Human antigen R mediated post-transcriptional regulation of inhibitors of apoptosis proteins in pancreatic cancer

AIM To determine the association of human antigen R(HuR) and inhibitors of apoptosis proteins(IAP1, IAP2) and prognosis in pancreatic cancer.METHODS Protein and mRNA expression levels of IAP1, IAP2 and HuR in pancreatic ductal adenocarcinoma(PDAC) were compared with normal pancreatic tissue. The correlations among IAP1/IAP2 and HuR as well as their respective correlations with clinicopathological parameters were analyzed. The Kaplan-Meier method and log-rank tests were used for survival analysis. Immunoprecipitation assay was performed to demonstrate HuR binding to IAP1, IAP2 mRNA. PANC1 cells were transfected with either anti-HuR siRNA or control siRNA for 72 h and quantitative reverse transcription polymerase chain reaction(RT-PCR), western blot analysis was carried out.RESULTS RT-PCR analysis revealed that HuR, IAP1, IAP2 mRNA expression were accordingly 3.3-fold, 5.5-fold and 8.4 higher in the PDAC when compared to normal pancreas(P < 0.05). Expression of IAP1 was positively strongly correlated with HuR expression(P < 0.05, r = 0.783). Western blot analysis confirmed RTPCR results. High IAP1 expression, tumor resection status, T stage, lymph-node metastases, tumor differentiation grade, perineural and lymphatic invasion were identified as significant factors for shorter survival in PDAC patients(P < 0.05).Immunohistological analysis showed that HuR was mainly expressed in the ductal cancer cell's nucleus and less so in cytoplasm. RNA immunoprecipitation analysis confirmed IAP1 and IAP2 post-transcriptional regulation by HuR protein. Following siHuR transfection, IAP1 mRNA and protein levels were decreased, however IAP2 expression levels were increased.CONCLUSION HuR mediated overexpression of IAP1 significantly correlates with poor outcomes and early progression of pancreatic cancer. Further studies are needed to assess the underlying mechanisms.

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression

AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.

Role of Extracelluar Regulated Protein Kinases in FTY720-induced Apoptosis of Leukemia Cell Lines HL-60 and U937

The effects of a novel immunosuppressive agent FTY720 on proliferation inhibition and apoptosis of acute leukemia cell lines HL 60 and U937, and the role of extracelluar regulated protein kinase (ERK) in the course of proliferation inhibition and apoptosis induced by FTY720 were studied. The proliferation inhibition rate of HL 60 and U937 cells by various concentrations of FTY720 was detected by MTT assay. Cell apoptosis was detected by DNA fragment analysis and flow cytometry. The phosphorylated ERK1/2 protein expression was observed by Western blotting. The change of intracellular distribution of ERK1/2 protein was identified by SP immunohistochemical staining. The results showed that FTY720 could inhibit the growth of HL 60 and U937 cells effectively in a dose dependent manner. After incubation with FTY720 for 24 h, apoptosis was observed in HL 60 and U937 cells. The intracellular expression of phosphorylated ERK1/2 protein was also down regulated and the distribution of ERK1/2 protein in cell nuclear was reduced during FTY720 induced apoptosis. So, that FTY720 inhibited ERK1/2 phosphorylation might mediate the role of FTY720 induced apoptosis and proliferation inhibition of leukemia cells.

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Killing effect of TNF-related apoptosis inducing ligand regulated by tetracycline on gastric cancer cell line NCI-N87

AIM: To clone the cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) into a tetracycline-regulated gene expression system, the RevTet-On system, transduce expression vectors into a gastric carcinoma cell line-NCI-N87 and examine the effects of controlled expression of TRAIL in vitro on the gastric carcinoma cells. METHODS: The full-length cDNA of TRAIL was inserted into a vector under the control of the tetracycline-responsive element (TRE) to obtain the plasmid pRevTRE-TRAIL, which was transfected into a packaging cell line PT67. In addition, vector pRev-Tet On and pRevTRE were also transfected into PT67 separately. After hygromycin and G418 selection, the viral titer was determined. The medium containing retroviral vectors was collected and used to transduce a gastric carcinoma cell line NCI-N87. The resulting cell line NCI-N87-Tet On TRE-TRAIL and a control cell line, NCI-N87 Tet On-TRE, were established. TRAIL expression in the cell line was induced by incubating cells with doxycycline (Dox), which is a tetracycline analogue. The killing effect on gastric carcinoma cells was analyzed after induction. RESULTS: The recombinant plasmid pRev-TRE-TRAIL was constructed. After hygromycin or G418 selection, the producer cell lines PT67-TRE, PT67-TRE-TRAIL and PT67-Tet On were obtained,with titers of about 10(8)CFU.L(-1). By transducing NCI-N87 cells with retroviral vectors from these cell lines, stable cell lines NCI-N87-Tet-On TRE-TRAIL (NN3T) and control cell line NCI-N87-Tet-On-TRE (NN2T) were established. The growth curves of the selected cell lines were the same with the wild type NCI-N87. When Dox was added, cell death was obvious in the test groups (29%-77%), whereas no difference was observed in control and wild type cell lines. With the addition of a medium from the test group, human leukemia cell line Jurkat was activated till death (83%), indicating the secretion of active TRAIL proteins from the test cells to the medium. CONCLUSION: With the use of the RevTet-On system, a regulated expression system for TRAIL was constructed. Using this system, the selected killing effect of TRAIL on gastric carcinoma cell line NCI-N87 could be observed.

XAF1 mediates apoptosis through an extracellular signal-regulated kinase pathway in colon cancer

Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.

Serine-threonine protein kinase activation may be an effective target for reducing neuronal apoptosis after spinal cord injury

The signaling mechanisms underlying ischemia-induced nerve cell apoptosis are poorly understood. We investigated the effects of apoptosis-related signal transduction pathways following ischemic spinal cord injury, including extracellular signal-regulated kinase（ERK）, serine-threonine protein kinase（Akt） and c-Jun N-terminal kinase（JNK） signaling pathways. We established a rat model of acute spinal cord injury by inserting a catheter balloon in the left subclavian artery for 25 minutes. Rat models exhibited notable hindlimb dysfunction. Apoptotic cells were abundant in the anterior horn and central canal of the spinal cord. The number of apoptotic neurons was highest 48 hours post injury. The expression of phosphorylated Akt（pAkt） and phosphorylated ERK（p-ERK） increased immediately after reperfusion, peaked at 4 hours（p-Akt） or 2 hours（p-ERK）, decreased at 12 hours, and then increased at 24 hours. Phosphorylated JNK expression reduced after reperfusion, increased at 12 hours to near normal levels, and then showed a downward trend at 24 hours. Pearson linear correlation analysis also demonstrated that the number of apoptotic cells negatively correlated with p-Akt expression. These findings suggest that activation of Akt may be a key contributing factor in the delay of neuronal apoptosis after spinal cord ischemia, particularly at the stage of reperfusion, and thus may be a target for neuronal protection and reduction of neuronal apoptosis after spinal cord injury.

Oncoprotein expression and inhibition of apoptosis during colorectal tumorigenesis

AIMS To study bcl-2 and P53 protein expression and inhibition of apoptosis during colorectal tumorigenesis. METHODS Expression of bcl -2 and p53 in 45 colorectal ade- nomas and 61 colorectal carcinomas was detected by immunohis- tochemical staining. RESULTS The bcl-2 and P53 protein expression was uniformly negative in normal mucosa,whereas bcl-2 and p53 positive rates were significantly higher in adenoma and carcinoma than in nor- reals(P<0.01 ).The area with strong bcl-2 expression was of- ten the area with severely dysplasia.In colorectal adenoma,ex- pression of p53 increased with the increasing size and dysplasia, in adenomas≥20 mm being higher than adenomas<10 mm(77, 8% vs 35.0%,P<0.05).p53 was relevant to differentiation and Duke's staging.A significant inverse correlation was found between bcl-2 and p53 in immunostaining in the adenomas,but not in the carcinomas.Furthermore,carcinomas with a high per- centage of bcl-2 positive cells were significantly more likely to have low rates of apoptosis. CONCLUSIONS These results suggest that bcl-2 gene appears to be an early event in colorectal tumorigenesis that can inhibit apoptosis,p53 expression plays an important role in the develop- ment and malignant change of colorectal adenoma,bcl-2 and p53 may be used as a good marker relating to cell apoptosis.

TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies

AIM:To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors,in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand(TRAIL),on overcoming TRAIL resistance in hepatocellular carcinoma(HCC)and to study the efficacy of agonistic TRAIL antibodies,as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS:Surface expression of TRAIL receptors (TRAIL-R1-4)and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL were analyzed by flow cytometry and Western blotting,respectively. Knock-down of MCL-1 and BCL-xL was performed by transfecting specific small interfering RNAs.HCC cellswere treated with kinase inhibitors and chemotherapeutic drugs.Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS:TRAIL-R1 and-R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However,treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates.Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002[inhibition of phosphoinositol- 3-kinase(PI3K)],AG1478(epidermal growth factor receptor kinase),PD98059(MEK1),rapamycin(mam- malian target of rapamycin)and the multi-kinase inhibitor Sorafenib.Furthermore,the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL play a major role in TRAIL resistance:knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells.Additionally, knock-down of MCL-1 and BCL-xL led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.CONCLUSION:Our data identify the blockage of survival kinases,combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

The involvement of p38 MAPK in transforming growth factor β1-induced apoptosis in murine hepatocytes

We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.

Gengnianchun recipe inhibits apoptosis of pheochromocytoma cells from beta-amyloid 25-35 insult, better than monotherapies and their compounds

This study aims to determine and compare the protective effects of Gengnianchun recipe drug serum and compounds of its representative drug monotherapies against sympathetic nerve pheochromocytoma cell line PC12 cells damaged by beta-amyloid 25-35 at the cellular apoptosis and related signal pathway levels. PC12 cells cultured with medicated rat serum showed enhanced cell viability and reduced cellular apoptosis rates compared with those of monotherapies and their compounds. Furthermore, Gengnianchun recipe up-regulated expressions of anti-apoptotic protein Bcl-2, estrogen receptor-beta and phosphorylated extracellular-signal-regulated kinase 1/2; and down-regulated expressions of pro-apoptotic proteins Bax and caspase-3. Gengnianchun recipe was superior to representative drug monotherapies, such as paeoniflorin, berberine, timosaponin A-III, icariine and their compounds in protecting PC12 cells. Mitogen-activated protein kinase blocker and estrogen receptor antagonist were found to reverse the above effects of Gengnianchun recipe. The experimental findings indicate that, Gengnianchun recipe protects PC12 cells from beta-amyloid 25-35 insult; its inhibitory effect on apoptosis may be achieved through the mitogen-activated protein kinase and estrogen receptor pathways.

Blockade of cholecystokinin-2 receptor and cyclooxygenase-2 synergistically induces cell apoptosis, and inhibits the proliferation of human gastric cancer cells in vitro

Gastrin and cyclooxygenase-2(COX-2) playimportant roles in the carcinogenesis and progression ofgastric cancer.However,it remains unknown whether the combination of cholecystokinin-2(CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic anti-tumor effects on human gastric cancer.Here,we demonstrated that the combination of AG-041R(a CCK-2 receptor antagonist) plus NS-398(a selective COX-2 inhibitor) treatment had synergistic effects on proliferation inhibition,apoptosis induction,down-regulation of Bcl-2 and up-regulation of Bax expression in MKN-45 cells.These results indicate that simultaneous targeting of CCK-2 receptor and COX-2 may inhibit gastric cancer development more effectively than targeting either molecule alone.(C)2008 Elsevier Ireland Ltd.All rights reserved.

Fermented Chinese formula Shuan-Tong-Ling attenuates ischemic stroke by inhibiting inflammation and apoptosis

The fermented Chinese formula Shuan-Tong-Ling is composed of radix puerariae（Gegen）,salvia miltiorrhiza（Danshen）,radix curcuma（Jianghuang）,hawthorn（Shanzha）,salvia chinensis（Shijianchuan）,sinapis alba（Baijiezi）,astragalus（Huangqi）,panax japonicas（Zhujieshen）,atractylodes macrocephala koidz（Baizhu）,radix paeoniae alba（Baishao）,bupleurum（Chaihu）,chrysanthemum（Juhua）,rhizoma cyperi（Xiangfu） and gastrodin（Tianma）,whose aqueous extract was fermented with lactobacillus,bacillus aceticus and saccharomycetes.ShuanTong-Ling is a formula used to treat brain diseases including ischemic stroke,migraine,and vascular dementia.Shuan-Tong-Ling attenuated H_2O_2-induced oxidative stress in rat microvascular endothelial cells.However,the potential mechanism involved in these effects is poorly understood.Rats were intragastrically treated with 5.7 or 17.2 m L/kg Shuan-Tong-Ling for 7 days before middle cerebral artery occlusion was induced.The results indicated Shuan-Tong-Ling had a cerebral protective effect by reducing infarct volume and increasing neurological scores.Shuan-Tong-Ling also decreased tumor necrosis factor-α and interleukin-1β levels in the hippocampus on the ischemic side.In addition,Shuan-Tong-Ling upregulated the expression of SIRT1 and Bcl-2 and downregulated the expression of acetylated-protein 53 and Bax.Injection of 5 mg/kg silent information regulator 1（SIRT1） inhibitor EX527 into the subarachnoid space once every 2 days,four times,reversed the above changes.These results demonstrate that Shuan-Tong-Ling might benefit cerebral ischemia/reperfusion injury by reducing inflammation and apoptosis through activation of the SIRT1 signaling pathway.

Overexpression of 14-3-3 protein protects pheochromocytoma cells against 1-methyl-4-phenylpyridinium toxicity

Objective To investigate the effects of 14-3-3 protein overexpression on the 1-methyl-4-phenylpyridinium （MPP^＋） induced pheochromocytoma （PC12） cell death and the potential mechanisms. Methods pcDNA3.1（＋）-14-3-3 plasmids, which could be expressed in mammalian cell, were constructed and transfected into PC 12 cells with Lipofectamine 2000. The expression of 14-3-3 protein, Bcl-2 protein, and BAD protein were determined by western blot. 3-（4,5- dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, microplate reader, and flow cytometric analysis were used to measure cell viability, the caspase activity, and apoptotic ratio respectively. Results （1） The expression of 14-3-3 protein increased significantly three weeks after pcDNA3.1（＋）-14-3-3 plasmids transfected into PC 12 cells. （2） MPP^＋ caused a decrease of cell viability in a dose-dependent manner. At 100μmol/L MPP^＋, cell viability reduced approximately 50%. （3） The caspase activity increased along with the MPP^＋ concentrations rising and reached its maximum value （0.34 μmol/mg protein） at 100 μmol/L MPP＊. However caspase activity decreased significantly when the MPP^＋ concentration exceeded 100 μmol/L. （4） Overexpression of 14-3-3 protein decreased the apoptosis ratio of PC 12 cells treated with 100μmol/L MPP^＋ from 26.5% to 8.6%. （5） Bcl-2 protein tended to decrease but BAD protein tended to increase after treatment of PC 12 cells with 100 μmol/L MPP^＋. Overexpression of 14-3-3 protein significantly increased the cellular level of Bcl-2 protein and decreased that of BAD protein. Conclusion Overexpression of 14-3-3 protein may reduce MPP^＋-induced apoptotic cell death in PC12 cells by up-regulating the Bcl-2 expression and down-regulating the BAD expression. These results may provide a promising target for treatment of Parkinson＇s disease.

冠心宁通过TNFAIP3-ASK1/JNK通路对动脉粥样硬化血管平滑肌细胞表型转换的调控作用

目的探讨冠心宁(GXN)调控动脉粥样硬化(AS)斑块稳定性的分子机制。方法制备含有GXN药物的血清,利用氧化低密度脂蛋白(ox-LDL)诱导血管平滑肌细胞(VSMC)构建AS体外模型,Western blot和qPCR检测VSMC表型转换情况。通过沉默肿瘤坏死因子α诱导蛋白3(TNFAIP3),检测GXN对VSMC表型转换的影响。构建凋亡信号调节激酶1(ASK1)过表达载体,并利用Lip3000转染进细胞,检测GXN是否通过凋亡信号调节激酶1/c-Jun氨基末端激酶(ASK1/JNK)通路发挥作用。结果与对照组比较,ox-LDL组的细胞表型转换,表现为I型胶原蛋白(COLIA1和COLIA2)、TNFAIP3和α-平滑肌肌动蛋白(α-SMA)的表达水平降低(均P<0.01),基质金属蛋白酶(MMP)2、MMP9、MMP13、骨桥蛋白(OPN)、ASK1磷酸化与ASK1比值(p-ASK1/ASK1)、JNK磷酸化与JNK比值(p-JNK/JNK)升高(均P<0.01);GXN处理后VSMC表型转换为收缩型,表现为与ox-LDL组相比COLIA1、COLIA2和α-SMA水平增高(均P<0.01),MMP2、MMP9、MMP13、OPN、TNFAIP3的表达水平、p-ASK1/ASK1以及p-JNK/JNK都降低(均P<0.01)。沉默TNFAIP3后,与ox-LDL+10%GXN+sh-NC组相比,COLIA1、COLIA2、TNFAIP3和α-SMA水平降低(均P<0.01),MMP2、MMP9、MMP13、OPN、p-ASK1/ASK1以及p-JNK/JNK都升高(均P<0.01)。过表达ASK1后,与ox-LDL+10%GXN+oe-NC组相比,COLIA1、COLIA2和α-SMA的表达水平降低(均P<0.01),MMP2、MMP9、MMP13、OPN、p-ASK1/ASK1以及p-JNK/JNK都升高(均P<0.01)。结论GXN可能通过TNFAIP3调控ASK1/JNK通路介导VSMC表型转换,从而起到稳定动脉硬化斑块的作用。

GPER agonist G1 suppresses neuronal apoptosis mediated by endoplasmic reticulum stress after cerebral ischemia/reperfusion injury

Studies have confirmed a strong association between activation of the endoplasmic reticulum stress pathway and cerebral ischemia/reperfusion(I/R) injury.In this study,three key proteins in the endoplasmic reticulum stress pathway(glucose-regulated protein 78,caspase-12,and C/EBP homologous protein) were selected to examine the potential mechanism of endoplasmic reticulum stress in the neuroprotective effect of G protein-coupled estrogen receptor.Female Sprague-Dawley rats received ovariectomy(OVX),and then cerebral I/R rat models(OVX+ I/R) were established by middle cerebral artery occlusion.Immediately after I/R,rat models were injected with 100 μg/kg E2(OVX + I/R +E2),or 100 μg/kg G protein-coupled estrogen receptor agonist G1(OVX + I/R + G1) in the lateral ventricle.Longa scoring was used to detect neurobehavioral changes in each group.Infarct volumes were measured by 2,3,5-triphenyltetrazolium chloride staining.Morphological changes in neurons were observed by Nissl staining.Terminal dexynucleotidyl transferase-mediated nick end-labeling staining revealed that compared with the OVX + I/R group,neurological function was remarkably improved,infarct volume was reduced,number of normal Nissl bodies was dramatically increased,and number of apoptotic neurons in the hippocampus was decreased after E2 and G1 intervention.To detect the expression and distribution of endoplasmic reticulum stress-related proteins in the endoplasmic reticulum,caspase-12 distribution and expression were detected by immunofluorescence,and mRNA and protein levels of glucose-regulated protein 78,caspase-12,and C/EBP homologous protein were determined by polymerase chain reaction and western blot assay.The results showed that compared with the OVX+ I/R group,E2 and G1 treatment obviously decreased mRNA and protein expression levels of glucose-regulated protein 78,C/EBP homologous protein,and caspase-12.However,the G protein-coupled estrogen receptor antagonist G15(OVX + I/R + E2 + G15) could eliminate the effect of E2 on cerebral I/R injury.These results confirm that E2 and G protein-coupled estrogen receptor can inhibit the expression of endoplasmic reticulum stress-related proteins and neuronal apoptosis in the hippocampus,thereby improving dysfunction caused by cerebral I/R injury.Every experimental protocol was approved by the Institutional Ethics Review Board at the First Affiliated Hospital of Shihezi University School of Medicine,China(approval No.SHZ A2017-171) on February 27,2017.

Mitomycin C induces apoptosis in human epidural scar fibroblasts after surgical decompression for spinal cord injury

Numerous studies have shown that topical application of mitomycin C after surgical decompression effectively reduces scar adhesion. However, the underlying mechanisms remain unclear. In this study, we investigated the effect of mitomycin C on the proliferation and apoptosis of human epidural scar fibroblasts. Human epidural scar fibroblasts were treated with various concentrations of mitomycin C （1, 5, 10, 20, 40 μg/mL） for 12, 24 and 48 hours. Mitomycin C suppressed the growth of these cells in a dose- and time-dependent manner. Mitomycin C upregulated the expression levels of Fas, DR4, DR5, cleaved caspase-8/9, Bax, Bim and cleaved caspase-3 proteins, and it downregulated Bcl-2 and Bcl-xL expression. In addition, inhibitors of caspase-8 and caspase-9 （Z-IETD-FMK and Z-LEHD-FMK, respectively） did not fully inhibit mitomycin C-induced apoptosis. Furthermore, mitomycin C induced endoplasmic reticulum stress by increasing the expression of glucose-regulated protein 78, CAAT/enhancer-binding protein homologous protein （CHOP） and caspase 4 in a dose-dependent manner. Salubrinal significantly inhibited the mitomycin C-induced cell viability loss and apoptosis, and these effects were accompanied by a reduction in CHOP expression. Our results support the hypothesis that mitomycin C induces human epidural scar fibroblast apoptosis, at least in part, via the endoplasmic reticulum stress pathway.