Correlation of AQPs family molecule expression with cell invasion and apoptosis in preeclampsia placenta

Objective: To study the correlation of aquaporins (AQPs) family molecule expression with cell invasion and apoptosis in preeclampsia placenta. Methods: The puerperae who were diagnosed with preeclampsia in Obstetrics Department of Mianyang Central Hospital between July 2014 and May 2017 were selected and divided into mild PE group and severe PE group according to the disease severity, and the healthy puerperae who received antenatal care and gave birth in Mianyang Central Hospital over the same period were selected as control group. The placenta was collected to determine the mRNA expression of AQPs family molecules, cell invasion molecules and apoptosis molecules. Results: AQP1, AQP3, AQP8, AQP9, TXNIP, NLRP3, Fas, HSP70 and Caspase-3 mRNA expression in placenta tissue of severe PE group and mild PE group were greatly higher than those of control group whereas LAMA4, GRP30, OPN, EMMPRIN, MMP2 and MMP9 mRNA expression were greatly lower than those of control group;AQP1, AQP3, AQP8, AQP9, TXNIP, NLRP3, Fas, HSP70 and Caspase-3 mRNA expression in placenta tissue of severe PE group were greatly higher than those of mild PE group whereas LAMA4, GRP30, OPN, EMMPRIN, MMP2 and MMP9 mRNA expression were greatly lower than those of mild PE group;AQP1, AQP3, AQP8 and AQP9 mRNA expression in placenta of PE were negatively correlated with LAMA4, GRP30, OPN, EMMPRIN, MMP2 and MMP9 mRNA expression, and positively correlated with TXNIP, NLRP3, Fas, HSP70 and Caspase-3 mRNA expression. Conclusion: Highly expressed AQP1, AQP3, AQP8 and AQP9 can inhibit the invasion of trophoblast cells and promote the apoptosis of trophoblast cells in the placenta of preeclampsia.

TuBG1 promotes hepatocellular carcinoma via ATR/P53-apoptosis and cycling pathways

Background:As reported,γ-tubulin(TuBG1)is related to the occurrence and development of various types of malignant tumors.However,its role in hepatocellular cancer(HCC)is not clear.The present study was to investigate the relationship between TuBG1 and clinical parameters and survival in HCC patients.Methods:The correlation between TuBG1 and clinical parameters and survival in HCC patients was ex-plored by bioinformatics analysis.Immunohistochemistry was used for the verification.The molecular function of TuBG1 was measured using colony formation,scratch assay,trans-well assay and flow cytometry.Gene set enrichment analysis(GSEA)was used to pick up the enriched pathways,followed by investigating the target pathways using Western blotting.The tumor-immune system interactions and drug bank database(TISIDB)was used to evaluate TuBG1 and immunity.Based on the TuBG1-related immune genes,a prognostic model was constructed and was further validated internally and externally.Results:The bioinformatic analysis found high expressed TuBG1 in HCC tissue,which was confirmed us-ing immunohistochemistry and Western blotting.After silencing the TuBG1 in HCC cell lines,more G1 arrested cells were found,cell proliferation and invasion were inhibited,and apoptosis was promoted.Furthermore,the silence of TuBG1 increased the expressions of Ataxia-Telangiectasia and Rad-3(ATR),phospho-P38 mitogen-activated protein kinase(P-P38MAPK),phospho-P53(P-P53),B-cell lymphoma-2 associated X protein(Bax),cleaved caspase 3 and P21;decreased the expressions of B-cell lymphoma-2(Bcl-2),cyclin D1,cyclin E2,cyclin-dependent kinase 2(CDK2)and CDK4.The correlation analysis of immunohistochemistry and clinical parameters and survival data revealed that TuBG1 was negatively corre-lated with the overall survival.The constructed immune prognosis model could effectively evaluate the prognosis.Conclusions:The increased expression of TuBG1 in HCC is associated with poor prognosis,which might be involved in the occurrence and development of HCC.

miR-24-3p promotes proliferation and inhibits apoptosis of porcine granulosa cells by targeting P27

Ovarian follicle development is associated with the physiological functions of granulosa cells(GCs),including proliferation and apoptosis.The level of miR-24-3p in ovarian tissue of high-yielding Yorkshire×Landrace sows was significantly higher than that of low-yielding sows.However,the functions of miR-24-3p on GCs are unclear.In this study,using flow cytometry,5-ethynyl-2′-de-oxyuridine(EdU)staining,and cell count,we showed that miR-24-3p promoted the proliferation of GCs increasing the proportion of cells in the S phase and upregulating the expression of cell cycle genes,moreover,miR-24-3p inhibited GC apoptosis.Mechanistically,on-line prediction,bioinformatics analysis,a luciferase reporter assay,RT-qPCR,and Western blot results showed that the target gene of miR-24-3p in proliferation and apoptosis is cyclin-dependent kinase inhibitor 1B(P27/CDKN1B).Furthermore,the effect of miR-24-3p on GC proliferation and apoptosis was attenuated by P27 overexpression.These findings suggest that miR-24-3p regulates the physiological functions of GCs.

The effect of celastrol in combination with 5-fluorouracil on proliferation and apoptosis of gastric cancer cell lines

Background:Despite the availability of chemotherapy drugs such as 5-fluorouracil(5-FU),the treatment of some cancers such as gastric cancer remains challenging due to drug resistance and side effects.This study aimed to investigate the effect of celastrol in combination with the chemotherapy drug 5-FU on proliferation and induction of apoptosis in human gastric cancer cell lines(AGS and EPG85-257).Materials and Methods:In this in vitro study,AGS and EPG85-257 cells were treated with different concentrations of celastrol,5-FU,and their combination.Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.The synergistic effect of 5-FU and celastrol was studied using Compusyn software.The DNA content at different phases of the cell cycle and apoptosis rate was measured usingflow cytometry.Results:Co-treatment with low concentrations(10%inhibitory concentration(IC10))of celastrol and 5-FU significantly reduced IC50(p<0.05)so that 48 h after treatment,IC50 was calculated at 3.77 and 6.9μM for celastrol,20.7 and 11.6μM for 5-FU,and 5.03 and 4.57μM for their combination for AGS and EPG85-257 cells,respectively.The mean percentage of apoptosis for AGS cells treated with celastrol,5-FU,and their combination was obtained 23.9,41.2,and 61.9,and for EPG85-257 cells 5.65,46.9,and 55.7,respectively.In addition,the 5-FU and celastrol-5-FU combination induced cell cycle arrest in the synthesis phase.Conclusions:Although celastrol could decrease the concentration of 5-fluorouracil that sufficed to suppress gastric cancer cells,additional studies are required to arrive at conclusive evidence on the anticancer effects of celastrol.

Prohibitin 1 inhibits cell proliferation and induces apoptosis via the p53-mediated mitochondrial pathway in vitro

BACKGROUND Prohibitin 1(PHB1)has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed,and it participates in a variety of essential cellular functions,including apoptosis,cell cycle regulation,prolifera-tion,and survival.Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma(HCC).However,the role of PHB1 in HCC is controversial.AIM To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro.METHODS HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria;then,PHB1 levels in the sera and liver tissues of these participates were determined using ELISA,RT-PCR,and immunohistoche-mistry.Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA(shRNA-PHB1)for 24-72 h.Cell prolif-eration was analysed with an MTT assay.Cell cycle progression and apoptosis were analysed using flow cytometry(FACS).The mRNA and protein expression levels of the cell cycle-related molecules p21,Cyclin A2,Cyclin E1,and CDK2 and the cell apoptosis-related molecules cytochrome C(Cyt C),p53,Bcl-2,Bax,caspase 3,and caspase 9 were measured by real-time PCR and Western blot,respectively.RESULTS Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals,and decreased PHB1 was positively correlated with low differentiation,TNM stage III-IV,and alpha-fetoprotein≥400μg/L.Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner.FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis.The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells.The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41%±1.06%,which was significantly greater than that of apoptotic control cells(3.65%±0.85%,P<0.01)and empty vector-transfected cells(4.21%±0.52%,P<0.01).Similar results were obtained with SMMC-7721 cells.Furthermore,the mRNA and protein expression levels of p53,p21,Bax,caspase 3,and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2,Cy-clin E1,CDK2,and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells.However,when PHB1 was upregulated in human HCC cells,Cyt C expression levels were increased in the cytosol and decreased in the mitochondria,which indicated that Cyt C had been released into the cytosol.Conversely,these effects were reversed when PHB1 was knocked down.CONCLUSION PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.

miR‑423 sponged by lncRNA NORHA inhibits granulosa cell apoptosis

Background Atresia and degeneration,a follicular developmental fate that reduces female fertility and is triggered by granulosa cell(GC)apoptosis,have been induced by dozens of miRNAs.Here,we report a miRNA,miR-423,that inhibits the initiation of follicular atresia(FA),and early apoptosis of GCs.Results We showed that miR-423 was down-regulated during sow FA,and its levels in follicles were negatively correlated with the GC density and the P4/E2 ratio in the follicular fluid in vivo.The in vitro gain-of-function experiments revealed that miR-423 suppresses cell apoptosis,especially early apoptosis in GCs.Mechanically speaking,the miR-423 targets and interacts with the 3’-UTR of the porcine SMAD7 gene,which encodes an apoptosis-inducing factor in GCs,and represses its expression and pro-apoptotic function.Interestingly,FA and the GC apoptosis-related lncRNA NORHA was demonstrated as a ceRNA of miR-423.Additionally,we showed that a single base deletion/insertion in the miR-423 promoter is significantly associated with the number of stillbirths(NSB)trait of sows.Conclusion These results demonstrate that miR-423 is a small molecule for inhibiting FA initiation and GC early apoptosis,suggesting that treating with miR-423 may be a novel approach for inhibiting FA initiation and improving female fertility.

3-Methyladenine potentiates paclitaxel-induced apoptosis and phosphorylation of cyclin-dependent kinase 1 at thr161 in nasopharyngeal carcinoma cell

Background:Nasopharyngeal carcinoma(NPC)exhibits a significant prevalence in the southern regions of China,and paclitaxel(PTX)is frequently employed as a medication for managing advanced NPC.However,drug resistance is typically accompanied by a poor prognosis.Exploring the synergistic potential of combining multiple chemotherapeutic agents may represent a promising avenue for optimizing treatment efficacy.Methods:This study investigated whether 3-Methyladenine(3-MA)could potentiated the effect of PTX and its potential molecular mechanism.Samples were divided into the following categories:Negative control(NC)with the solvent dimethyl sulfoxide(DMSO,0.5%v/v),PTX(400 nM),3-MA(4 mM),and PTX(400 nM)+3-MA(4 mM).The viability of NPC cells was assessed using both the cell counting kit-8(CCK-8)assay and the colony formation assay.Microscopic observation was performed to identify morphological cell changes.Flow cytometry was used to assess cell cycle status,mitochondrial membrane potential(MMP),and apoptotic cells.Western blotting was conducted to quantify the protein expression.Results:3-MA enhanced PTX-specific inhibition of NPC cell proliferation.PTX,either alone or in combination with 3-MA,caused cell cycle halt at the G2/M phase in the majority of NPC cells,and the combination treatment of PTX with 3-MA induced a higher rate of NPC cell death compared to PTX alone.Western blotting results revealed the combination of PTX with 3-MA heightened activation of cyclin-dependent kinase 1(CDK1),a key molecule in shifting cells from mitotic arrest to apoptosis,led to a reduction in Myeloid Cell Leukemia 1(MCL-1)expression and an increase in Poly(ADP-ribose)polymerase(PARP)cleavage.Conclusion:The concurrent administration of PTX with 3-MA effectively enhances PTX’s inhibitory impact on NPC and activates the apoptosis signal regulated by CDK1.

Hydrangea serrata extract exerts tumor inhibitory activity against hepatocellular carcinoma HepG2 cells via inducing p27/CDK2-mediated cell cycle arrest and apoptosis

Objective:To examine the inhibitory effect of Hydrangea serrata extract against hepatocellular carcinoma HepG2 cells and its underlying mechanisms.Methods:The effects of Hydrangea serrata extract on growth inhibition of tumor cells and spheroids were assessed using MTT and 3D culture assays.Quantitative real-time PCR and Western blot analyses were employed to investigate the changes in mRNA and protein expression levels of molecules related to cell cycle and apoptosis.Results:Hydrangea serrata extract effectively inhibited the growth of both tumor cells and spheroids.The extract also significantly upregulated p27 mRNA expression and downregulated CDK2 mRNA expression,leading to cell cycle arrest.Moreover,increased BAX/Bcl-2 ratio as well as caspase-9 and-3 were observed after treatment with Hydrangea serrata extract,indicating the induction of tumor cell apoptosis.Conclusions:Hydrangea serrata extract has the potential to alleviate tumors by effectively modulating cell-cycle-related gene expressions and inducing apoptosis,thereby inhibiting tumor growth.

Paclitaxel induces human KOSC3 oral cancer cell apoptosis through caspase pathways

Background:Paclitaxel is a compound derived from Pacific yew bark that induces various cancer cell apoptosis.However,whether it also has anticancer activities in KOSC3 cells,an oral cancer cell line,is unclear.Methods:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,flow cytometry,and western blotting assays were carried out to assess cell viability,subG1 phase of the cell cycle,and apoptosis-related protein expression,respectively.Results:Ourfindings indicate that paclitaxel could inhibit cell viability and increase the expression of apoptotic markers,including plasma membrane blebbing and the cleavage of poly ADP-ribose polymerase in KOSC3 cells.Also,the treatment with paclitaxel remarkably elevated the percentage of the subG1 phase in KOSC3 cells.In addition,treatment with a pan-caspase inhibitor could recover paclitaxel-inhibited cell viability.Moreover,caspase-8,caspase-9,caspase-7,and BH3 interacting domain death agonist(Bid)were activated in paclitaxel-treated KOSC3 cells.Conclusions:Paclitaxel induced apoptosis through caspase cascade in KOSC3 cells.

Effect of acacetin on inhibition of apoptosis in Helicobacter pyloriinfected gastric epithelial cell line

BACKGROUND Helicobacter pylori(H.pylori)infection can cause extensive apoptosis of gastric epithelial cells,serving as a critical catalyst in the progression from chronic gastritis,gastrointestinal metaplasia,and atypical gastric hyperplasia to gastric carcinoma.Prompt eradication of H.pylori is paramount for ameliorating the pathophysiological conditions associated with chronic inflammation of the gastric mucosa and the primary prevention of gastric cancer.Acacetin,which has multifaceted pharmacological activities such as anti-cancer,anti-inflammatory,and antioxidative properties,has been extensively investigated across various domains.Nevertheless,the impact and underlying mechanisms of action of acacetin on H.pylori-infected gastric mucosal epithelial cells remain unclear.AIM To explore the defensive effects of acacetin on apoptosis in H.pylori-infected GES-1 cells and to investigate the underlying mechanisms.METHODS GES-1 cells were treated with H.pylori and acacetin in vitro.Cell viability was assessed using the CCK-8 assay,cell mortality rate via lactate dehydrogenase assay,alterations in cell migration and healing capacities through the wound healing assay,rates of apoptosis via flow cytometry and TUNEL staining,and expression levels of apoptosis-associated proteins through western blot analysis.RESULTS H.pylori infection led to decreased GES-1 cell viability,increased cell mortality,suppressed cell migration,increased rate of apoptosis,increased expressions of Bax and cle-caspase3,and decreased Bcl-2 expression.Conversely,acacetin treatment enhanced cell viability,mitigated apoptosis induced by H.pylori infection,and modulated the expression of apoptosis-regulatory proteins by upregulating Bcl-2 and downregulating Bax and cleaved caspase-3.CONCLUSION Acacetin significantly improved GES-1 cell viability and inhibited apoptosis in H.pylori-infected GES-1 cells,thereby exerting a protective effect on gastric mucosal epithelial cells.

Effects of Quercetin Extracted from Flower Buds of Sophora japonica cv.jinhuai on Proliferation and Apoptosis of Human Breast Cancer MCF-7 Cells

[Objectives]To investigate the effects of quercetin extracted from flower buds of Sophora japonica cv.jinhuai on the proliferation,apoptosis and migration of human breast cancer MCF-7 cells.[Methods]MTT assay,inverted microscope observation,hoechst33342 staining,flow cytometry(FCM)and wound healing assay were adopted to investigate the proliferation,morphological changes,apoptosis level and cell migration ability of human breast cancer MCF-7 cells,respectively.[Results]The morphological changes of cells in the treatment groups included gradually decreased number,reduced volume,vague cell contour,loose intercellular connection,uneven cytoplasm distribution and increased cell debris.With the increase of drug concentration,quercetin significantly inhibited the proliferation of human breast cancer MCF-7 cells(P<0.05).The number of apoptotic bodies increased gradually.When the concentration reached 100μmol/L,a large number of nuclear fragments appeared,and the level of apoptosis was statistically different(P<0.05).The mobility and migration ability of cells showed a decreasing trend,and the differences were statistically significant(P<0.05).[Conclusions]This study can provide experimental basis for clinical application of quercetin against breast cancer.

SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo

AIM:To explore the effect of silent information regulator factor 2-related enzyme 1(SIRT1)on modulating apoptosis of human lens epithelial cells(HLECs)and alleviating lens opacification of rats through suppressing endoplasmic reticulum(ER)stress.METHODS:HLECs(SRA01/04)were treated with varying concentrations of tunicamycin(TM)for 24h,and the expression of SIRT1 and C/EBP homologous protein(CHOP)was assessed using real-time quantitative polymerase chain reaction(RT-PCR),Western blotting,and immunofluorescence.Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8(CCK-8)assay,respectively.In the SRA01/04 cell apoptosis model,which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation,the expression levels of SIRT1,CHOP,glucose regulated protein 78(GRP78),and activating transcription factor 4(ATF4)were examined.The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid(4-PBA;an ER stress inhibitor)was investigated.In vivo,age-related cataract(ARC)rat models were induced by sodium selenite injection,and the protective role of SIRT1,activated by SRT1720 intraperitoneal injections,was evaluated through morphology observation,hematoxylin and eosin(H&E)staining,Western blotting,and RT-PCR.RESULTS:SIRT1 expression was downregulated in TMinduced SRA01/04 cells.Besides,in SRA01/04 cells,both cell apoptosis and CHOP expression increased with the rising doses of TM.ER stress was stimulated by TM,as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model.Inhibition of SIRT1 by siRNA knockdown increased ER stress activation,whereas SRT1720 treatment had opposite results.4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis.In vivo,SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models.CONCLUSION:SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress.These findings suggest a novel strategy for cataract treatment focused on targeting ER stress,highlighting the therapeutic potential of SIRT1 modulation in ARC development.

Mechanism of stilbene glycosides on apoptosis of SH-SY5Y cells via regulating PI3K/AKT signaling pathway

Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.

Treadmill exercise exerts a synergistic effect with bone marrow mesenchymal stem cell-derived exosomes on neuronal apoptosis and synaptic-axonal remodeling

Treadmill exercise and mesenchymal stem cell transplantation are both practical and effective methods for the treatment of cerebral ischemia.However,whether there is a synergistic effect between the two remains unclear.In this study,we established rat models of ischemia/reperfusion injury by occlusion of the middle cerebral artery for 2 hours and reperfusion for 24 hours.Rat models were perfused with bone marrow mesenchymal stem cell-derived exosomes(MSC-exos)via the tail vein and underwent 14 successive days of treadmill exercise.Neurological assessment,histopathology,and immunohistochemistry results revealed decreased neuronal apoptosis and cerebral infarct volume,evident synaptic formation and axonal regeneration,and remarkably recovered neurological function in rats subjected to treadmill exercise and MSC-exos treatment.These effects were superior to those in rats subjected to treadmill exercise or MSC-exos treatment alone.Mechanistically,further investigation revealed that the activation of JNK1/c-Jun signaling pathways regulated neuronal apoptosis and synaptic-axonal remodeling.These findings suggest that treadmill exercise may exhibit a synergistic effect with MSC-exos treatment,which may be related to activation of the JNK1/c-Jun signaling pathway.This study provides novel theoretical evidence for the clinical application of treadmill exercise combined with MSC-exos treatment for ischemic cerebrovascular disease.

Cryptotanshinone induces apoptosis of activated hepatic stellate cells via modulating endoplasmic reticulum stress

BACKGROUND Cryptotanshinone(CPT)has wide biological functions,including anti-oxidative,antifibrosis,and anti-inflammatory properties.However,the effect of CPT on hepatic fibrosis is unknown.AIM To investigate the effects of CPT treatment on hepatic fibrosis and its underlying mechanism of action.METHODS Hepatic stellate cells(HSCs)and normal hepatocytes were treated with different concentrations of CPT and salubrinal.The CCK-8 assay was used to determine cell viability.Flow cytometry was used to measure apoptosis and cell cycle arrest.Reverse transcription polymerase chain reaction(RT-PCR)and Western blot analyses were used to measure mRNA levels and protein expression of endoplasmic reticulum stress(ERS)signaling pathway related molecules,respectively.Carbon tetrachloride(CCL4)was used to induce in vivo hepatic fibrosis in mice.Mice were treated with CPT and salubrinal,and blood and liver samples were collected for histopathological examination.RESULTS We found that CPT treatment significantly reduced fibrogenesis by modulating the synthesis and degradation of the extracellular matrix in vitro.CPT inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in cultured HSCs.Furthermore,we found that CPT promoted apoptosis of activated HSCs by upregulating expression of ERS markers(CHOP and GRP78)and activating ERS pathway molecules(PERK,IRE1α,and ATF4),which were inhibited by salubrinal.Inhibition of ERS by salubrinal partially eliminated the therapeutic effect of CPT in our CCL4-induced hepatic fibrosis mouse model.CONCLUSION CPT can promote apoptosis of HSCs and alleviate hepatic fibrosis through modulating the ERS pathway,which represents a promising strategy for treating hepatic fibrosis.

miR-221/222/PUMA Axis Promotes Oral Squamous Cell Carcinoma Apoptosis

Background: To investigate the therapeutic activity of the miR-221/222 inhibitor against OSCC in vitro and in vivo. Materials and Methods: HSC3 and HSC6 were treated with miR-221/222 inhibitor and the empty vector respectively. After the recombinants were transfected into HSC3 and HSC6 with LipofectamineTM MAX, the expression of miR-221/222 and PUMA was analyzed by RT-PCR. The proliferation and migration of HSC3 and HSC6 were detected by CCK-8 assay and Wound-healing assay. Cell cycle and apoptosis were detected by flow cytometry. The effect of the miR-221/222 inhibitor was also assessed in OSCC xenografts in BALB/c-nu mice. Results: Transfection of the miR-221/222 inhibitor increased cell apoptosis and upregulated PUMA expression in OSCC cell lines HSC3 and HSC6 with the significantly reduced expression of miR-221 and miR-222. Furthermore, the miR-221/222 inhibitor suppressed cell growth and invasion and blocked the cell cycle at the G1 phase. Obvious anti-tumor activity was achieved in BALB/c-nu mice by treatment with the miR-221/222 inhibitor, together with the upregulation of PUMA protein in tumors retrieved from the mice. Conclusions: There was a significant inhibitory effect of the miR-221/222 inhibitor on the growth of OSCC both in vitro and in vivo, and there might be a regulatory loop between miR-221/222 and PUMA. These findings demonstrated that downregulation of miR-221/222 could induce cell apoptosis, and it might be considered as a candidate target for gene therapy of OSCC.

Synergism of calycosin and bone marrow-derived mesenchymal stem cells to combat podocyte apoptosis to alleviate adriamycininduced focal segmental glomerulosclerosis

BACKGROUND Bone marrow-derived mesenchymal stem cells(MSCs)show podocyte-protective effects in chronic kidney disease.Calycosin(CA),a phytoestrogen,is isolated from Astragalus membranaceus with a kidney-tonifying effect.CA preconditioning enhances the protective effect of MSCs against renal fibrosis in mice with unilateral ureteral occlusion.However,the protective effect and underlying mechanism of CA-pretreated MSCs(MSCsCA)on podocytes in adriamycin(ADR)-induced focal segmental glomerulosclerosis(FSGS)mice remain unclear.AIM To investigate whether CA enhances the role of MSCs in protecting against podocyte injury induced by ADR and the possible mechanism involved.METHODS ADR was used to induce FSGS in mice,and MSCs,CA,or MSCsCA were administered to mice.Their protective effect and possible mechanism of action on podocytes were observed by Western blot,immunohistochemistry,immunofluorescence,and real-time polymerase chain reaction.In vitro,ADR was used to stimulate mouse podocytes(MPC5)to induce injury,and the supernatants from MSC-,CA-,or MSCsCA-treated cells were collected to observe their protective effects on podocytes.Subsequently,the apoptosis of podocytes was detected in vivo and in vitro by Western blot,TUNEL assay,and immunofluorescence.Overexpression of Smad3,which is involved in apoptosis,was then induced to evaluate whether the MSCsCA-mediated podocyte protective effect is associated with Smad3 inhibition in MPC5 cells.RESULTS CA-pretreated MSCs enhanced the protective effect of MSCs against podocyte injury and the ability to inhibit podocyte apoptosis in ADR-induced FSGS mice and MPC5 cells.Expression of p-Smad3 was upregulated in mice with ADR-induced FSGS and MPC5 cells,which was reversed by MSCCA treatment more significantly than by MSCs or CA alone.When Smad3 was overexpressed in MPC5 cells,MSCsCA could not fulfill their potential to inhibit podocyte apoptosis.CONCLUSION MSCsCA enhance the protection of MSCs against ADR-induced podocyte apoptosis.The underlying mechanism may be related to MSCsCA-targeted inhibition of p-Smad3 in podocytes.

Alu antisense RNA ameliorates methylglyoxal-induced human lens epithelial cell apoptosis by enhancing antioxidant defense

AIM:To determine whether an antisense RNA corresponding to the human Alu transposable element(Aluas RNA)can protect human lens epithelial cells(HLECs)from methylglyoxal-induced apoptosis.METHODS:Cell counting kit-8(CCK-8)and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays were used to assess HLEC viability.HLEC viability/death was detected using a Calcein-AM/PI double staining kit;the annexin V-FITC method was used to detect HLEC apoptosis.The cytosolic reactive oxygen species(ROS)levels in HLECs were determined using a reactive species assay kit.The levels of malondialdehyde(MDA)and the antioxidant activities of total-superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)were assessed in HLECs using their respective kits.RT-q PCR and Western blotting were used to measure m RNA and protein expression levels of the genes.RESULTS:Aluas RNA rescued methylglyoxal-induced apoptosis in HLECs and ameliorated both the methylglyoxalinduced decrease in Bcl-2 m RNA and the methylglyoxalinduced increase in Bax m RNA.In addition,Aluas RNA inhibited the methylglyoxal-induced increase in Alu sense RNA expression.Aluas RNA inhibited the production of ROS induced by methylglyoxal,restored T-SOD and GSHPx activity,and moderated the increase in MDA content after treatment with methylglyoxal.Aluas RNA significantly restored the methylglyoxal-induced down-regulation of Nrf2 gene and antioxidant defense genes,including glutathione peroxidase,heme oxygenase 1,γ-glutamylcysteine synthetase and quinone oxidoreductase 1.Aluas RNA ameliorated methylglyoxal-induced increases of the m RNA and protein expression of Keap1 that is the negative regulator of Nrf2.CONCLUSION:Aluas RNA reduces apoptosis induced by methylglyoxal by enhancing antioxidant defense.

Valproate reduces retinal ganglion cell apoptosis in rats after optic nerve crush

The retinal ganglion cells of the optic nerve have a limited capacity for self-repair after injury.Valproate is a histone deacetylase inhibitor and multitarget drug,which has been demonstrated to protect retinal neurons.In this study,we established rat models of optic nerve-crush injury and injected valproate into the vitreous cavity immediately after modeling.We evaluated changes in the ultrastructure morphology of the endoplasmic reticulum of retinal ganglion cells over time via transmission electron microscope.Immunohistochemistry and western blot assay revealed that valproate upregulated the expression of the endoplasmic reticulum stress marker glucose-regulated protein 78 and downregulated the expression of transcription factor C/EBP homologous protein,phosphorylated eukaryotic translation initiation factor 2α,and caspase-12 in the endoplasmic reticulum of retinal ganglion cells.These findings suggest that valproate reduces apoptosis of retinal ganglion cells in the rat after optic nerve-crush injury by attenuating phosphorylated eukaryotic translation initiation factor 2α-C/EBP homologous protein signaling and caspase-12 activation during endoplasmic reticulum stress.These findings represent a newly discovered mechanism that regulates how valproate protects neurons.

Influence of Photodynamic Therapy on Apoptosis and Invasion of Human Cholangiocarcinoma QBC939 Cell Line

Objective To investigate the effect of photodynamic therapy(PDT) mediated by hematoporphyrin derivative(HPD) on apoptosis and invasion of cholangiocarcinoma QBC939 cell lines. Methods In vitro cultured cholangiocarcinoma QBC939 cell line was exposed to 2, 4, 6, 8, 10, 12, and 14 μg/ml HPD with 5, 10, and 15 J/cm2 light intensity, respectively. The optical density at 450 nm of the QBC939 cells was measured by CCK8 assay and its growth inhibition ratio was calculated. Flow cytometry and transwell migration assay were applied to detect cell apoptosis and invasion respectively. RT-PCR and immunocytochemistry analyses were used to detect expressions of vascular endothelial growth factor-C(VEGF-C), cyclooxygenase-2(COX-2), and proliferating cell nuclear antigen(PCNA). Enzyme-linked immunosorbent assay(ELISA) was carried out to examine the secretion of VEGF-C and COX-2 in QBC939 cells. Results Exposure to HPD-PDT can significantly suppress the growth of QBC939 cells(all P<0.05). HPD-PDT can promote apoptosis of QBC939 cells at the early stage. When the concentration of HPD was 2 μg/ml and light irradiation was 5 J/cm2, HPD-PDT had no obvious inhibitory effect on QBC939 cell growth, but can obviously inhibit cell invasion, and significant difference was observed between the HPD-PDT and control groups(P<0.01). The HPD-PDT can reduce the m RNA and protein expressions of VEGF-C, COX-2, and PCNA, and decrease the secretion of VEGF-C and COX-2 in QBC939 cells. Conclusion PDT could promote apoptosis and inhibit growth and invasion of cholangiocarcinoma cells QBC939 in vitro.