Effects of rapamycin on expression of Bcl-2 and Bax in human lens epithelial cells and cell cycle in rats

The effects of rapamycin on the expression of Bcl-2 and Bax protein in in vitro cultured human lens epithelial cells(LECs) and cell cycle were investigated in order to provide the theoretical basis for the development of new inhibitory drugs for clinical prevention and treatment of after-cataract.The cultured LECs of second and third passages were collected and treated with rapamycin.The LECs were transferred into 96-well culture plates and divided into 6 groups,and each group was set to have 8 duplicate wells.In the negative control group,the LECs were given culture medium only,and in the blank control group,only culture medium was given.In the four rapamycin-treated groups,different concentrations(20,40,60 and 80 ng/mL) of rapamycin were given.After treatment for 24,48 and 72 h,the absorbance(A) values in each well were determined by MTT assay.The cell cycles of all groups were detected by using flow cytometry.Real-time fluorescent quantitative polymerase chain reaction(RFQ-PCR) and Western blot were used to detect the mRNA and protein expression of Bcl-2 and Bax respectively.MTT assay showed that rapamycin could inhibit proliferation of LECs in a time-and dose-dependent manner.Flow cytometry revealed that rapamycin could block the conversion of LECs from G1 phase to S phase,resulting in the increase of cells in G1 phase and the decrease of the cells in S phase.RFQ-PCR indicated that rapamycin could down-regulate the expression of Bcl-2 mRNA,but up-regulate the expression of Bax mRNA,suggesting it could induce apoptosis of LECs.Western blot demonstrated that rapamycin could suppress the expression of Bcl-2 protein,but promote the expression of Bax protein.It is concluded that rapamycin could inhibit proliferation of LECs probably not only by blocking the progression of cell cycle,but also by promoting the induction of apoptosis.     

Renal protective activity of Hsian-tsao extracts in diabetic rats

Objective To investigate the renal protective activity of Hsian-tsao Mesona procumbens Hemsl. water extracts in diabetic rats. Methods Thirty Sprague-dawley female rats were randomly divided into three groups （n=10 each）, ＂control group＂ with intraperitoneal saline injection, ＂diabetic group＂ with 60 mg of intraperitoneal streptozotocin injection per kg of body weight and ＂Hsian-tsao group＂ with intragastric administration of Hsian-tsao extraction everyday for 4 weeks after intraperitoneal streptozotocin injection. The body weight and blood sugar were measured before and after model induction in the three groups. Thrombospondin-1 （TSP-1） expressions in the kidney were monitored by immunohistochemistry. Kidney ultrastructural changes were also analyzed by using transmission electron microscopy. Results Before diabetic model induction, there were no significant differences among the three groups in body weight and blood sugar. Four weeks after the induction of diabetes, the differences became statistically significant. Electron microscopy also revealed disruption of the foot processes of the podocytes and other damages in diabetic group. These damages were significantly less severe in Hsian-tsao group when compared with the diabetic group. TSP-1 expressions in the kidney were significantly increased in both the diabetic group and Hsian-tsao group, but it was relatively lower in Hsian-tsao group than in diabetic group. Conclusion Our results showed that Hsian-tsao treatment in the diabetic rats effectively prevented the pathological alterations in the kidney and decreased the TSP- 1 expression. It was suggested that Hsian-tsao had protective effect on the kidneys of the diabetic rats.     

Role of reactive oxygen species in triptolide-induced apoptosis of renal tubular cells and renal injury in rats

This study investigated the role of reactive oxygen species(ROS) in the pathogenesis of triptolide-induced renal injury in vivo.Rats were randomly divided into 4 groups(n=5 in each):triptolide group in which the rats were intraperitoneally injected with triptolide solution at a dose of 1 mg/kg of body weight on day 8;control group in which the rats received a single intraperitoneal injection of 0.9% physiological saline on day 8;vitamin C group in which the rats were pretreated with vitamin C by gavage at a dose of 250 mg/kg of body weight per day for 7 days before the same treatment as the control group on day 8;triptolide+vitamin C group in which the rats were first subjected to an oral administration of vitamin C at a dose of 250 mg/kg of body weight per day for 7 days,and then to the same treatment as the triptolide group on day 8.All the rats were sacrificed on day 10.Blood samples were collected for detection of plasma creatinine(Pcr) and plasma urea nitrogen(PUN) concentrations.Both kidneys were removed.The histological changes were measured by haematoxylin-eosin(HE) staining.The production of ROS was determined by detecting the fluorescent intensity of the oxida-tion-sensitive probe rhodamine 123 in renal tissue.Renal malondialdehyde(MDA) content was meas-ured to evaluate lipid peroxidation level in renal tissue.TUNEL staining was performed to assess apop-tosis of renal tubular cells.Renal expression of apoptosis-related proteins Bcl-2,Bax,Bid,Bad,Fas and FasL,as well as corresponding encoding genes were assessed by Western Blotting and real-time PCR.The results showed that triptolide treatment promoted the generation of a great amount of ROS,up-regulated the expression of Bax,Bid,Bad,Fas and FasL at both protein and mRNA levels,as well as the ratio of Bax to Bcl-2,and caused the apoptosis of renal tubular cells and renal injury.However,pretreatment with an antioxidant,vitamin C,significantly reduced the generation of ROS and effectively inhibited the triptolide-induced apoptosis of renal tubular cells and renal injury.It was concluded that ROS plays a critical role in triptolide-induced apoptosis of renal tubular cells and renal injury.The protective administration of vitamin C may help alleviate triptolide-induced renal injury and nephrotoxicity.     

Berberine ameliorates renal injury in streptozotocin-induced diabetic rats by suppression of both oxidative stress and aldose reductase

Background Berberine is one of the main constituents of Coptidis rhizoma （CR） and Cortex phellodendri. In this study, we investigated the beneficial effects of berberine on renal function and its possible mechanisms in rats with diabetic nephropathy （DN）.
Methods Male Wistar rats were divided into three groups： normal, diabetic model, and berberine treatment groups. Rats in the diabetic model and berberine treatment groups were induced to diabetes by intraperitonal injection with streptozotocin （STZ）. Glomerular area, glomerular volume, fasting blood glucose （FBG）, blood urea nitrogen （BUN）, serum creatinine （Cr） and urine protein for 24 hours （UP24h） were measured using commercially available kits. Meanwhile, the activity of superoxide dismutase （SOD）, content of malondialdehyde （MDA） in serum, activity of aldose reductase （AR） and the expression of AR mRNA and protein in kidney were detected by different methods.
Results The results showed that oral administration of berberine （200 mg·kg^-1·d^-1） significantly ameliorated the ratio of kidney weight to body weight. Glomerular area, glomerular volume, FBG, BUN, Cr and UP24h were significantly decreased in the berberine treatment group compared with the diabetic model group （P〈0.05）. Berberine treatment significantly increased serum SOD activity and decreased the content of MDA compared with diabetic model group （P 〈0.05）. AR activity as well as the expression of AR mRNA and protein in the kidney was markedly decreased in the berberine treatment group compared with diabetic model group （P 〈0.05）.
Conclusion These results suggested that berberine could ameliorate renal dysfunction in DN rats through controlling blood glucose, reduction of oxidative stress and inhibition of the activation of the polyol pathway.     

Study on norcantharidin-induced apoptosis in SMMC-7721 cells through mitochondrial pathways

Objective: To investigate the mechanism of norcantharidin (NCTD)-induced SMMC-7721 hepatoma cell apoptosis. Methods: SMMC-7721 cell growth inhibition was measured by the MTT method. Apoptosis was detected by Annexin Ⅴ/propidium iodide staining. The mitochondrial membrane potential was measured by flow cytometry. Western blot analysis was used to evaluate the level of cytochrome c, caspase-3, AIF, Bcl-2 and Bax expression. Results: NCTD inhibited SMMC-7721 cell growth in a time-and dose-dependent manner. The cells treated with NCTD showed the loss of mitochondrial membrane potential. The activities of caspase-3, cytochrome c, AIF, and Bax were up-regulated after NCTD treatment at different doses. The expression of Bcl-2 was decreased after treatment with NCTD. Conclusions: NCTD could induce SMMC-7721 cell apoptosis. The activation of the mitochondrial pathway was involved in the process of NCTD-induced SMMC-7721 cell apoptosis.     

Mesenchymal stem cells transplantation mildly ameliorates experimental diabetic nephropathy in rats

Background Diabetic nephropathy is a common complication of diabetes mellitus. This study aimed to explore whether mesenchymal stem cells （MSCs） transplantation could attenuate diabetic nephropathy in experimental diabetic rats. Methods Sprague-Dawley rats received a single intraperitoneal injection of streptozotocin （STZ） （60 mg/kg）. Diabetic rats were randomized to four groups： diabetes control group （DC）, ciclosporin A group （CsA）, MSC group, and MSC＋CsA group （MSCA）. Bone marrow mesenchymal stem cells were cultured, identified and labeled by 5-bromo-2＇-deoxyuridine （BrdU） in vitro. Then they were transplanted to diabetic rats via introcardiac infusion. Ciclosporin A was administered daily at 5 mg/kg. At 1,2, 4, 8 weeks after transplantation, random blood glucose, urine albumin/creatinine ratio （AIb/Cr）, endogenous creatinine clearance rate and renal mass index were tested. Renal morphology and labeled cells were examined. Results Cultured MSCs expressed mesenchymal cell phenotype, and could be multidifferentiated to osteogenic and adipogenic cells. Labeled MSCs could be detected in the kidney of nephropathic rats, mainly in renal interstitium, but they did not propagate after engrafting in kidney. Over the course of the experiment, MSCA group showed a significant decrease in blood glucose compared with MSC group, CsA group and DC group （P 〈0.05, respectively）. The AIb/Cr in MSCA group and MSC group were significantly lower than CsA group and DC group （P〈0.05）. And the AIb/Cr in MSCA group showed a significant decrease compared with MSC group （0.74 vs 0.84, P 〈0.05）. There was a significant difference in renal mass index between the MSCA group and DC group （5.66 vs 6.37, P 〈0.05）. No significant difference was found in creatinine clearance rate among 4 groups （P 〉0.05）. Treatment with MSC＋CsA significantly ameliorated the morphology of diabetic kidney. Conclusion MSC could mildly ameliorate diabetic nephropathy by decreasing blood glucose, AIb/Cr ratio and renal mass index.     

Realgar-induced apoptosis of cervical cancer cell line Siha via cytochrome c release and caspase-3 and caspase-9 activation

Objective:To explore the molecular mechanism of realgar-induced apoptosis of cervical cancer cells.Methods:The cervical cancer cell line Siha was used to determine the cell viability and apoptosis after treatment with realgar using MTT assay and flow cytometry.The activities of caspase-3,-8,and-9 were detected by fluorescence resonance energy transfer technology and colorimetric assay,while the levels of Bcl-2,cytochrome c,and Bax were detected by Western blot method.Results:Induction of apoptosis by realgar was detected in Siha cell line in a dose-dependent manner.The apoptosis was accompanied by a significant increase in cytochrome c release and activation of caspase-3 and caspase-9 but not caspase-8.Further,the realgar-induced apoptosis was inhibited by a broad-spectrum caspase inhibitor,a caspase-3 inhibitor,and a caspase-9 inhibitor but not by a caspase-8 inhibitor.Bcl-2 and Bax protein expressions were not changed by realgar.Conclusion:The induction of apoptosis by realgar is mediated through a cytochrome c-dependent pathway,which sequentially activates caspase-9 and caspase-3.     

Effect of down-regulation of TRPC6 on the puromycin aminonucleoside-induced apoptosis of mouse podocytes

Eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside （PAN）-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN treatment＋shRNA transfection group, and PAN treatment＋negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.     

Effects of mycophenolate mofetil, valsartan and their combined therapy on preventing podocyte loss in early stage of diabetic nephropathy in rats

Background Podocyte has inflammatory role in the development of diabetic nephropathy (DN). Mycophenolate mofetil (MMF), an anti-inflammatory agent, can suppress macrophage infiltration and reduce renal injury in streptozotocin-induced diabetic rats. Angiotensin II receptor blocker (ARB), another renal protecting agent, can decrease podocyte loss in DN. In this study, we detected the expression levels of monocyte chemoattractant protein-1 (MCP-1) and nephrin to evaluate podocyte’s role in inflammatory reaction in DN, observe and compare the effect of MMF alone and in combination with valsartan, on preventing podocyte loss in streptozotocin (STZ) induced diabetic rats. Methods Diabetic model was constructed in uninephrectomized male Wistar rats by single peritoneal injection of STZ (65 mg/kg). The successfully induced diabetic rats were randomly divided into four groups: diabetes without treatment group (DM), valsartan treated group (DMV), MMF treated group (DMM), and combined therapy group (DMVM). Normal rats of the same sibling were chosen as control (NC). At the end of the 8th week, serum biochemistry, 24-hour urinary protein (UP) and the ratio of kidney weight/body weight (RWK/B) were measured. The rats were sacrificed for the observation of renal histomorphology through light and electron microscope. Nephrin, desmin and MCP-1 levels were detected by semi-quantitative immunohistochemical assays. Real-time quantitative PCR was used to detect the mRNA levels of nephrin and MCP-1.Results Compared with group NC, serum glucose level, 24-hour UP and RWK/B in group DM were significantly higher (P<0.01), and the nephrin mRNA level in DM group was significantly lower (P<0.05). The nephrin mRNA expression levels in group DMV, DMM and DMVM were all higher than that of DM group (P<0.05) and no significant differences were found among the three treatment groups (P>0.05). Treatment with MMF, valsartan or their combination could significantly decrease the 24-hour UP and RWK/B, and suppress glomerulosclerosis and interstitial fibrotic lesions in diabetic rats. In diabetic rats, the high expressions of desmin and MCP-1 in kidney were suppressed by valsartan, MMF or their combination.Conclusions Podocytes are involved in the inflammatory reaction of diabetic rats. MMF could suppress MCP-1 and desmin expression, enhance nephrin expression, and attenuate proteinuria in diabetic rats. The combined therapy of valsartan and MMF did not show any superiority over monotherapies on renal protection. MMF may have renoprotective effect in early stages of diabetic nephropathy through preventing podocytes loss and anti-inflammatory activity.     

Effects of Livin gene RNA interference on apoptosis of cervical cancer Hela cells and enhanced sensitivity to cisplatin

The recombinant plasmids pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into Hela cells and cisplatin was added with different concentrations in order to study the inhibitory effects of Livin gene, increase the apoptosis induced by cisplatin, and detect the expression ofBcl-2, Bax, caspase-3, and survivin genes. The pGenesil-1-BIRC71 and pGenesil-1-BIRC72 were transfected into Hela cells, and the expression levels of Livin, Bcl-2, Bax, caspase-3, and survivin genes were detected by using fluorescence quantitative real-time PCR. Then cisplatin at different concentrations （3.0, 6.0 and 9.9 μg/mL） was added into the transfected Hela cells, and 24, and 48 h later, the apoptosis rate was measured by flow cytometry. After transfection of pGenesil-l-BIRC71 and pGenesil-1-BIRC72 into Hela cells, the expression level of Livin gene was obviously reduced, and the apoptosis rate was significantly increased in transfection group as compared with control group （P〈0.05）. Cisplatin could increase the apoptosis rate in a dose- and time-dependent manner. After cisplatin was added, the expression levels of Bcl-2 mRNA were reduced, and those of Bax, caspase-3, and survivin mRNA were increased in transfection group as compared with those in control group （P〈0.05）. It was concluded that shRNA expression vector targeting Livin gene could inhibit the expression of Livin gene in Hela cells and enhance the apoptosis induced by cisplatin, which was related to the decreased expression of Bcl-2 and activation of Bax and caspase-3. Survivin might play an important role as an antagonist in the process of apoptosis induction.     

Cytoprotective Effect of Silymarin against Diabetes-Induced Cardiomyocyte Apoptosis in Diabetic Rats

Objective The beneficial effects of silymarin have been extensively studied in the context of inflammation and cancer treatment, yet much less is known about its therapeutic effect on diabetes. The present study was aimed to investigate the cytoprotective activity of silymarin against diabetes-induced cardiomyocyte apoptosis.
Methods Rats were randomly divided into: control group, untreated diabetes group and diabetes group treated with silymarin (120 mg/kg·d) for 10 d. Rats were sacrificed, and the cardiac muscle specimens and blood samples were collected. The immunoreactivity of caspase-3 and Bcl-2 in the cardiomyocytes was measured. Total proteins, glucose, insulin, creatinine, AST, ALT, cholesterol, and triglycerides levels were estimated.
Results Unlike the treated diabetes group, cardiomyocyte apoptosis increased in the untreated rats, as evidenced by enhanced caspase-3 and declined Bcl-2 activities. The levels of glucose, creatinine, AST, ALT, cholesterol, and triglycerides declined in the treated rats. The declined levels of insulin were enhanced again after treatment of diabetic rats with silymarin, reflecting a restoration of the pancreaticβ-cells activity.
Conclusion The findings of this study are of great importance, which confirmed for the first time that treatment of diabetic subjects with silymarin may protect cardiomyocytes against apoptosis and promote survival-restoration of the pancreaticβ-cells.     

Protective Effect of Sodium Tanshinone ⅡA Sulfonate on Injury of Small Intestine in Rats with Sepsis and Its Mechanism

Objective:To explore the protective effect of sodium tanshinoneⅡA sulfonate(STS) on small intestine injury in rats with sepsis and its possible mechanism.Methods:According to a random number table, 24 Tats were randomly divided into 3 groups:sham operation group(sham group),sepsis model group(model group) and STS treatment group(STS group),with 8 Tats in each group.A rat model of sepsis was induced by cecal ligation and puncture(CLP) for 5 h.STS(1 mg/kg) was slowly injected through the right external jugular vein after CLP.The histopathologic changes in the intestine tissue were observed under a light microscope,and the intestinal epithelial cell apoptosis was evaluated by terminal deoxynucleoddyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL) method.The expressions of Bcl-2,Bax and nuclear factorκB(NF-κB) p65 in the intestinal tissue was determined by Western blot.The levels of tumor necrosis factorα(TNF-α) and interleukin 6(IL-6) in the intestinal tissue were determined using enzyme-linked immuno-sorbent assay(ELISA). Results:Obvious injuries were observed in the intestinal tissue in the CLP group compared with the sham group. The expression of NF-κB p65 and the levels of TNF-αand IL-6 were up-regulated after CLP,the apoptosis of intestinal epithelial cells was increased after CLP,and the ratio of Bcl-2 to Bax was decreased.STS posttreatment could attenuate the injury on the intestinal tissue induced by CLP,decrease the apoptosis of intestinal epithelial cells and the levels of NF-κB p65,TNF-αand IL-6,and increase the ratio of Bcl-2 to Bax.Conclusion: STS can protect the small intestine in rats with sepsis,and the mechanism may be associated with the inhibition of intestinal epithelial apoptosis and the reduction of activation of inflammatory cytokines.     

Effects of <Emphasis Type="Italic">Panax notoginoside</Emphasis> on the expression of TGF-β1 and Smad-7 in renal tissues of diabetic rats

In order to explore the effects of Panax notoginoside (PNS) on the expression of transforming growth factor β1 (TGF-β1) and Smad-7 in renal tissues of diabetes, a rat model of diabetic nephropathy was set up by intravenous injection of streptozotocin (STZ). Wistar rats were randomly divided into normal group, diabetic control group, group treated by PNS at low-dosage (PL), group treated by PNS at high-dosage (PH) and group treated by catopril (C), respectively. Fasting blood glucose (FBG), renal index, endogenous creatinine clearance rate (CCr) and urinary albumin (UAlb) in 24 h were examined after 6 weeks. Meanwhile, the expressions of TGF-β1 and Smad7 in renal tissues were immunohistochemically dectected. At the end of the sixth week, FBG, renal index, Ccr, UAlb were all elevated significantly in control group (P<0.01). The expression of TGF-β1 protein was increased while Smad7 protein decreased in renal tissue (P<0.01). However, the treatment with PNS reversed the aforementioned changes in renal tissues of diabetic rats. These results indicate that PNS possess a protective effect on the kidney of diabetic rats and it might protect kidney by inhibiting the expression of TGF-β1 protein and enhancing the expression of Smad7 protein.     

Huaiqihuang Granules (槐杞黄颗粒) Reduce Proteinuria by Enhancing Nephrin Expression and Regulating Necrosis Factor κB Signaling Pathway in Adriamycin-Induced Nephropathy

Objective: To investigate the effects of Huaiqihuang Granules(槐杞黄颗粒, HQH), a mixture of Chinese herbs including Trametes robiniophila Murr, Fructus Lycii and Polygonatum sibiricum, on adriamycininduced nephropathy(ADRN) in rats and its underlying mechanisms. Methods: Rats with ADRN were divided into four groups: the sham group, the model group(distilled water), the low-dose HQH-treated(2 g/kg) group, and the high-dose HQH-treated(4 g/kg) group. Body weight and 24-h urinary protein(Upro) were checked every week. After 5-week intervention, at the end of the study, the rats were sacrificed and blood samples were collected for examination of biochemical parameters, including glomerular morphological makers, podocyte shape, cellular apoptosis, expressions of nephrin, inflammatory and apoptosis markers. Results: HQH ameliorated the rat's general status, proteinuria, renal morphological appearance and glomerulosclerosis. The decreased expression of nephrin in ADRN rats was increased by HQH, as well as the impaired podocyte foot process fusion. Cytosolic levels of p65 and inhibitor of nuclear factor κBα(IκBα) were decreased in ADRN rats, and recovered by the treatment of HQH. Consistently, the induced expression of tumor necrosis factor α(TNF-α), phosphorylated nuclear factor κB p65(p-NFκB p65) and IκBα in ADRN were markedly suppressed by HQH. In addition, induction of Bax, cleaved caspase-3 and cytochrome C in ADRN rats were suppressed by HQH, indicating the amelioration of apoptosis. Conclusion: HQH could ameliorate renal impairments in ADRN rats by increasing nephrin expression, inhibiting NF-κB signaling pathway via the down-regulation of p-NF-κB p65 and p-IκBα, and suppression of glomerular and tubular apoptosis.     

Effect of Bushenyisui Formula on brain tissue apoptosis and Bcl-2 in beta-amyloid protein-induced Alzheimer’s disease rat models

OBJECTIVE:To investigate the effect of Bushenyisui Formula on cell apoptosis and positive B cell lymphoma(Bcl-2) in the Brain of rat models of Alzheimer’s disease(AD) induced by beta-amyloid protein(Aβ) and the mechanism underlying the effect.METHODS:Total of 40 SD rats,20 females and 20 males,were randomly assigned to 4 groups,controlled group(A),model group(B),conventional treatment group(C) and Bushenyisui Formula treatment(BYFT) group(D),10 rats in each group.Aβ 1-42 was injected into the bilateral hippocampus of the rats in group B,C and D to create the models of AD.Sham operation was performed on the rats of group A in the same way by injecting equal volume of 0.9% sodium chloride solution into their bilateral hippocampus.5 days after operation,Bushenyisui Formula was intraperitoneally administered at a dose of 450 mg/kg to the rats of group D(QD) for 20 days.Equalvolumeof0.9%sodiumchloridesolution wasintraperitoneallyinjectedintotheratsofgroupB withthesameprocedure.C suspension(20 mg/mL) was intraperitoneally injected into the rats of group B with the same procedure.The number of apoptotic cells in Brain and the positive Bcl-2 were counted.The changes of learning and memory abilities were evaluated usingY-maze.RESULTS:Right after the establishment of the models,group B,C and D compared to group A respectively,the outcomes of Y-maze were significantly different from that of group A,which suggested obvious learning and memory disorder in those groups(P<0.01).After treatment,the times of electronic shocks of group C and D were significantly less than that of group B(P<0.05),and the numbers of apoptotic cells and positive Bcl-2 were significantly different from those of group B,apoptotic sells’ number of group C and D smaller than that of group B and the number of positive Bcl-2 greater than that of group B.CONCLUSION:Bushenyisui Formula could increase the number of Bcl-2 in brain,which improved the function of nervous system pertaining to learning and memory abilities.     

Effects of Local Testicular Heating on Bcl-2 and Bax Protein Expression in Spermatogenic Cells in Rats

Objective To investigate the expression of Bcl-2 and Bax in spermatogenic cells induced by local testicular heating in rats Methods Forty adult male SD rats were divided into experimental group and control group at random. According to day 0.5, 1, 3 and 6 after local testicular heating, each group was divided into 4 subgroups： experimental subgroup （n=6） and control subgroup （n=4）. The expression of Bcl-2 and Bax in the spermatogenic cells was detected on day 0.5, 1, 3 and 6 after heat exposure by using immunohistochemistry. Results Compared with control groups, the ratio of positive cells and content of Bcl-2 positive cells significantly decreased in all experimental subgroups （P〈0.01）. The content of Bax positive cells increased in all experimental subgroups （P〈0.01）, the ratio of positive cells which had no significant difference （P〉0.05） except 6 d group decreased （P〈0.01 ）. Redistribution of Bax from a cytoplasmic to perinuclear or nuclear localization could be observed after heating. Conclusions Expression of Bcl-2 would decrease and Bax would increase with redistribution in spermatogenic cells in rats after heating. The change of Bcl-2 and Bax expression in spermatogenic cells would be correlated with the spermatogenic cell apoptosis induced by heating.     

Influence of silencing soluble epoxide hydrolase with RNA interference on cardiomyocytes apoptosis induced by doxorubicin

In order to investigate the influence of silencing soluble epoxide hydrolase(sEH) with double-stranded small interfering RNA(siRNA) on cardiomyocytes apoptosis induced by doxorubicin(DOX),two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents.The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively,and the plasmids that silenced sEH most significantly were selected,and renamed EH-R.The plasmids carrying a nonspecific siRNA coding sequence(PCN) served as the negative control.Cardiomyocytes were divided into four groups:control group,DOX group,PCN+DOX group,and EH-R+DOX group.Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 μmol/L.Apoptosis rate of cardiomyocytes was determined by flow cytometery.The protein expression levels of Bcl-2 and Bax were detected by Western blotting.The results showed that the expression of sEH was down-regulated by EH-R plasmid.The expression levels of sEH mRNA and protein in the EH-R+DOX group were significantly decreased as compared with other groups(P<0.01).As compared with the control group,the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased,the expression levels of Bax increased,and those of Bcl-2 decreased(P<0.01).However,the expression levels of Bax were decreased,those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obvi-ously decreased in EH-R+DOX group when compared with those in the DOX group and the PCN+DOX group(P<0.01 for each).It was concluded that the recombinant plasmids could be successfully constructed,and transfected into the primary cultured cardiomyocytes.They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.     

LOW MOLECULAR WEIGHT HEPARIN DECREASES THE ALBUMINURIA AND RENAL PATHOLOGICAL CHANGES IN DIABETIC NEPHROPATHY RATS

Objective To investigate the renoprotection effect of low molecular weight heparin（LMWH）in diabetic rats.Methods Diabetic rats were induced by injection of steptozotocin（STZ）.Rats were randomly divided into three groups：control group,diabetic model group,and LMWH treatment group.The levels of total cholesterol（TC）,triglyceride（TG）,prothrombin time（PT）,kaolin partial thromboplastin time（KPTT）,24-hour urinary albumin excretion rate（UAER）,and serum soluble P-selectin were determined after 8 weeks.Glomerular morphology was observed by light microscopy.Results The levels of TG,UAER,and P-selectin in LWMH treatment group were lower than those in model group（all P〈0.01）.The levels of PT and KPTT in LWMH treatment group were shorter than those in model group.And LWMH improved the histological changes of diabetic rats.Conclusion LMWH has some renal protective effects in diabetic rats,partly through down-regulating the expression of P-selectin.     

Effects of Ethyl Pyruvate on Myocardial Apoptosis and Expression of Bcl-2 and Bax Proteins after Ischemia-reperfusion in Rats

In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion （I/R） in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups （n=8 in each group）： control group was perfused for 120 min. In the I/R group, after 30 min stabilization the injury was induced by 30 min global ischemia followed by 60 min reperfusion. Ethyl pyruvate （EP） group was set up with the same protocol as I/R group except that it was supplied with 2 mmol/L EP 15 rain before ischemia and throughout reperfusion. Myocardial malonaldehyde （MDA） content was measured. Myocardial apoptotic index （AI） was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling （TUNEL） method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group （P〈0.05）. These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.