RXRα agonist bexarotene inhibits atherosclerosis in ApoE-/- mice by regulating TGF-β1/Smad2 signaling pathway

AIM To observe the effect of retinoid X receptor α （RXRα） agonist bexarotene （Bex） on the proliferation of transforming growth factor β1 （TGF-β1）-induced vascular smooth muscle cells （VSMCs） and atherosclerosis in apolipoprotein E knockout （ApoE^-/-） mice, and to explore the underlying mechanism. METHODS Ten C57BL/6 mice were selected as normal control group, and 30 ApoE^-/- mice were randomly divided into 3 groups： ApoE^-/- group, ApoE^-/-+Bex5 （5 mg·kg^-1·d^-1 Bex） group and ApoE^-/-+Bex10 （10 mg·kg^-1·d^-1 Bex） group. Bex was intragastrically given once a day for 8 weeks. The levels of triglyceride （TG） and total cholesterol （TC） were determined by oxidase method, and select masking method was used to determine serum levels of low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C）. The protein levels of TGF-β1, p-Smad2 and Smad2 were determined by Western blot. HE staining was used to observe the intima of the thoracic aorta. The VSMCs were cultured with tissue patch method, and the proliferation of VSMCs was measured by BrdU incorporation method. RESULTS The serum levels of TG, TC and LDL-C, and the expression of TGF-β1 and p-Smad2 in thoracic aorta in ApoE^-/- group were significantly higher than those in C57BL/6 group （P<0.01）. Bex increased p-Smad2 protein level in thoracic aorta in a dose-dependent manner, inhibited the intimal plaque formation and vascular medial proliferation, and decreased the plaque area in ApoE^-/- mice （P<0.01）. No significant difference in serum levels of TG, TC, HDL-C and LDL-C, and TGF-β1 and Smad2 expression in thoracic aorta among ApoE^-/- group, ApoE^-/-+Bex5 group and ApoE^-/-+Bex10 group was observed. TGF-β1 （0.1~10 μg/L） promoted the proliferation of VSMCs, while Bex （10^-9~10^-7 mol/L） inhibited TGF-β1 （5 μg/L）-induced proliferation of VSMCs in a concentration-dependent manner. Bex （10^-7 mol/L） synergistically promoted the protein level of p-Smad2 in VSMCs induced by TGF-β1 （P<0.01）, but inhibited TGF-β1-induced nuclear translocation of p-Smad2. CONCLUSION RXRα agonist Bex inhibits the formation of atherosclerosis in ApoE^-/- mice, and its mechanism may be related to the regulation of TGF-β1/Smad2 pathway.     

Aβ1-40 induces inflammation and phenotypic switching via activation of MAPKs signaling pathway in vascular smooth muscle cells

AIM To investigate the effects of amyolid β-protein 1-40 （Aβ1-40） on inflammation, viability, migration and phenotypic switching in vascular smooth muscle cells （VSMCs）, and to analyze the underlying mechanisms. METHODS The VSMCs were treated with Aβ1-40 at different concentration gradients for appropriate time. CCK-8 and Transwell assays were performed to evaluate the viability and migration ability of VSMCs. The levels of inflammatory factors including interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α）, phenotypic switching-related proteins including α?smooth muscle actin （α?SMA）, osteopontin （OPN） and Krüppel?like factor 4 （KLF4）, and mitogen-activated protein kinases （MAPKs） signaling pathway-related proteins including p-p38 MAPK, p-ERK1/2 and p-JNK were determined by Western bolt. RESULTS After Aβ1-40 treatment, the levels of inflammatory factors IL-1β and TNF-α in the VSMCs were significantly increased （P<0.05）, and the expression of phenotypic switching-related proteins was altered, as indicated by down-regulation of α?SMA and up-regulation of OPN and KLF4 （P<0.05）. Treatment with Aβ1-40 within a certain concentration range promoted the viability and migration of the VSMCs. In addition, the protein levels of p-p38 MAPK, p-ERK1/2 and p-JNK were significantly increased by Aβ1-40 treatment （P<0.05）. Furthermore, pretreatment with specific inhibitors of MAPKs pathway significantly reduced the levels of IL-1β and TNF-α （P<0.05）, and inhibited the phenotypic switching, as indicated by up-regulation of α?SMA and down-regulation of OPN and KLF4 （P<0.05）. CONCLUSION Treatment with Aβ1-40 induces the inflammation and phenotypic switching in VSMCs via activation of MAPKs signaling pathway.     

Effect of HOCs implantation on protein expression of TGF-β/Smad signaling pathway in liver tissue of hepatic fibrosis rats

AIM: To investigate the effect of hepatic oval cells (HOCs) on the protein expression of TGF-β/Smad signaling pathway in the liver tissue of hepatic fibrosis rats.METHODS: The SD rat models of liver fibrosis were made by treating with carbon tetrachloride and combined factors. The HOCs was isolated from the model rats. HOCs suspension (0.5 mL at a density of 1×1012cells/L) were transplanted via portal vein into the hepatic fibrosis rats at 8th week and observed continuously for 30 days. Meanwhile, WuLing capsules were used for positive control. The blood samples were collected through trail vein at 8th day, 15th day, 23th day and 30th day after transplantation of HOCs. The levels of aspartate aminotransferase (AST) and alamine aminotransferase (ALT) in serum were determined by enzyme method. The morphological changes of hepatic tissues were observed under microscope with HE and Musson staining. The protein levels of collagen type I (Col-Ⅰ), extracellular-signal regulated protein kinase (ERK), phosphory-lation extracellular regulatedprotein kinases (p-ERK), TGF-β receptor type Ⅰ (TβRⅠ), TGF-β receptor type Ⅱ (TβRⅡ), mothers against decapentaplegic homolog 2/3 (Smad 2/3) and mothers against decapentaplegic homolog (Smad 7) were assessed in liver tissues by Western blotting. RESULTS: In HOCs and WuLing capsules treated groups, the levels of ALT and AST decreased significantly at 15th day, 23th day and 30th day after the transplantation of HOC. The damage degree of hepatic fiber hyperplasia of the liver histological structure reduced notably. The expression levels of Col-Ⅰ, ERK, p-ERK, TβRⅠ and TβRⅡ in liver tissues of hepatic fibrosis rats were down-regulated obviously while the expression of Smad 7 increased significantly.CONCLUSION: The implantation of HOCs prevents the progress of liver fibrosis in rats. The mechanism of action is to inhibit the protein expression of p-ERK, TβRⅠ, TβR Ⅱ for TGF-β/Smad signaling pathway of liver tissue.     

Role of danshensu on TGF-β signal transduction in rat’s hepatic stellate cells

AIM: To investigate the role of danshensu on Smad signal transduction in rat hepatic stellate cells (HSCs) stimulated with transforming growth factor (TGF-β1). METHODS: The rat HSCs was isolated with collagenase by in situ-liver recirculation perfusion and cultured in vitro. MTT colorimetric assay was used to detect proliferation of HSCs treated with different concentration of danshensu. The expressions of α-SMA and TβR I and II were observed by immunocytochemitry, indirect immunofluorescent staining and Western blotting when HSCs stimulated with TGF-β1 and with different concentrations of danshensu for 24 h. RESULTS: (1) Danshensu at the concentration from 0.0625 mmol/L to 1 mmol/L prevented the proliferation of HSCs in a dose-dependent manner (P<0.05). Danshensu also inhibited the proliferation of HSCs induced by TGF-β1 in a dose-dependent manner (P<0.05). (2) At concentration of 0.25 mmol/L, danshensu down-regulated α-SMA protein expression in HSCs with or without stimulation of TGF-β1 (P<0.05), and the activation of HSCs was inhibited also. (3) Danshensu down-regulated the protein expression of TβR I and II in HSCs stimulated with TGF-β1 (P<0.05, or P<0.01), these effects were correlated with the concentration. (4) TGF-β1 increased the mRNA level of Smad2, 3, and 7 in HSCs (P<0.01). Danshensu down-regulated the mRNA level of Smad2, 3 (P<0.05) and up-regulate the mRNA level of Smad7 in HSCs induced by TGF-β1 (P<0.05). CONCLUSION: Danshensu inhibits the activation and proliferation of HSCs through down-regulating the expression of TβR I and II located in cellular membrane of HSCs. Danshensu suppresses the activation of HSCs, and also inhibits the activation of HSCs stimulated by TGF-β1 through up-regulation of Smad7 mRNA and down-regulation of Smad2, Smad3 mRNA expression in HSCs.     

Effects of platelet-rich plasma on chondrocyte apoptosis and NLRP3/IL-1β signaling pathway in rabbits with osteoarthritis

AIM To explore the effect of platelet-rich plasma (PRP) on rabbit osteoarthritis and its possible mechanism. METHODS The rabbits with knee osteoarthritis were prepared and then divided into model group, sodium hyaluronate (SH) group and PRP group, and another sham operation group was set up, with 6 rabbits in each group. The gross morphological changes of rabbit cartilage were observed. HE staining was used to evaluate the pathomorphological changes of the cartilage. TUNEL staining was used to detect the apoptosis of chondrocytes. The expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/interleukin-1β （IL-1β） signaling pathway-related molecules was observed by immunohistochemical staining, and the protein levels of caspase-3, Bcl-2 and Bax were determined by Western blot. Chondrocytes were isolated and processed according to grouping, and the NLRP3 and IL-1β levels of the cells were measured by ELISA. RESULTS Compared with sham operation group, Pelletier score, Mankin score, chondrocyte apoptotic rate, the positive protein expression rates of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax in model group were increased significantly (P<0.05), while the protein expression of Bcl-2 was decreased significantly (P<0.05). Compared with model group, Pelletier score, Mankin score, the apoptotic rate of chondrocytes, the positive protein expression rates of NLRP3, ASC, caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax in SH group and PRP group were decreased significantly (P<0.05), while the protein expression of Bcl-2 was increased significantly (P<0.05). In PRP group, Pelletier score, Mankin score, the apoptotic rate of chondrocytes, the positive protein expression rates of NLRP3, ASC, caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax were lower than those in SH group, while the protein expression of Bcl-2 was higher than that in SH group (P<0.05). Compared with control group, the expression of NL?RP3 and IL-1β in MCC950 (NLRP3 ihibitor) group were significantly reduced (P<0.05), the expression of NLRP3 in eucalyptol (IL-1β inhibitor) group was not significantly changed (P>0.05), and the expression of IL-1β was significantly reduced (P<0.05). CONCLUSION Platelet-rich plasma promotes the repair of cartilage in osteoarthritis rabbits, which has better effect than SH. The mechanism may be related to the inhibition of NLRP3/IL-1β pathway and the reduction of chondrocyte apoptosis.     

PCDH10 inhibits proliferation of breast cancer MDA-MB-231 cells by NF-κB/cyclin D1 signaling pathway

AIM To investigate the inhibitory effect of protocadherin 10 （PCDH10） on proliferation of human breast cancer cells and its possible mechanism. METHODS RT-PCR was used to measure the mRNA expression levels of PCDH10 in 4 human breast cancer cell lines and normal mammary epithelial MCF-10A cells. The breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed by recombinant lentivirus （pLV-PCDH10） infection, and blank control （blank） group and negative control （pLV-NC） group were also set up. The cell proliferation ability was detected using methyl thiazolyl tetrazolium （MTT） and colony formation experiments. The cell cycle was detected by flow cytometry. Western blot was used to determine the expression of cyclin D1, cyclin-dependent kinase 4 （CDK4）, nucear factor-κB p65 subunit （NF-κB p65） and NF-κB inhibitor α （IκBα）. RESULTS The mRNA expression levels of PCDH10 in 4 breast cancer cell lines were lower than that in normal mammary epithelial MCF-10A cells （P<0.05）. A breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed successfully. Compared with negative control group, PCDH10 overexpression significantly inhibited cell proliferation, induced cell cycle arrest at G₁ phase, down-regulated the expression of cyclin D1 and CDK4, and decreased phosphorylation of NF-κB p65 and IκBα（P<0.05）. CONCLUSION PCDH10 inhibits the proliferation and blocks cell cycle progression of breast cancer cells by targeting NF-κB/cyclin D1 signaling pathway.     

RIP1-mediated necroptosis regulates inflammatory response of renal tubular epithelial cells via NF-κB signaling pathway

AIM To investigate the effect of receptor-interacting protein 1 (RIP1)-mediated necroptosis on human kidney proximal tubular cell inflammation and its related mechanisms. METHODS Human kidney proximal tubular HK-2 cells were cultured in vitro, and stimulated with tumot tumor necrosis factor-α (TNF-α) and Z-VAD-FMK for 24 h. Lactate dehydrogenase (LDH) cytotoxicity assay was used to detect the percentage of necrosis. Western blot was used to detect the protein expression of RIP1, IKK-α and NF-κB p65. The protein levels of interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were determined by Western blot and ELISA. Real-time PCR was used to detect the mRNA expression level of NF-κB p65. Furthermore, the RIP1 inhibitor necrostatin-1 (Nec-1) and the NF-κB specific inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) were used, and the above indicators were also detected. RESULTS Compared with control group, the protein level of RIP1 was increased in TNF-α combined with Z-VAD-FMK stimulation group (T/Z group). The protein levels of IKK-α and NF-κB p65 were obviously increased, and the release of LDH was increased (P<0.01). Western blot and ELISA showed that the expression levels of IL-1β and MCP-1 were significantly increased (P<0.01). Real-time PCR showed that the mRNA expression level of NF-κB p65 was also obviously increased. After Nec-1 or PDTC stimulation (T/Z+N group or T/Z+P group), the release of LDH, and the expression levels of inflammation-related indicators IL-1β and MCP-1 were significantly decreased. The protein expression levels of IL-1β and MCP-1 were further reduced after treatment with the above 2 stimulati (T/Z+P/N group). CONCLUSION Under T/Z condition, RIP1-mediated necroptosis plays an important role in renal tubular inflammatory response, which may be partly achieved by regulating the activation of NF-κB signaling pathway.     

Effects of IOX1 on proliferation,apoptosis and extracellular matrix-related protein expression in LX2 cells induced by TGF-β

AIM To investigate the effects of histone demethylase inhibitor IOX1 (5-carboxy-8-hydroxyquinoline) on the proliferation, apoptosis and extracellular matrix (ECM)-related protein expression in transforming growth factor-β (TGF-β)-induced human hepatic stellate LX2 cells. METHODS The proliferation and apoptosis of the LX2 cells were determined by real-time cell analysis and flow cytometry, respectively. The level of histone H3 lysine 9 dimethylation (H3K9me2) and the protein expression of ECM-related molecules [α-smooth muscle actin (α-SMA), collagen type I (Col I), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1)] in the LX2 cells were detected by Western blot. RESULTS Treatment with IOX1 at 50~300 μmol/L significantly inhibited LX2 cell proliferation, and 300 μmol/L IOX1 significantly promoted the apoptosis of the LX2 cells. In addition, different concentrations of IOX1 increased the levels of H3K9me2 and MMP-1, and down-regulated the expression of α-SMA, Col I and TIMP-1 in TGF-β-induced LX2 cells (P<0.05). CONCLUSION Treatment with IOX1 inhibits the proliferation of LX2 cells induced by TGF-β, promotes the cell apoptosis, and regulates the synthesis and metabolism of ECM by elevating H3K9me2 level, thus attenuating hepatic fibrosis.     

Sphingosine 1-phosphate inhibits TGF-β-induced endothelial-to-mesenchymal transition in human umbilical vein endothelial cells

AIMTo investigate the effect of sphingosine 1-phosphate (S1P) on endothelial-to-mesenchymal transition (End-MT) in human umbilical vein endothelial cells (HUVECs) induced by transforming growth factor-β (TGF-β) in vitro. METHODSThe HUVECs in different groups were treated with TGF-β, S1P or sphingosine 1-phosphate receptor 1(S1PR1) inhibitor VPC23019. Western blot was used to detect the protein levels of endothelial cell markers (CD31 and VE-cadherin), mesenchymal cell markers (α-smooth muscle actin and fibroblast-specific protein 1), S1PR1 and p-Smad3. Immunofluorescence staining was used to analyze the nuclear translocation of Smad3. RESULTSCompared with TGF-β group, the process of End-MT was significantly inhibited, and the phosphorylation and nuclear translocation of Smad3 were significantly reduced in TGF-β+S1P group (P<0.05). However, the above effects of SP1 were reversed after the addition of S1PR1 inhibitor (P<0.05). CONCLUSION S1P inhibits TGF-β-induced End-MT via S1PR1 in HUVECs. This effect may be associated with decreases in Smad3 phosphorylation and nuclear translocation.     

Effects of rhTGF-β1 and TGF-β1 gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro

AIM: To investigate the effects of rhTGF-β₁ and TGF-β₁ gene transfection on the proliferation of cultured rabbit corneal endothelial cells in vitro. METHODS: Cell growth induced by various concentrations of rhTGF-β₁ was determined by MTT proliferation assay. Under the induction of liposomes, recombinant pSecTag2-TGF-β₁MP vectors were transferred into the corneal endothelial cells. Morphological changes of transfected cells were observed by HE staining. The expression levels of TGF-β₁ were assessed by ELISA. Cell cycle analysis was assessed by flow cytometry. DNA fragment analysis was used to confirm the presence of apoptosis. RESULTS: rhTGF-β₁ in concentrations of 5-20 μg/L showed a significant suppressive effect on the proliferation of corneal endothelial cells, 0.5-1 μg/L had no effect, 0.05-0.1 μg/L facilitated cell growth, as compared with negative controls. The morphous of transfected corneal cells had no significant abnormality compared with normal cells. According to the result of ELISA, the concentration of TGF-β₁ in the supernatant was calculated to be (98±3) ng/L. Flow cytometry assay showed that S and G₂/M phase of transfected cells decreased significantly compared with that of control group, but the cell cycle recovered normally after adding 10 μg/L EGF into the culture medium. Agarose electrophoresis didn′t show marked ladders in transfected group. CONCLUSION: Effects of rhTGF-β₁ on the proliferation of corneal endothelial cells are different with various concentrations. TGF-β₁ gene transfection shows suppressive effect on the proliferation of cultured corneal endothelial cells, but does not induce cell apoptosis. EGF is the antagonist of this suppressive effect.     

Baicalin attenuates pancreatic fibrosis by inhibiting TGF-β1/TAK-NF-κB signaling pathway in chronic pancreatitis mice

AIM: To explore the role of transforming growth factor-β1 (TGF-β1)/TGF-β-activated kinase (TAK)-nuclear factor-κB (NF-κB) signaling pathway in chronic pancreatitis (CP) mice and the effect of baicalin on pancreatic fibrosis in the mice. METHODS: Kunming mice (n=58) were randomly divided into 3 groups, including control group, CP group and baicalin group. The mice in CP group and baicalin group were intraperitoneally injected with 20% L-arginine. After 2 weeks of CP, the mice in baicalin group were intraperitoneally injected with baicalin (100 mg/kg, once a day). At 2 weeks, 4 weeks and 6 weeks after modeling, the mice were anesthetized and sacrificed. The morphological changes of the pancreas were observed by HE and Masson staining. The serum level of TGF-β1 was analyzed by ELISA. The expression of fibronectin (FN) and NF-κB in the pancreas was observed by immunohistochemistry staining. The protein levels of transforming growth factor-β receptor type Ⅰ (TGF-βRⅠ), phosphorylated TAK1 (p-TAK1) and NF-κB in the pancreas were determined by Western blot. The mRNA expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloprotease-1 (TIMP-1) was detected by real-time PCR. RESULTS: At 2 weeks, 4 weeks and 6 weeks after intraperitoneal injection of L-arginine, the pancreatic tissues were obviously injured and exhibited different degrees of fibrosis, and FN expression was significantly increased. After treatment with baicalin, the degrees of pancreatic injury and fibrosis were significantly attenuated and the expression of FN was reduced (P<0.01). Compared with control group, the protein levels of TGF-β1, TGF-βRⅠ, p-TAK1, NF-κB and TIMP-1 in the pancreas of CP group were significantly increased, and the expression of MMP-1 was decreased at each time point. In baicalin group, the protein levels of TGF-β1, TGF-βRⅠ, p-TAK1, NF-κB and TIMP-1 were significantly decreased, and the expression of MMP-1 was markedly increased at the corresponding time points compared with CP group (P<0.01). CONCLUSION: Baicalin effectively atte-nuates pancreatic fibrosis by inhibiting the activation of TGF-β1/TAK-NF-κB signaling pathway and regulating the balance of MMP-1/TIMP-1.     

Role of TGF-β1/Smads and ERK expression in vascular remodeling induced by high-salt diet in Wistar rats

AIM: To investigate the role of transforming growth factor β₁ (TGF-β₁)/Smads and extracellular signal-regulated kinase(ERK) expression in vascular remodeling induced by high-salt diet in Wistar rats. METHODS: Wistar rats were randomly divided into 3 groups: normal control group (n=13), high salt (8%) model group and high salt+telmisartan group (n=13). Tail-cuff arterial pressure was determined every 2 weeks. After 24 weeks, the rats in high salt model group were divided into model animals with hypertension group (MH, n=12) and model animals without hypertension group (MN, n=12). The remodeling of aorta and mesenteric artery was observed by HE and Masson staining. In addition, the techniques of immunohistochemistry and real-time PCR were applied to detect the expression of proliferating cell nuclear antigen (PCNA), TGF-β₁, p-Smad2/3, p-ERK1/2 and Smad7 at both protein and mRNA levels. RESULTS: Compared with normal control group, blood pressure in MH group was much higher, and media thickness (MT) and collagen volume fraction (CVF) of arteries in MH and MN groups were higher.The mRNA expression of TGF-β₁, Smad2 and Smad7 in the aorta was significantly increased, and the protein levels of PCNA, p-ERK1/2, TGF-β₁ and p-Smad2/3 in the aorta and mesenteric artery media were elevated, but Smad7 decreased. After telmisartan treatment, MT and CVF were much lower,and the protein levels of PCNA, TGF-β₁, p-Smad2/3 and p-ERK1/2 were significantly reduced, whereas Smad7 was increased. CONCLUSION: The abnormal expression of TGF-β₁/Smads and ERK may be involved in the mechanism of remodeling of aorta and mesenteric artery induced by high-salt diet. Telmisartan prevents the vascular remodeling via regulating TGF-β₁/Smads and ERK signal pathways mediated by angiotensinⅡ type 1 (AT₁) receptor, at least in part.     

Effect of Kang Xianling decoction on TGF-β1-Smad pathway in a unilateral ureteral obstruction rat model

AIM: To study the effect of Kang Xianling decoction,comprised of dahuang,danshen,taoren,niuxi and danggui,on TGF-β₁-Smad pathway in unilateral ureteral obstruction rat model.METHODS: Eighteen male SD rats were divided into 3 groups,sham group,model group and model group treated with Kang Xianling decoction randomly.Renal interstitial fibrosis model was established in rats by unilateral ureteral obstruction (UUO).After treatment for additional 14 d,parameters of hydroxyproline in obstructed kidney from 3 groups were analyzed.Rats were sacrificed and the pathological statuses of their kidneys were checked by HE staining and electron microscopy.Transforming growth factor-β₁ (TGF-β₁) mRNA in kidney tissue was determined by RT-PCR.TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),phosphorylated Smad2 and Smad2 protein were determined by Western blotting.RESULTS: Parameters of hydroxyproline in animals of model group were significantly increased than those in sham operation group (P<0.05).The mRNA expression of TGF-β₁ and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2 in kidney tissue of animals in model group were significantly up-regulated.After intervention with Kang Xianling decoction,the above-mentioned up-regulated parameters,except TGF-β₁,were all significantly inhibited.Compared to model group,the pathological changes in renal tissues in treatment group were remarkable improved.CONCLUSION: Kang Xianling decoction inhibits the TGF-β₁-Smad pathway and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2,so as to decrease the level of collagen in obstructed kidney and to alleviate the renal interstitial fibrosis in UUO rats.     

Effect of salvianolic acid B on expression of TGF-β1 receptors and Smad2 in activated mesangial cells

AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β₁ (TGF-β₁) receptors and Smad2 in TGF-β₁-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β₁ was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10^-6 mol/L and 10^-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β₁,and TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β₁ for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10^-6 mol/L and 10^-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β₁-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.     

Metformin attenuates nerve injury in stroke rats by regulating SIRT1/PGC-1α signaling pathway via miR-29c

AIM To explore the inhibitory effect of metformin （MET） on nerve injury in rats with stroke and its mechanism. METHODS SD rats were randomly divided into sham group （n=15）, model group （n=30）, MET group （n=30）, MET+agomir-NC group （n=30） and MET+agomir group （n=30）. The modified Puisinelli four-vessel occlusion method was used to prepare the model of global ischemic stroke, while the blood vessels in sham rats were isolated without clamping the common artery. One week before modeling, the rats in MET group, MET+agomir-NC group and MET+agomir group were given intraperitoneal injection of 100 mg·kg^-1·d^-1 MET, 100 mg·kg^-1·d^-1 MET+40 nmol/d agomir-NC, 100 mg·kg^-1·d^-1 MET+40 nmol/d miR-29c agomir, respectively, and the rats in sham group and model group were given intraperitoneal injection of the same amount of normal saline. Each treatment in the above groups was given once a day, 0.2 mL each time, for 7 consecutive days. The neurological deficit scores were measured 24, 48 and 72 h after operation. HE staining was used to observe the morphological changes of the hippocampus, and the living neurons were counted. RT-qPCR was used to detect the expression level of miR-29c, and the mRNA levels of silent information regulator 1 （SIRT1） and peroxisome proliferator-activated receptor γ coactivator 1α （PGC-1α） in hippocampus. The protein expression levels of SIRT1 and PGC-1α were determined by Western blot. RESULTS At the same time point, compared with model group, the neurological deficit score in MET group was significantly decreased, and the survival rate of the neurons was significantly increased （P<0.05）. Compared with MET+agomir-NC group, the neurological deficit score in MET+agomir group was increased, and the survival rate of the neurons was significantly decreased （P<0.05）. With the prolongation of time, except for sham group, the neurological deficit score was increased and the survival rate of the neurons was decreased. At 72 h after operation, compared with sham group, the expression of miR-29c in hippocampus of model group was significantly increased, and the mRNA and protein expression levels of SIRT1 and PGC-1α were significantly decreased （P<0.05）. Compared with model group, the expression of miR-29c in hippocampus of MET group was significantly decreased, and the expression of SIRT1 and PGC-1α at mRNA and protein levels was significantly increased （P< 0.05）. Compared with MET+agomir-NC group, the expression of miR-29c in hippocampus of MET+agomir group was significantly increased, and the mRNA and protein expression of SIRT1 and PGC-1α was significantly decreased （P<0.05）. CONCLUSIONS MET alleviates nerve injury in stroke rats, which may be related to down-regulation of miR-29c and promotion of SIRT1/PGC-1α signaling pathway activation.     

Role of PI3K/Akt signaling pathway in P12 cells apoptosis induced by β-amyloid peptide

AIM:To investigate the role of PI3K/Akt signaling pathway on cell apoptosis induced by β-amyloid peptide in PC12 cells. METHODS: The viability of PC12 cells and the levels of p-AktSer473 and p-GSK-3βSer9 were detected by MTT and Western blotting, respectively. Cell apoptosis was determined by Annexin V-PI staining. RESULTS: Aβ25-35 treatment decreased the viability of PC12 cells in a time-depended manner (P<0.05). Annexin V-PI staining demonstrated that Aβ25-35 induced PC12 cells apoptosis in a time-depended manner (P<0.05). Western blotting showed that Aβ25-35 induced an immediately decreased expression of p-AktSer473 and p-GSK-3βSer9 at 30 min (P<0.05), but increased at 3 h and decreased again at 6 h. However, after 12 h of peptide treatment, the p-GSK-3βSer9 increased while the p-AktSer473 remained decrease. CONCLUSION:PI3K/Akt signaling pathway is involved in cell apoptosis induced by β-amyloid peptide, which provides a new clue for the study of pathogenesis and management of AD.