved for delaying or reversing retinal neurodegeneration. Huperzine A, a natural alkaloid isolated from Huperzia serrata, displays neuroprotective and antiapoptotic effects in treating neurodegenerative disorders. Our study aims to investigate the effect of huperzine A in preventing retinal neurodegeneration of diabetic retinopathy and its possible mechanism., Methods: Diabetic retinopathy model was induced by streptozotocin. H&E staining, optical coherence tomography, immunofluorescence staining and angiogenic factors were used to determine the degree of retinal pathological injury. The possible molecular mechanism was unrevealed by network pharmacology analysis and further validated by biochemical experiments., Results: In our study, we demonstrated that huperzine A has a protective effect on the diabetes retina in a diabetic rat model. Based on the network pharmacology analysis and biochemical studies, huperzine A may treat diabetic retinopathy via key target HSP27 and apoptosis-related pathways. Huperzine A may modulate the phosphorylation of HSP27 and activate the antiapoptotic signalling pathway., Conclusion: Our findings revealed that huperzine A might be a potential therapeutic drug to prevent diabetic retinopathy. It is the first-time combining network pharmacology analysis with biochemical studies to explore the mechanism of huperzine A in preventing diabetic retinopathy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Cholangiocarcinoma (CCA) is a malignant tumor with a poor prognosis. The specific mechanism of photodynamic therapy (PDT) in treating CCA remains unclear. This study aims to investigate the mechanisms of PDT in the treatment of CCA and try to improve the therapeutic effect of PDT by intervening associated signaling pathways., Methods: The Cell Counting Kit-8 (CCK-8) was used to examine the cytotoxicity of CCA cell lines following PDT. Apoptosis and reactive oxygen species (ROS) levels were measured by flow cytometry. A transmission electron microscope was used to study the changes in cell mitochondria after PDT. The levels of glutathione (GSH), malondialdehyde (MDA), ferrous iron (Fe 2+ ), lactate dehydrogenase (LDH), and lipid peroxide (LPO) were determined. Changes in the expression of apoptosis and ferroptosis-related proteins were determined using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Xenograft tumor models were developed to investigate the effects of PDT on tumor proliferation, apoptosis, and ferroptosis in vivo., Results: PDT inhibited tumor proliferation and induced apoptosis both in vivo and in vitro. This treatment led to swelling and damage of the mitochondria in affected cells. Furthermore, ROS levels rose, accompanied by an increase in the proportion of apoptotic-positive cells. The expressions of Bax and Caspase-3 were upregulated, while the Bcl-2 was downregulated. Meanwhile, PDT triggered ferroptosis, marked by decreased expressions of GPX4 and SLC7A11, and reduced GSH levels. This was accompanied by upregulation of P53 expression and heightened levels of Fe 2+ , LPO, MDA, and LDH. After inducing the ferroptosis pathway, the therapeutic effect of PDT was enhanced, the tumor tissue was further reduced, and the degree of malignancy was reduced., Conclusion: PDT promotes apoptosis and ferroptosis of cholangiocarcinoma cells by activating the P53/SLC7A11/GPX4 signaling pathway and inhibits the growth of cholangiocarcinoma. Inducing ferroptosis can enhance the effectiveness of photodynamic therapy., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Previous studies reveal that hydrogen sulphide (H 2 S) exerts neuroprotection against neurotoxin-induced Parkinson's disease (PD), but the underlying mechanism remains elusive. The present study was aimed to investigate whether H 2 S inhibits neuronal apoptosis of substantia nigra with the involvement of autophagy via promoting leptin signalling in 6-hydroxydopamine (6-OHDA)-induced PD rats. In this study, neuronal apoptosis was analysed by TUNEL staining, the activity of caspase-3 was measured by Caspase-3 fluorometric assay kit, the expressions of Bax, Bcl-2, Beclin-1, LC3II, P62 and leptin were determined by Western blot analysis, and the numbers of autophagosomes and autolysosomes were assessed by transmission electron microscopy. Results showed that NaHS, a donor of exogenous H 2 S, mitigates 6-OHDA-induced the increases in the numbers of TUNEL-positive cells, the activity of caspase-3 and the expression of Bax, and attenuates 6-OHDA-induced a decrease in the expression of Bcl-2 in substantia nigra of rats. In addition, 6-OHDA enhanced the expressions of Beclin-1, LC3-II and P62, increased the number of autophagosomes, and decreased the number of autolysosomes in the substantia nigra, which were also blocked by administration of NaHS. Furthermore, NaHS reversed 6-OHDA-induced the down-regulation of leptin expression in the substantia nigra, and treatment with leptin-OBR, a blocking antibody of leptin receptor, attenuated the inhibition of NaHS on neuronal apoptosis and the improvement of NaHS on the blocked autophagic flux in substantia nigra of 6-OHDA-treated rats. Taken together, these results demonstrated that H 2 S attenuates neuronal apoptosis of substantia nigra depending on restoring impaired autophagic flux through up-regulating leptin signalling in PD., (© 2021 John Wiley & Sons Australia, Ltd.)

Purpose: Cervical carcinoma is the second most common malignancy in females and most of the cases are found in developing countries. The objectives of the present study were (a): to demonstrate the antiproliferative and apoptotic effects of Achillea falcata (A.falcata) extract in human cervical cancer cells (HeLa), and (b): to study the effect of the extract on cellular morphology, cell cycle phase distribution and mitochondrial membrane potential., Methods: MTT assay was used to evaluate the anticancer effect of the extract on HeLa cells. Phase contrast, fluorescence microscopy and transmission electron microscopy (TEM) were used to investigate the morphological changes in these cancer cells after extract treatment. Flow cytometry was used to evaluate the effects of the extract on cell cycle and mitochondrial membrane potential., Results: The results revealed that A. falcata extract led to a significant antiproliferative effect in HeLa cancer cells. The extract induced cellular shrinkage, chromatin condensation and appearance of apoptotic bodies which are the hallmarks of cellular apoptosis. TEM results showed that extract-treated cells had nuclear membrane which was hemispherical and the nuclear chromatin was concentrated and bundled on the inner border of karyotheca. The endoplasmic reticulum also became enlarged in the inner segment. The extract also induced G2/M phase cell cycle arrest along with loss of mitochondrial membrane potential., Conclusion: Achillea falcata extract induced potent antiproliferative and apoptotic effects in HeLa cells. This was accompanied by cellular shrinkage, chromatin condensation, G2/M phase cell cycle arrest and a loss of mitochondrial membrane potential in these cancer cells.

Background: Osteoporosis is caused by excessive bone resorption (by osteoclasts) over bone formation (by osteoblasts). Monocytes are important to osteoporosis by serving as progenitors of osteoclasts and produce cytokines for osteoclastogenesis., Aim: To identify osteoporosis-related genes, we performed microarray analyses of monocytes using Affymetrix 1.0 ST arrays in 42 (including 16 pre- and 26 postmenopausal) high hip BMD (bone mineral density) vs. 31 (including 15 pre- and 16 postmenopausal) low hip BMD Caucasian female subjects. Here, high vs. low BMD is defined as belonging to top vs. bottom 30% of BMD values in population., Method: Differential gene expression analysis in high vs. low BMD subjects was conducted in the total cohort as well as pre- and post-menopausal subjects. Focusing on the top differentially expressed genes identified in the total, the pre- and the postmenopausal subjects (with a p <5E-03), we performed replication of the findings in 3 independent datasets of microarray analyses of monocytes (total N = 125)., Results: We identified (in the 73 subjects) and successfully replicated in all the 3 independent datasets 2 genes, DAXX and PLK3. Interestingly, both genes are apoptosis induction genes and both down-regulated in the low BMD subjects. Moreover, using the top 200 genes identified in the meta-analysis across all of the 4 microarray datasets, GO term enrichment analysis identified a number of terms related to induction of apoptosis, for which the majority of component genes are also down-regulated in the low BMD subjects. Overall, our result may suggest that there might be a decreased apoptosis activity of monocytes in the low BMD subjects., Conclusion: Our study for the first time suggested a decreased apoptosis rate (hence an increased survival) of monocytes, an important osteoclastogenic cell, as a novel mechanism for osteoporosis.

Kashin-Beck disease (KBD) is a serious osteoarthropathia, mainly characterized by excessive chondrocyte necrosis and apoptosis. The molecular signaling pathways underlying KBD excessive chondrocyte apoptosis remain unclear, leading to a lack of effective medical interventions now. To clarify whether expression quantitative trait loci (eQTLs) contribute to excessive chondrocyte apoptosis of Kashin-Beck disease through regulating the expression of apoptosis pathways. We conducted a genome-wide eQTLs based pathway association analysis of KBD using Affymetrix Human SNP Array 6.0 in 1717 Chinese Han subjects. PLINK software was used for genome-wide association study (GWAS) of KBD. A modified gene set enrichment algorithm was applied for pathway association analysis based on GWAS results. The KBD-associated pathways were compared with abnormally expressed pathways in KBD articular cartilage, identified by microarray study of KBD. We identified 4 eQTLs pathways, which were not only significantly associated with KBD, but also abnormally expressed in KBD articular cartilage, including REACTOME&#95;INTRINSIC&#95;PATHWAY&#95;FOR&#95;APOPTOSIS (P=0.008), MAHAJAN &#95;RESPONSE&#95;TO&#95;IL1A&#95;UP (P=0.010), KEGG&#95;PEROXISOME (P=0.005) and MARKS&#95;HDAC&#95;TARGETS&#95;UP (P=0.006). Our results suggest that eQTLs contributed to KBD excessive chondrocyte apoptosis through regulating the expression of apoptosis related pathways. This study provides novel insight into the genetic susceptibility and therapeutic rationale of KBD., (Copyright © 2014. Published by Elsevier B.V.)

Since abnormal post-translational modifications or gene mutations of tau have been detected in over twenty neurodegenerative disorders, tau has attracted widespread interest as a target protein. Among its various post-translational modifications, phosphorylation is the most extensively studied. It is recognized that tau hyperphosphorylation is the root cause of neurodegeneration in Alzheimer's disease (AD); however, it is not clear how it causes neurodegeneration. Based on the findings that tau hyperphosphorylation leads to the escape of neurons from acute apoptosis and simultaneously impairs the function of neurons, we have proposed that the nature of AD neurodegeneration is the consequence of aborted apoptosis induced by tau phosphorylation. Therefore, proper manipulation of tau hyperphosphorylation could be promising for arresting AD neurodegeneration. In this review, the neuroprotective and neurodegenerative effects of tau hyperphosphorylation and our thoughts regarding their relationship are presented.

Objective: To investigate the effect of culture supernatant of Toxoplasma gondii on the proliferation and Toxoplasma gondii tachyzoites with seed counts of 2 x 10(7)/ml, apoptosis of human gastric cancer BGC-823 cells., Methods: 4 x 10(7)/ml, and 8 x 10(7)/ml harvested from infected mice were cultured for 24 h, and then the culture supernatant was collected. BGC-823 cells (5 x 10(4)/ml) at mid-exponential phase were incubated with different concentrations of culture supernatants of Toxoplasma gondii tachyzoites. While in control group, the same volume of DMEM was given. At 24, 48 and 72 h after incubation, the measurement of tumor cell growth inhibition rate was performed by using CCK-8 kit. Cell apoptosis was observed under a fluorescence microscope after 24 h incubation. The DNA ladder zone of apoptosis cells was analyzed with the method of agarose gel electrophoresis. Flow cytometric analysis was used to analyze cell cycle for cell proliferation index and the expression of p53 and Bcl-2., Results: The culture supernatant of Toxoplasma tachyzoites inhibited the proliferation of BGC-823 cells and the growth inhibition rates increased with the time and the concentration. The highest rate reached at the 72nd hour when the concentration of Toxoplasma tachyzoites was 8 x 10(7)/ml. Apoptotic bodies were found in experimental group. The amount of apoptotic body was positively associated with the tachyzoite concentration. DNA fragment in the treated cells after 24 h incubation was revealed by agarose electrophoresis. When the proportion of BGC-823 cells in G0/G1 phase increased, the proportion of cells in S phase decreased. Cell proliferation index decreased with the increase of the concentration of tachyzoites. The proliferation index (0.36) of the cells cultured with culture supernatant of 8 x 10(7)/ml tachyzoites was significantly lower than that of control group (0.6, P < 0.05). p53 protein expression was higher in experiment group than that of the control, whereas Bcl-2 protein expression in experiment group was lower than that of the control (P < 0.05)., Conclusion: The culture supernatant of Toxoplasma gondii can inhibit the proliferation of BGC-823 cells and cause apoptosis of BGC-823 cells, which may be related with up-regulating p53 expression and down-regulating Bcl-2 expression.

Lung cancer remains a deadly disease with unsatisfactory overall survival. Resveratrol (Res) has the potential to inhibit growth of several types of cancer such as prostate and colorectal examples. In the current study, we evaluated in vitro and in vivo anticancer efficiency of Res in a xenograft model with A549 cells. Cell inhibition effects of Res were measured by MTT assay. Apoptotis of A549 cells was assessed with reference to caspase-3 activity and growth curves of tumor volume and bodyweight of the mice were measured every two days. In vitro cytotoxicity evaluation indicated Res to exert dose-dependent cell inhibition effects against A549 cells with activation of caspase-3. In vivo evaluation showed Res to effectively inhibit the growth of lung cancer in a dose- dependent manner in nude mice. Therefore, we believe that Res might be a promising phytomedicine for cancer therapy and further efforts are needed to explore this potential therapeutic strategy.

Lung cancer is one of the most common human cancers and the leading cause of cancer-related death worldwide. The identification of lung cancer associated genes is essential for the diagnosis and treatment of this disease. Lung cancer metastasis-related protein 1 (LCMR1) is a novel gene cloned by differential display polymerase chain reaction in our laboratory. This study was designed to investigate the role of LCMR1 in the generation of apoptosis in lung cancer cells. RNA interference (RNAi) approach was used to knock down LCMR1 in lung cancer cells. The impacts of LCMR1 knockdown on the apoptosis of lung cancer cells were assessed using flow cytometry, quantitative real-time RT-PCR and western blotting. The results demonstrated that the inhibition of LCMR1 by RNAi enhanced apoptotic activity in lung cancer cells and this process required the involvement of p53. In addition, LCMR1 knockdown resulted in the upregulation of Bax and downregulation of Mcl-1. In conclusion, LCMR1 suppresses apoptosis in lung cancer cells and this effect is associated with multiple apoptosis-related proteins, including p53, Bax and Mcl-1. These results provide new insights into the potential role of LCMR1 in the regulation of lung cancer carcinogenesis.

The neural dysfunction in Alzheimer's disease (AD) could arise from endoplasmic reticulum (ER) stress and deficits of the unfolded protein response (UPR). To explore whether tau hyperphosphorylation, a hallmark of AD brain pathologies, plays a role in ER stress-induced alterations of cell viability, we established cell lines with stable expression of human tau (HEK293/tau) or the vector (HEK293/vec) and treated the cells with thapsigargin (TG), an ER stress inducer. We observed that the HEK293/tau cells were more resistant than the HEK293/vec cells to the TG-induced apoptosis, importantly, a time dependent increase of tau phosphorylation at Thr205 and Thr231 sites was positively correlated with the inhibition of apoptosis. We also observed that expression of tau upregulated phosphorylation of PERK, eIF2 and IRE1 with an increased cleavage of ATF6 and ATF4. The potentiation of UPR was also detected in HEK293/tau cells treated with other ER stress inducers, including staurosporine, camptothecin and hydrogen peroxide, in which a suppressed apoptosis was also shown. Our data suggest that tau hyperphosphorylation could attenuate the ER stress-induced apoptosis with the mechanism involving upregulation of UPR system.

Phosphorylation of tau, a major microtubule-associated protein, has been recently discovered to affect cell apoptosis. While the phosphorylation of tau is dynamically regulated, the role of tau dephosphorylation in cell viability is elusive. Here, we observed that the cells bearing high level of the dephosphorylated tau at Tau-1 epitope were more vulnerable to the apoptosis induced by staurosporine, camptothecin, and hydrogen peroxide, though the general outcome of tau expression was still anti-apoptotic. Further studies demonstrate that co-expression of tau and protein phosphatase 2A catalytic subunit (PP2Ac), the most active tau phosphatase, potentiates cell apoptosis with a correlatively increased dephosphorylation of tau and phosphorylation of Bcl-2 at Ser87 (pS87-Bcl2, the inactive form of the anti-apoptotic factor), whereas expression of PP2Ac alone in the absence of tau decreases the levels of pS87-Bcl2 and cleaved PARP, markers of early apoptosis. Finally, both tau and Bcl-2 were co-immunoprecipitated with PP2Ac, but the binding level of Bcl-2 with PP2Ac decreased prominently when tau was co-expressed. These data suggest that tau dephosphorylation by PP2Ac facilitates cell apoptosis with the mechanisms involving a failed dephosphorylation/activation of Bcl-2.

It has been a puzzle why the tangle-bearing neurons in Alzheimer's disease (AD) brain do not die preferentially of apoptosis even though they are actually challenged by multiple proapoptotic factors. Recently, we have reported that phosphorylation of tau can antagonize apoptosis induced by exogenous apoptotic inducers. Amyloid-beta (Abeta), a recognized endogenous proapoptotic factor, is significantly increased in the AD brains, however, it is not known whether tau could abate the Abeta-potentiated apoptosis. Here, we observed that the cells bearing high level of Abeta were more vulnerable than the controls to H2O2-induced apoptosis, and this effect of Abeta was associated with decrease of Bcl-2, elevation of Bax and cytosolic cytochrome-c, as well as activation of caspase-3, suggesting that Abeta could potentiate the oxidant-induced cell apoptosis with involvement of mitochondria-caspase-3 pathway. More importantly, we also found that expression of tau that became hyperphosphorylated could reduce the Abeta-potentiated apoptosis with simultaneous preservation of Bcl-2 and suppression of Bax, cytosolic cytochrome-c, and caspase-3 activity, implying that overexpression of tau that became hyperphosphorylated can attenuate the Abeta-potentiated cell apoptosis through mitochondria-caspase-3 pathway. These findings provide an explanation of the chronic nature of neurodegeneration of neurons with neurofibrillary pathology of abnormal hyperphosphorylated tau in AD and related tauopathies.

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, showed anti-oxidant neuroprotective property in our previous study. However, it is still unknown whether PCA have effects on the cultured neural stem cells (NSCs). In this study, we investigated the roles of PCA in the survival and apoptosis of rat NSCs under normal conditions. NSCs obtained from 13.5-day-old rat embryos were propagated as neurospheres and cultured under normal conditions with or without PCA for 4 and 7 days. The cell viability was determined by the cell counting kit-8 (CCK-8) test, while cell proliferation was assayed by bromodeoxyuridine (BrdU) labeling. PCA increased the cellular viability of NSCs and stimulated cell proliferation in a dose- and time-dependent manner. Apoptotic cells were detected after 4 days by observing the nuclear morphological changes and flow cytometric analysis. Compared with the control on both culture days, treatment with PCA effectively reduced the levels of apoptosis of NSCs. At the same time, the reactive oxygen species (ROS) level in NSCs was depressed. In addition, PCA also significantly decreased the activity of elevated caspase-3, indicating that PCA may inhibit apoptosis of NSCs via suppression of the caspase cascade. These results suggest that PCA may be a potential growth inducer and apoptosis inhibitor for NSCs.

Hyperphosphorylated tau is the major protein subunit of neurofibrillary tangles in Alzheimer's disease (AD) and related tauopathies. It is not understood, however, why the neurofibrillary tangle-containing neurons seen in the AD brains do not die of apoptosis but rather degeneration even though they are constantly awash in a proapoptotic environment. Here, we show that cells overexpressing tau exhibit marked resistance to apoptosis induced by various apoptotic stimuli, which also causes correlated tau hyperphosphorylation and glycogen synthase kinase 3 (GSK-3) activation. GSK-3 overexpression did not potentiate apoptotic stimulus-induced cell apoptosis in the presence of high levels of tau. The resistance of neuronal cells bearing hyperphosphorylated tau to apoptosis was also evident by the inverse staining pattern of PHF-1-positive tau and activated caspase-3 or fragmented nuclei in cells and the brains of rats or tau-transgenic mice. Tau hyperphosphorylation was accompanied by decreases in beta-catenin phosphorylation and increases in nuclear translocation of beta-catenin. Reduced levels of beta-catenin antagonized the antiapoptotic effect of tau, whereas overexpressing beta-catenin conferred resistance to apoptosis. These results reveal an antiapoptotic function of tau hyperphosphorylation, which likely inhibits competitively phosphorylation of beta-catenin by GSK-3beta and hence facilitates the function of beta-catenin. Our findings suggest that tau phosphorylation may lead the neurons to escape from an acute apoptotic death, implying the essence of neurodegeneration seen in the AD brains and related tauopathies.

Objective: To explore the role of exogenous carbon monoxide (CO) in apoptosis of polymorphonuclear leukocyte (PMN) stimulated by ischemia-reperfusion (IR)., Methods: PMNs isolated from the venous blood of a healthy volunteer, incubated, put in 24-well plates, and randomly divided into 4 groups of 8 wells: control group (exposed to 5% CO2), control + CO group (exposed to 0.025% CO and 5% CO2 for I hour and then serum of healthy person was used to replace the culture fluid), IR group (exposed to 5% CO2 and then IR serum was used to replace the culture fluid), and IR + CO group (exposed to 0.025% CO and 5% CO2 for I hour and then serum of healthy person was used to replace the culture fluid). The IR serum was obtained from 8 male patients with osteoarthritis of knee undergoing knee replacement. After 24-hour incubation the PMNs underwent flow cytometry and electrophoresis to examine the apoptosis of PMNs. Electrophoretic mobility shift assay (EMSA) was used to detect the NF-kappaB binding activity., Results: The PMN apoptotic rate of the IR + CO group was 9.38% +/- 1.58%, significantly higher than that of the control group (4.18% +/- 1.02%, P < 0.05). The PMN apoptotic rate of the IR group was 2.15% +/- 1.02%, significantly lower than that of the control group (P < 0.05). However, the PMN apoptotic rate of the control + CO group was 4.16% +/- 1.12%, not significantly different from that of the control group (P > 0.05). Electrophoresis showed that PMN apoptosis DNA ladder was seen in the control, control + CO, and IR + CO groups, but not in the IR group. EMSA showed that after co-incubation of PMN nuclear extract and isotope- labeled NF-kappaB probe in term of the strength of radiation self-development band the result the IR group was significantly greater than that of the control group, and the result of the IR + CO group was significantly lower than that of the IR group, however, there was no significant difference between the control and control + CO groups., Conclusion: Exogenous CO improves the inhibitory effect of IR blood on the PMN apoptosis with a mechanism of suppressing the NF-kappaB binding activity.

Survival of mesenchymal stem cells in the bone marrow is essential for bone marrow microenvironment homeostasis, but the molecular mechanisms remain poorly understood. RIPK1 has emerged as a critical molecule of programmed cell death in tissue homeostasis. However, little is known about the regulation of RIPK1 on bone marrow mesenchymal stem cells (MSCs). Here, we have investigated for the first time the role of RIPK1 in bone marrow MSCs. We have found that RIPK1 knockdown suppressed proliferation, differentiation, and migration in bone marrow MSCs. Furthermore, RIPK1 knockdown resulted in the opening of mitochondrial permeability transition pore (mPTP) and mtDNA damage, leading to mitochondrial dysfunction, and consequently induced apoptosis and necroptosis in bone marrow MSCs. Moreover, we identified that the p53-PUMA axis pathway was involved in mitochondrial dysfunction in RIPK1-deficient bone marrow MSCs. Together, our findings highlighted that RIPK1 was indispensable for bone marrow MSC survival. [ABSTRACT FROM AUTHOR]

We recently reported that necroptosis contributed to compression-induced nucleus pulposus (NP) cells death. In the current study, we investigated the regulative effect of necroptosis inhibitor Necrostatin-1 on NP cells apoptosis and autophagy. Necrostatin-1, autophagy inhibitor 3-Methyladenine and apoptosis inhibitor Z-VAD-FMK were employed, and NP cells were exposed to 1.0 MPa compression for 0, 24 and 36 h. Necroptosis-associated molecules were measured by Western blot and RT-PCR. Autophagy and apoptosis levels were evaluated by Western blot and quantified by flow cytometry after monodansylcadaverine and Annexin V-FITC/propidium iodide staining, respectively. The cell viability and cell death were also examined. Furthermore, we measured mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (MPTP) and indices of oxidative stress to assess mitochondrial dysfunction. The results established that Necrostatin-1 blocked NP cells autophagy, and 3-Methyladenine had little influence on NP cells necroptosis. The Necrostatin-1+3-Methyladenine treatment exerted almost the same role as Necrostatin-1 in reducing NP cells death. Necrostatin-1 restrained NP cells apoptosis, while Z-VAD-FMK enhanced NP cells necroptosis. The Necrostatin-1+Z-VAD-FMK treatment provided more prominent role in blocking NP cells death compared with Necrostatin-1, consistent with increased MMP, reduced opening of MPTP and oxidative stress. In summary, the synergistic utilization of Necrostatin-1 and Z-VAD-FMK is a very worthwhile solution in preventing compression-mediated NP cells death, which might be largely attributed to restored mitochondrial function. [ABSTRACT FROM AUTHOR]

Abstract: Menopause, a risk factor for brain dysfunction in women, is characterized by neuropsychological symptoms including depression and dementia, which are closely related to alterations in different brain regions after menopause. However, little is known about the variability in pathophysiologic changes associated with menopause in the brain. Here, we observed that menopause in rats induced by bilateral ovariectomy (OVX) showed depressive and dementia‐related behaviors along with neuronal loss in the prefrontal cortex (PFC), hippocampus (HIP), hypothalamus (HYP), and amygdala (AMY) by Nissl staining. Meanwhile, by immunohistochemical staining, increased microglia in the HIP and AMY and increased astrocytes in the PFC, HYP, and AMY were shown. Using quantitative proteomics, we identified 146 differentially expressed proteins in the brains of OVX rats, for example, 20 in the PFC, 41 in the HIP, 17 in the HYP, and 79 in the AMY, and performed further detection by western blotting. A link between neuronal loss and apoptosis was suggested, as evidenced by increases in adenylate kinase 2 (AK2), B‐cell lymphoma 2 associated X (Bax), cleaved caspase 3, and phosphorylated p53 and decreases in Huntingtin‐interacting protein K, hexokinase, and phosphorylated B‐cell lymphoma 2 (Bcl‐2), and apoptosis might be triggered by endoplasmic reticulum stress (probed by increased glucose‐regulated protein 78 (GRP78), cleaved caspase 12, phosphorylated protein kinase R (PKR)‐like endoplasmic reticulum kinase, inositol‐requiring enzyme‐1 and activating transcription factor 6), and mitochondrial dysfunction (probed by increased cytochrome c and cleaved caspase 3 and decreased sideroflexin‐1 (SFXN1) and NADH dehydrogenase (ubiquinone) 1 α subcomplex 11 (NDUFA11)). Activation of autophagy was also indicated by increased autophagy‐related 7, γ‐aminobutyric acid (GABA) receptor‐associated protein‐like 2, and oxysterol‐binding protein‐related protein 1 and confirmed by increased microtubule‐associated protein light chain 3 (LC3II/I), autophagy‐related 5, and Beclin1 in the HIP and AMY. In the AMY, which is important in emotion, higher GABA transporter 3 and lower vesicular glutamate transporter 1 levels indicated an imbalance between excitatory and inhibitory neurotransmission, and the increased calretinin and decreased calbindin levels suggested an adjustment of GABAergic transmission after OVX. In addition, cytoskeletal abnormalities including tau hyperphosphorylation, dysregulated Ca²+ signals, and glutamic synaptic impairments were observed in the brains of OVX rats. Collectively, our study showed the changes in different brain regions related to depression and dementia during menopause. [ABSTRACT FROM AUTHOR]

An injectable new-type temperature-sensitive hydrogel consisted of chitosan- glycerophosphate-collagen (C-GP-Co) was prepared and the biocompability for adipose tissue-derived stem cells (ADSCs) was detected. The C-GP-Co hydrogel was prepared as follows: chitosan solution (2. 2%, volume fraction) and j3-glycerophosphate solution (50%, mass fraction) were mixed at a series of proportion, and then, the collagen solution (2 mg/mL) was added into the previous mixture to get the C-GP-Co hydrogel. The gelation time was measured at 37 `C, and the Osmotic pressure of the media within the hydrogels was measured, and then, the micro structure analysis was carried out by SEM. The isolated, cultured and identified ADSCs were seeded into C-GP-Co hydrogels and cell growth status was observed under inverted microscope. The cell apoptosis was detected using Live/ Dead Viability-Cytotoxicity Kit, while the proliferation of ADSCs was determined by CCK-8 kits. The experimental results show that the gelation time is about 12 mm at 37 °C, the osmotic pressure of media in the hydrogel is in the normal range of 310-330 mmol/kg and the inner structure of hydrogel is spongy porous. And later, the biocompability of the C-GP-Co hydrogel was further examined. After 7 days' culture, cells within C-GP-Co hydrogels display adherent cell morphology and high cell viability. It is demonstrated that the C-GP-Co hydrogel has excellent cell compatibility and is a proper scaffold for ADSCs proliferation. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific protease 12 (USP12) plays a significant role in tumor cell apoptosis and cell cycle progression. However, the regulatory mechanism of USP12 in human cervical carcinoma HeLa cell growth is unknown. In this study, we showed that knockdown of USP12 effectively induced cell cycle arrest in HeLa cells and decreased BMI-1, c-Myc and cyclin D2 transcription levels. By contrast, unlike the inactive C48S mutant, over-expression of USP12 and the deubiquitinase activity enhanced L153S and R237C mutants, had the opposite effects. Interestingly, compared to wild-type, the L153S mutant resulted in a more effective cell cycle-promotion and increased BMI-1, c-Myc and cyclin D2 transcript levels. In addition to BMI-1, USP12 R237C exhibited a functional resemblance to the wild-type by involving c-Myc and cyclin D2. The effect of USP12 on HeLa cell apoptosis was not observed in our study. These results suggest that USP12 may be responsible for HeLa cell growth by affecting cell cycle progression. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific protease 22 (USP22) can regulate the cell cycle and apoptosis in many cancer cell types, while it is still unclear whether the deubiquitinating enzyme activity of USP22 is necessary for these processes. As little is known about the impact of USP22 on the growth of HeLa cell, we observed whether USP22 can effectively regulate HeLa cell growth as well as the necessity of deubiquitinating enzyme activity for these processes in HeLa cell. In this study, we demonstrate that USP22 can regulate cell cycle but not apoptosis in HeLa cell. The deubiquitinating enzyme activity of USP22 is necessary for this process as confirmed by an activity-deleted mutant (C185S) and an activity-decreased mutant (Y513C). In addition, the deubiquitinating enzyme activity of USP22 is related to the levels of BMI-1, c-Myc, cyclin D2 and p53. Our findings indicate that the deubiquitinating enzyme activity of USP22 is necessary for regulating HeLa cell growth, and it promotes cell proliferation via the c-Myc/cyclin D2, BMI-1 and p53 pathways in HeLa cell. [ABSTRACT FROM AUTHOR]