Effect of resveratrol on autophagy and renal interstitial fibrosis in kidney of diabetic mice

AIMTo observe the effect of resveratrol (Res) on renal autophagy level and renal interstitial fibrosis in the mice with diabetes mellitus (DM), and to discuss the possible mechanism. METHODSThe wild-type C57BL/6 mice were randomly divided into 3 groups, including normal control (NC) group, DM group and Res group (8 in each group). The diabetic mouse model was established by injection of streptozotocin. After 8 weeks of successful replication of the diabetic model, Res was given to the mice in Res group by continuous gavage for 12 weeks, and then the mice in each group were sacrificed to detect the relevant biochemical parameters. The pathological changes of the kidney tissues were observed by HE staining and Masson staining. The levels of the proteins related to autophagy, epithelial-mesenchymal transition (EMT) and fibrosis were determined by Western blot. The mRNA expression of collagen type IV (Col IV), α-smooth muscle actin (α-SMA) and E-cadherin were detected by real-time PCR. RESULTSCompared with NC group, fasting blood glucose (FBG), kidney index (KI), serum creatinine, 24-hour urinary albumin excretion rate and 24-hour urine total protein were remarkably increased in DM group (P<0.05). The results of HE and Masson staining indicated that renal tissue presented fibrosis in DM group. The protein levels of E-cadherin, beclin-1, microtubule-associated protein 1 light chain 3-II (LC3-II) were reduced in DM group, while the levels of α-SMA, Col IV and Snail1 were increased (P<0.05). After intervention with Res for 12 weeks, all the relevant biochemical parameters and KI were reduced (P<0.05) except FBG (P>0.05), and renal fibrosis lesions were obviously alleviated. Compared with DM group, the protein levels of E-cadherin, beclin-1 and LC3-II were increased in Res group, but the protein expression levels of α-SMA, Col IV, Snail1 were reduced (P<0.05). Compared with DM group, the mRNA level of E-cadherin was increased in Res group , but the mRNA levels of Col IV and α-SMA were reduced (P<0.05). CONCLUSION Resveratrol significantly inhibits EMT and reduces renal interstitial fibrosis in diabetic mice, and its mechanism may be related to the promotion of renal autophagy.     

circRNA-42398 inhibits activation of hepatic stellate cells via TGF-?β1/Smads signaling pathway modulation

AIM To study the effect of mouse circular RNA-42398 (mmu_circ_42398) over-expression on the activation of hepatic stellate cells. METHODS Mouse hepatic stellate JS1 cells were cultured and randomly divided into control group, vector group and mmu_circ_42398 over-expression group.mmu_circ_42398 over-expression plasmid vector was constructed, and then transiently transfected into JS1 cells using Lipofectamine 2000. The cells were collected 48 h after transfection. Expression of mmu_circ_42398 was detected by RT-qPCR.The backsplice site of PCR products was verified by sequencing. The protein levels of α-smooth muscle actin (α-SMA), collagen type I （Col I), transforming growth factor β1(TGF-β1), Smad2, Smad3, p-Smad2 and p-Smad3 in the cells were determined by Western blot. RESULTS RT-qPCR results showed that the expression of mmu_circ_42398 was significantly increased after mmu_circ_42398 over-expression vector was transiently transfected into the JS1 cells (P<0.01). The protein expression levels of α-SMA and Col I were significantly decreased(P<0.01), and the phosphorylation levels of Smad2 and Smad3 were decreased significantly in mmu_circ_42398 over expression group (P<0.01). However, the protein expression levels of TGF-β1, Smad2 and Smad3 had no significant change (P>0.05). CONCLUSION mmu-circ-42398 inhibits the activation of hepatic stellate cells via TGF-β1/Smads signaling pathway modulation.     

Relationship between rat hepatic fibrosis and regulation of hepatic stellate cell autophagy by microRNA-181a

AIM To observe the changes of liver structure, the levels of transforming growth factor-β1 （TGF-β1）, microRNA-181a, LC3-II/-I, beclin-1 and collagen deposition in hepatic fibrosis （HF） rats induced by carbon tetrachloride （CCl₄）, and the effect of microRNA-181a on autophagy of rat hepatic stellate cells （HSCs） induced by TGF-β1, and to explore the possible mechanism of microRNA-181a in regulating HSC activation and HF. METHODS Wistar rats （n=40） were randomly divided into 5 groups （with 8 in each）： control group （subcutaneous injection of olive oil, 3 mL/kg, twice a week）, and CCl₄-induced HF groups of 2, 4, 6 and 8 weeks （subcutaneous injection of 40% CCl₄, 3 mL/kg, twice a week for 2, 4, 6 and 8 weeks, respectively）. Masson staining was used to evaluate the changes of HF in rats. The levels of TGF-β1 in serum and liver tissue of the rats were measured by ELISA. The level of microRNA-181a in rat liver tissues was detected by RT-qPCR. The protein levels of LC3-II/-I, beclin-1, α-smooth muscle actin （α-SMA）, collagen type I （Col I） and collagen type Ⅲ （Col Ⅲ） in rat liver tissues were measured by Western blot. HSC-T6 cells were transfected with microRNA-181a inhibitor, or pretreated with the autophagy inhibitor 3-methyladenine （3-MA）, before treatment with TGF-β1 to stimulate autophagy. The expression of microRNA-181a, LC3-II/-I, beclin-1, α-SMA, Col I and Col Ⅲ in HSC-T6 cells were determined by RT-qPCR and Western blot. RESULTS The levels of TGF-β1, microRNA-181a, LC3-II/-I ratio and beclin-1 in liver tissues showed an overall trend of increasing with the progression of HF, and microRNA-181a expression showed a positive correlation with autophagy-associated proteins （P<0.01）. MicroRNA-181a level was significantly increased, which was associated with TGF-β1-induced autophagy and activation of HSC-T6 cells.MicroRNA-181a expression was significantly down-regulated in the HSC-T6 cells transfected with microRNA-181a inhibitor, along with suppression of autophagy and cell activation （P<0.01）, which were similar to the effects of 3-MA treatment. CONCLUSION CCl₄ promotes rat HF, the microRNA-181a expression of liver tissue, and autophagy in a time-dependent manner. Reducing the expression of microRNA-181a in HSC-T6 cells inhibits the autophagy of HSCs-T6 cells induced by TGF-β1. The regulation of HSC autophagy by microRNA-181a may be involved in rat HF.     

Effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE-/- mice

AIM To observe the effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE^-/- mice. METHODS Thirty-two ApoE^-/- mice were randomly divided into model group, high-dose (60 mg/kg) tanshinone ⅡA group, low-dose (30 mg/kg) tanshinone ⅡA group and simvastatin group, and C57BL/6J mice (n=8) were used as normal control group. The mice in normal control group were given the basic feeding, while the others were given high-fat diet. The mice in tanshinone ⅡA groups and simvastatin group were given corresponding drugs. The mice in normal control group and model group were intraperitoneally injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by automatic biochemistry analyzer. The liver tissues were stained with HE and oil red O. The contents of reactive oxygen species (ROS) and glutathione (GSH) in liver tissues of the mice were measured by commercially available kits. The liver glutathione peroxidase 4 (GPX4) and p53 were detected by immunohistochemical method. The protein and mRNA expression levels of ferroptosis-related factors GPX4, xCT/SLC7A11, p53 and ferritin heavy chain 1 (FTH1) were determined by Wes automatic Western blot quantitative analysis system and RT-qPCR. RESULTS Compared with normal control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.05 or P<0.01), and HDL-C did not change significantly. The fat vacuoles were clearly visible in liver tissue. The content of ROS in liver tissue was increased significantly,and GSH was decreased significantly (P<0.01). The mRNA and protein expression levels of p53 were increased significantly, and GPX4, xCT/SLC7A11 and FTH1 were decreased significantly (P<0.05 or P<0.01). Compared with model group, tanshinone ⅡA significantly decreased the serum levels of TC, TG and LDL-C (P<0.05 or P<0.01), and HDL-C did not change significantly. High-dose and low-dose tanshinoneⅡA also significantly decreased the degree of steatosis, and the size of lipid droplets. The content of ROS in liver tissues was decreased significantly, and GSH was increased significantly (P<0.01). The mRNA and protein expression levels of GPX4, xCT/SLC7A11 and FTH1 were increased significantly, and p53 were decreased significantly (P<0.05 or P<0.01). CONCLUSION Tanshinone ⅡA reduces liver lipid deposition and lipid peroxidation damage in ApoE^-/- mice, which may be related to the intervention of ferroptosis-related proteins in the liver cells.     

Notch1 regulates activation of pancreatic stellate cells by non-classical Notch signaling pathway

AIM: To investigate the effect of Notch1 on the activation of pancreatic stellate cells (PSCs). METHODS: The expression of Notch1 in pancreatic duct adenocarcinoma (PDAC) tissues was detected by the immunohistochemical and immunofluorescence double staining. The PSCs were isolated, cultured, and identified by oil red O staining, Western blot and RT-qPCR. The expression of Notch1 and HES1 was detected by Western blot and RT-qPCR. After transfection of Notch1 siRNA to PSCs, Western blot was used to detect the protein expression of α-smooth muscle actin (α-SMA), fibronectin and collagen type Ⅰ (ColⅠ) in activated PSCs. The expression of Notch1 and HES1 was also detected by Western blot. The effects of Notch1 siRNA on migration ability and viability of PSCs were determine by scratch test and CCK-8 assay. RESULTS: The results of immunohistochemical and immunofluorescence double staining showed that Notch1 expressed in α-SMA positive cells in PDAC stroma. The mouse PSCs were successfully cultured, and the expression of α-SMA, fibronectin, ColⅠ, Notch1 and HES1 in activated PSCs were significantly increased compared with unactivated PSCs (P<0.01). After transfection of Notch1 siRNA to mouse PSCs, the expression of α-SMA and ColⅠ was significantly reduced compared with negative groups, but the expression of fibronectin and HES1 did not change significantly. After knock-down of Notch1 expression in activated PSCs, the migration ability and viability of PSCs were significantly reduced compared with negative group. CONCLUSION: Notch1 is involved in regulating the activation of PSCs. Knock-down of Notch1 expression inhibits the expression of the markers of activated PSCs, α-SMA and ColⅠ, reduces the activation of PSCs, and attenuates the migration capacity and viability of PSCs. Notch1 regulates the activation of PSCs without relying on the classic Notch signaling pathway.     

Experimental research on cardiac fibrosis induced by recombinant mouse interleukin-11 in mice

AIM To observe the effect of recombinant mouse interleukin-11 （rmIL-11）injected subcutaneously into mice on heart structure and function and to determine its pro-fibrotic effect. METHODS C57BL/6 mice were randomly divided into experimental group and control group.The mice in experimental group were injected subcutaneously with recombinant mouse IL-11 at the dose of 100 μg·kg^-1·d^-1 for 3 consecutive weeks, while the control group were given equal volume of normal saline in the same way. After the experiment was finished, the parameters of heart function were measured by echocardiography.The heart weight was weighed and the cardiac weight index (CWI) was calculated. HE staining and Masson's trichrome staining were performed to observe the pathological changes and the extent of myocardial fibrosis in mouse myocardia respectively, and the cardiac collagen volume fraction (CVF) was calculated. The expression levels of extracellular matrix proteins in the myocardial tissues of mice, including type Ⅰ collagen, type Ⅲ collagen and fibronectin, were determined by Western blot. RESULTS Left ventricular ejection fraction and left ventricular fraction shortening in experimental group were obviously lower than those in control group (P<0.01), however left ventricular end-diastolic diamension and left ventricular end systolic dimension were significantly higher than those in control group (P<0.05).Compared with control group, the CWI was increased (P<0.01), the myocardial arrangement was disorder, the necrosis of cardiac myocytes was increased, and excessive deposition of collagen was observed in the myocardial tissues in experimental group. Correspondingly, the CVF and protein levels of type Ⅰ collagen, type Ⅲ collagen and fibronectin in the left ventricle in experimental group were increased significantly (P<0.05). CONCLUSION Injection of rmIL-11 into the mice subcutaneously induces fibrogenesis in the heart, which implies that IL-11 is likely a novel pro-fibrotic factor.     

NKT cells inhibit Ang II-induced phenotypic transformation of mouse vascular smooth muscle cells by down-regulating PDGFRβ

AIM To investigate the role of natural killer T (NKT) cells in the phenotypic transformation of vascular smooth muscle cells induced by hypertension in mice. METHODS The hypertension model in wild-type and CD1d gene knockout mice was established by continuous infusion of angiotensin II (Ang II) at a dose of 490 ng·kg^-1·min^-1 for 14 d, and the NKT cell specific agonist α-galactosylceramide (α-GC) was given at a dose of 100 μg/kg in wild-type mice. The blood pressure of the mice was monitored by non-invasive tail cuff method. The thickness of aortic wall was measured by HE staining, and the degree of aortic fibrosis was observed by Masson staining. The expression levels of α-smooth muscle actin (α-SMA), osteopontin (OPN) and platelet-derived growth factor receptor β (PDGFRβ) in aortic tissue were detected by Western blot. RESULTS Compared with control group, the blood pressure, the aortic wall thickness, the degree of aortic fibrosis, the secretory marker protein OPN expression and the PDGFRβ expression were all increased significantly after infusion of Ang II for 14 d (P<0.05), while the expression of contractile marker protein α-SMA was decreased (P<0.05). The above changes were aggravated after CD1d gene knockout (P<0.05), but were attenuated after administration of α-GC (P<0.05). CONCLUSION NKT cells reduces Ang II-induced phenotypic transformation of mouse vascular smooth muscle cells by reducing the expression of PDGFRβ, increasing the expression of contractile protein and decreasing the expression of secretory protein.     

Changes of histone modification levels in rats with liver fibrosis

AIM: To observe the changes of histone modifications in the liver of rats with hepatic fibrosis and its possible role in the development of hepatic fibrosis. METHODS: Male Wistar rats (n=20) were randomly divided into liver fibrosis group and normal control group. The liver fibrosis model was established by hypodermic injection of CCl₄, and the rats in normal control group were injected with vegetable oils. At the end of the 8th week, all rats were killed. Liver function indexes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and liver fibrosis indexes including haluronic acid (HA), laminin (LN), collagen type IV (Col Ⅳ) and procollagen type Ⅲ (PCⅢ) were determined by biochemical and RIA methods. The liver index was analyzed, and the liver fibrosis degree and the morphological change of the liver were detected by HE and Masson staining. The levels of α-smooth muscle actin (α-SMA), collagen type Ⅰ (ColⅠ), H3K4me2, H3K9me2, acH3K9 and acH4K12 were detected by Western blot. RESULTS: After hypodermic injection of CCl₄ for 8 weeks, the liver index, ALT, AST, HA, LN, Col Ⅳ and PCⅢ of the rats in liver fibrosis group were higher than those in control group (P<0.05). Compared with control group, the level of acH4K12 was decreased (P<0.05), while acH3K9, H3K9me2, α-SMA and ColⅠ were increased (P<0.05), but H3K4me2 had no significant change.CONCLUSION: The levels of acH4K12, acH3K9 and H3K9me2 may play essential roles in the pathogenesis of liver fibrosis, and these histone modifications may regulate gene expression associated with extracellular matrix metabolism.     

Effect of formononetin on viability,migration and invasion of ovarian cancer cells in vitro

AIM To observe the effect of formononetin on the viability, migration and invasion of ovarian cancer cells, and to explore its mechanism. METHODS Human ovarian serous cystadenocarcinoma SKOV-3 cells were cultured in vitro. The cells were treated with formononetin at 0, 25, 50 and 100 μmol/L for 48 h. The cell viability was measured by MTS assay. The migration and invasion abilities of the SKOV-3 cells were detected by scratch wound assay and Transwell assay. RT-qPCR and Western blot were used to detect the mRNA and protein levels of E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS The viability of SKOV-3 cells was decreased with the increase in the formononetin concentration compared with control group (P<0.01). The wound migration distance of the cells in 50 μmol/L formononetin group was less than that in control group (P<0.01). The number of invasive SKOV-3 cells across the Transwell sub-compartment was significantly decreased in 50 μmol/L formononetin group compared with control group (P<0.01). The mRNA and protein levels of E-cadherin in 50 μmol/L formononetin group were significantly higher than those in control group (P<0.01), while the mRNA and protein levels of MMP-9 in 50 μmol/L formononetin group were significantly lower than those in control group (P<0.01). CONCLUSION Formononetin inhibits the migration and invasion abilities of ovarian cancer SKOV-3 cells by increasing expression of E-cadherin and decreasing expression of MMP-9.     

CD36/Src/ERK pathway is involved in process of monocyte differentiation into macrophages

AIM To investigate the role of fatty acid translocase (FAT/CD36) on differentiation of monocytes to macrophages. METHODS Human monocyte THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) at 0, 100 and 200 μg /L. Small interfering RNA (siRNA) targeting CD36 (siCD36) was employed to knock down the expression of CD36 in THP-1 cells. The CD36 over-expression (CD36OE) cell line was constructed by transfection with a recombinant lentivirus containing CD36 cDNA. Optical microscopy and crystal violet staining were used to detect the monocyte morphological changes and adhesion ability. The protein expression of CD36 was measured by flow cytometry and Western blot. The mRNA levels of CD36, CD11b and CD80 were detected by real-time PCR. The protein levels of extracellular signal-regulated kinase (ERK） and Src tyrosine kinase were determined by Western blot. RESULTS The cellular adhesiveness of THP-1 cells was elevated in the process of monocytes differentiation, and the expression of CD36 was increased in this process as well (P<0.01). siCD36 was transfected into the THP-1 cells (CD36i group) and the silencing efficiency was approximately 80%. The cell surface area and cellular adhesiveness were significantly decreased in CD36i group compared with scrambled siRNA (NCi) group (P<0.01). The mRNA levels of CD11b and CD80 were decreased in CD36i group compared with NCi group (P<0.01). The cell surface area and cellular adhesiveness were increased in CD36OE group compared with empty vector (vector) group (P<0.05). The mRNA levels of CD11b and CD80 were increased in CD36OE group compared with vector group (P<0.01). The phosphorylation levels of ERK and Src were decreased in CD36i group compared with NCi group (P<0.05). CONCLUSION CD36 promotes the differentiation of human monocyte THP-1 cells to macrophages by increasing the phosphorylation of Src and further activating ERK.     

Role of BMP-7/Smads pathway in regulation of EndoMT in rats with HHPH

AIM: To investigate the role of bone morphogenetic protein 7 (BMP-7)/Smads pathway in the re-gulation of endothelial-mesenchymal transition (EndoMT) in rats with hypoxia-hypercapnia pulmonary hypertension (HHPH). METHODS: The rat pulmonary artery endothelial cells (RPAECs) were divided into normoxic control (Con) group, hypoxia-hypercapnia (HH) group, BMP receptor agonist rhBMP-7 group, BMP receptor inhibitor DMH-1 group and solvent DMSO group. After given the corresponding drugs in each group, the cells in Con group were cultured in a normal-oxygen incubator, and the cells in the remaining 4 groups were cultured in a low-oxygen and high-carbon-dioxide incubator. The expression levels of CD31 and α-smooth muscle actin (α-SMA) were observed by immunofluorescence staining. The mRNA and protein expression levels of α-SMA, CD31 and Smad1/5/8 were detected by RT-PCR and Western blot, respectively. The cell viability was measured by CCK-8 assay. The cell migration ability was detected by Transwell chamber assay. RESULTS: Compared with Con group, the expression of α-SMA at mRNA and protein levels in HH group was increased, the expression levels of CD31 mRNA and protein, Smad1/5/8 mRNA and p-Smad1/5/8 protein were decreased, the cell viability was decreased and the number of migratory cells was increased (P<0.05). Compared with HH group, the expression of α-SMA at mRNA and protein levels in rhBMP-7 group was decreased, the expression levels of CD31 mRNA and protein, Smad1/5/8 mRNA and p-Smad1/5/8 protein were increased, the cell viability was increased and the number of migratory cells was reduced (P<0.05). Compared with rhBMP-7 group, the expression of α-SMA at mRNA and protein in DMH-1 group was increased, the expression levels of CD31 mRNA and protein, Smad1/5/8 mRNA and p-Smad1/5/8 protein were decreased, the cell viability was decreased and the number of migratory cells was increased (P<0.05). CONCLUSION: Hypoxia-hypercapnia conditions promote EndoMT in RPAECs, which promotes the development of hypoxia-hypercapnia pulmonary hypertension, and the underling mechanism may be related to the inhibition of BMP-7/Smads pathway.     

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.     

Effect of Jieyuwan on dendritic spines in prefrontal cortex and hippocampus in WKY depression-like rats

AIM To observe the changes of dendritic spines in prefrontal cortex and hippocampus of Wistar-Kyoto (WKY) depression-like rats, and to explore the effects of Jieyuwan (JYW) on them. METHODS The male WKY rats were selected as the experimental group, and the same strain of Wistar rats were selected as the control group. Firstly, sucrose preference test, open-field experiment and forced swimming test were used to detect the behavior changes in the rats as their baseline. Then, all WKY rats were randomly divided into model (WKY+NaCl) group, WKY+JYW group and WKY+citalopram group. All WKY rats and Wistar rats (Wistar+NaCl group) were administered intragastrically for 21 d, and the changes of behavior after administration were detected by the same behavioral methods. Golgi staining was used to observe the pathological characteristics of dendritic spines in the prefrontal cortex and hippocampus, and Western blot was used to detect the protein expression level of postsynaptic density protein-95 (PSD-95) in the prefrontal cortex and hippocampus. RESULTS Before administration, WKY rats clearly showed depression-like behavior, the density of dendritic spines in the prefrontal cortex and hippocampus decreased significantly (P<0.01), and the protein expression level of PSD-95 was significantly reduced (P<0.01). After treatment with the drugs, the depression-like behavior of WKY rats was significantly attenuated, the density of dendritic spines in the prefrontal cortex and hippocampus increased (P<0.01), and the protein expression level of PSD-95 also increased (P<0.01). CONCLUSION Jieyuwan significantly attenuates the depression-like behavior of WKY rats, and affects the structural changes of dendritic spines and the expression of PSD-95 protein, which further proves that dendritic spines may be one of the importantearly structural changes in depression.     

IL-33-modified BMSCs attenuate acute kidney injury induced by sepsis in rats through MyD88

AIM To investigate the effect of interleukin-33 (IL-33)-modified bone marrow mesenchymal stem cells (BMSCs) on sepsis-induced acute kidney injury (AKI) in rats and the expression of myeloid differentiation factor 88(MyD88). METHODS A septic rat model was established by cecal ligation and puncture. The SD rats (n=80) were randomly divided into control group, model group, negative transfection group (transplanting untransfected BMSCs) and IL-33 transfection group (transplanting BMSCs transfected with IL-33), with 20 in each group. Survival rates of the rats within 72 h in the 4 groups were compared. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured before, and 24, 48 and 72 h after transplantation. The kidney pathological damage was observed by HE staining, and the apoptosis of renal cells was detected by TUNEL method 72 h after transplantation. Western blot was used to detect the protein expression levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), Toll-like receptor 4 (TLR4) and MyD88. RESULTS The survival rate of the rats in model group was significantly lower than that in control group (P<0.05). The survival rate of the rats in IL-33 transfection group was higher than that in model group and negative transfection group (P<0.05). The levels of SCr and BUN in model group were higher than those in control group (P<0.05). The levels of SCr and BUN in IL-33 transfection group were significantly reduced after transplantation, and were lower than those in model group and negative transfection group (P<0.05). The renal tissue pathological injury score in model group was significantly higher than that in control group (P<0.05). Compared with model group and negative transfection group, the renal tissue pathological injury score in IL-33 transfection group was significantly reduced (P<0.05). The proportion of apoptotic cells in the kidney tissues in model group were higher than that in control group (P<0.05). Compared with model group and negative transfection group, the proportion of apoptotic cells in the kidney tissues in IL-33 transfection group was significantly reduced (P<0.05). The protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in model group were significantly higher than those in control group (P<0.05). Compared with model group and negative transfection group, the protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in IL-33 transfection group were significantly decreased (P<0.05). CONCLUSION IL-33 gene-modified BMSCs significantly improve the renal function of AKI rats with sepsis. The mechanism may be related to IL-33 regulating TLR4/MyD88 signaling pathway and inhibiting renal inflammatory response.     

Expression of histone chaperone ASF1B in prostate cancer cells and its effect on cell viability in vitro

AIM To investigate the expression of histone chaperone anti-silencing function 1B （ASF1B） in prostate cancer cells and its effect on cell viability in vitro. METHODS Human prostate cancer PC-3 cells were used, and knockdown of ASF1B was conducted by small interfering RNA （siRNA） transfection into the cells. The cells were divided into control group, siRNA negative control vector （mock） group and siRNA-ASF1B group. The viability of the PC-3 cells treated with ASF1B-siRNA for 12, 24 and 48 h was measured by MTT assay. Flow cytometry was used to analyze cell apoptosis and cell cycle distribution. The mRNA expression of apoptosis-related molecules was detected by RT-qPCR, and the expression levels of MAPK/JNK/ERK signaling pathway-related proteins were determined by Western blot. RESULTS The protein level of ASF1B in the normal cells （benign prostatic hyperplasia） was significantly lower than that in the PC-3 cells （P<0.01）. Compared with control group and mock group, the protein expression level of ASF1B in the PC-3 cells transfected with siRNA-ASF1B plasmid and the viability of the PC-3 cells were significantly decreased （P<0.01）. The apoptotic rate of the PC-3 cells transfected with siRNA-ASF1B plasmid was significantly higher than that in control group （P<0.01）, and the cell cycle was arrested at G₁ phase. The mRNA levels of p53, caspase-3, Bax and PARP-1 in the PC-3 cells transfected with siRNA-ASF1B plasmid were up-regulated compared with those in control group and Mack group （P<0.01）. In addition, the protein levels of MAP2K4 and p-JNK in the PC-3 cells in siRNA-ASF1B group were significantly higher than those in mock group （P<0.01）, while the protein level of p-ERK was significantly lower than that in mock group （P<0.01）. CONCLUSION ASF1B silencing induces G₁ arrest and promotes apoptosis of PC-3 cells. Activating MAPK/JNK/ERK signaling pathway may be a possible contributor to the anti-prostate cancer effect of siRNA-ASF1B.     

Inhibitory effect of Wendan decoction on atherosclerotic plaque formation in ApoE-/- mice by regulating expression of membrane proteins involved in reverse cholesterol transport

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on reverse cholesterol transport. METHODS Eight-week-old apolipoprotein E gene knockout （ApoE^-/-） mice with high-fat diet and daily drug gavage were randomly divided into model group, simvastatin group, and low-, middle- and high-dose Wendan decoction groups, with 15 mice in each group. The C57BL/6 mice of the same age served as control group. The mice were weighed once every week. After 10 weeks, the mice were anesthetized with chloral hydrate. The serum were collected for lipid level examination. The atherosclerotic plaque buildup in aortic root and whole aorta was observed by HE staining and oil red O staining, respectively. The levels of proteins related to cholesterol transport, ATP-binding cassette transporter A1 （ABCA1） and caveolin-1 in the aorta, and scavenger receptor class B type I （SR-BI） and CD36 in the liver, were quantified by Western blot. RESULTS Wendan decoction at middle dose inhibited the increase in the body weight of ApoE^-/- mice fed with high-fat diet （P<0.05）. Wendan decoction at different doses significantly reduced the serum levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol in the ApoE^-/- mice （P<0.05 or P<0.01）, but had no effect on serum high-density lipoprotein cholesterol level （P>0.05）. Wendan decoction at different doses inhibited the formation of atherosclerotic plaques in whole aorta of the ApoE^-/- mice （P<0.05 or P<0.01）. Middle- and high-dose Wendan decoction significantly inhibited the formation of atherosclerotic plaques in the aortic root （P<0.05）. Bedsides, Wendan decoction at different doses increased the protein level of ABCA1 and decreased the protein level of caveolin-1 in the aorta of the ApoE^-/- mice （P<0.01）. Middle- and high-dose Wendan decoction increased the liver protein level of SR-BI in the ApoE^-/- mice （P<0.01）. However, Wendan decoction at different doses had no effect on the liver protein level of CD36 in the ApoE^-/- mice （P>0.05）. CONCLUSION Wendan decoction reduces the body weight, serum lipid levels and formation of atherosclerotic plaques in ApoE^-/- mice fed with high-fat diet, and its mechanism is related to up-regulation of ABCA1 protein level in the aorta and SR-BI protein level in the liver as well as down-regulation of caveolin-1 protein level in the aorta.     

Ligustilide inhibits angiogenesis and epithelial-mesenchymal transition in human hemangioendothelial cells by reducing VEGF levels

AIM To investigate the effect of ligustilide on human hemangioendothelial cells (HemECs) and to analyze its mechanism. METHODS The effect of ligustilide at different concentrations on the viability of HemECs was measured by CCK-8 assay. The HemECs were divided into control group and ligustilide （10, 25 and 50 μmol/L） treatment groups, and the proliferation of HemECs was detected by EdU staining. The effects of ligustilide on the angiogenesis of HemECs was tested by microtubule formation experiment. The protein expression of vascular endothelial growth factor (VEGF) and epithelial-mesenchymal transition (EMT)-related markers in HemECs cells was determined by Western blot. RESULTS There was no significant difference in the viability of the cells treated with ligustilide at the concentrations between 0.1~50 μmol/L compared with control cells. Compared with control group, ligustilide at 25 and 50 μmol/L significantly reduced the number of EdU-positive cells and microtubule-like structures (P<0.05), reduced the protein expression level of VEGF (P<0.05), increased the protein expression of E-cadherin, and decreased the protein expression of vimentin and β-catenin (P<0.05). Compared with control group, the expression of VEGF and vimentin was significantly up-regulated, and the protein expression of E-cadherin was significantly down-regulated in VEGF overexpression group (P<0.05). Compared with VEGF overexpression group, the expression of VEGF and vimentin in 50 μmol/L ligustilide-treated VEGF-overexpressing cells were significantly reduced (P<0.05), and the protein expression of E-cadherin was significantly increased (P<0.05). CONCLUSION Ligulide inhibits the proliferation of HemECs, and also inhibits the angiogenesis and EMT process of HemECs by reducing the level of VEGF.     

Expression relevance of GATA-1 and miR-451a in erythroid differentiation of K562 cells under hypoxia

AIM To investigate the expression relevance of GATA binding protein-1 (GATA-1) and microR?NA-451a (miR-451a) in erythroid differentiation of human chronic myeloid leukemia K562 cells under hypoxia. METHODS The K562 cells were divided into 2 groups: normoxia group and hypoxia (1% O₂) group, and 40 μmol/L hemin chloride was used to induce K562 cell differentiation for 48 and 72 h. The mRNA expression of γ-globin was detected by RT-qPCR, hemoglobin production was observed by benzidine staining, and flow cytometry was used to detect CD235a expression for verifying erythroid differentiation model. The protein expression of GATA-1 during K562 cell differentiation under normoxia and hypoxia was determined by Western blot. RT-qPCR was used to detect the mRNA expression of GATA-1 and the expression level of miR-451a, and their correlation was analysis. The K562 cells were infected by lentivirus for over-expression or knock-down of GATA-1. Meanwhile, the morphological changes of the cells in the above groups were analyzed by Wright-Giemsa staining method to clarify the erythroid differentiation of K562 cells. The expression miR-451a was detected by RT-qPCR after GATA-1 over-expression or knock-down. REULTS: Under normoxia and hypoxia conditions, the expression levels of γ?-globin and CD235a and the positive rate of benzidine staining at 48 and 72 h were significantly higher than those at 0 h (P<0.05).At 72 h, the expression levels of γ?-globin and CD235a and the benzidine staining positive rate in hypoxia group were significantly higher than normoxia group (P<0.05). The expression of GATA-1 mRNA and miR-451a under hypoxia showed an upward trend during the erythroid differentiation of K562 cells, and was significantly higher than that in normoxia group at 72 h (P<0.05). Correlation analysis showed that the mRNA expression of GATA-1 was positively correlated with miR-451a expression under hypoxia (P<0.01). After over-expression of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the positive rate of benzidine staining, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly higher than those in negative control group (P<0.05). After knock-down of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the benzidine staining positive rate, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly lower than those in negative control group (P<0.05). Compared with negative control group under hypoxia, the expression of miR-451a was significantly increased after GATA-1 over-expression (P<0.05), while the expression of miR-451a was significantly decreased after GATA-1 knock-down (P<0.05). CONCLUSION Hypoxia increases the expression of GATA-1 and then up-regulates miR-451a to promote erythroid differentiation of K562 cells.