[The regulatory mechanism of Raf/MEK/ERK pathway on the rat cardiac hypertrophy induced by transverse aortic constriction].

OBJECTIVE: To investigate the mRNA, protein expression levels and the phosphorylation levels of key factors in rapidly accelerated fibrosarcoma/mitogen-activated protein kinase kinase/extracellular regulated protein kinases (Raf/MEK/ERK) pathway, and to clarify the regulatory function of Raf/MEK/ERK pathway in myocardial hypertrophy. METHODS: Twenty SD rats were divided into sham-operated group and model group. The myocardial hypertrophy model was established by transverse aortic constriction (TAC). At 12 weeks after TAC, blood samples were collected from the submandibular vein, and the serum was separated to detect the content of N terminal pro B type natriuretic peptide (NT-proBNP). After that, the rats were subjected to echocardiography and hemodynamic measurement. Then the pathological changes of myocardial tissue were observed. And the levels of mRNA, protein expression and the phosphorylation of key factors in Raf/MEK/ERK pathway were detected in myocardial tissue. RESULTS: Compared with sham-operated group, left ventricular end-diastolic interventricular septal thickness (IVSd), left ventricular end-systolic interventricular septal thickness (IVSs), left ventricular end-diastolic posterior wall thickness (LVPWd) and left vebtricular end-systolic posterior wall thickness (LVPWs) in TAC model group were increased significantly (P＜0.05,P＜0.01), left ventricular end-systolic diameter (LVIDs) was decreased significantly (P＜0.01), LV Mass and LW(LV Mass/Weight)were increased significantly (P＜0.05, P＜0.01). The levels of heart rate (HR), left ventricular pressure maximal rate of rise (+dp/dtmax), left ventricular pressure maximal rate of fall (-dp/dtmax) were decreased significantly (P＜0.01). The serum level of NT-proBNP in TAC rat was increased significantly (P＜0.01). The myocardial cells in TAC model group were arranged disorderly, myocardial cell hypertrophy, cytoplasm were increased significantly, and inflammatory cells infiltrated. A large amount of collagen fibers were deposited and large area of myocardial cells were stained blue in TAC rat. The expression levels of phospho-c-Raf (Ser259) and phospho-c-Raf (Ser338) in myocardial tissue were significantly increased (P＜0.01), meanwhile the expression levels of phospho- MEK1/2(Ser217/Ser221) and phospho-ERK1/2 (Thr202/Tyr204) were also significantly increased (P＜0.01). CONCLUSION: The regulatory role of Raf / MEK / ERK pathway in cardiac hypertrophy may be through the activation of phosphorylation of c-raf, MEK1, Mek2, ERK1 and ERK2 at specific sites.

Effect of Total Flavone of Haw Leaves on Nuclear Factor Erythroid-2 Related Factor and Other Related Factors in Nonalcoholic Steatohepatitis Rats.

OBJECTIVE: To investigate the effect of total flavone of haw leaves (TFHL) on the expression of nuclear factor erythroid-2 related factor (Nrf2) and other related factors in nonalcoholic steatohepatitis (NASH) rats induced by high-fat diet and then to further discuss the mechanism of TFHL's prevention against NASH. METHODS: High-fat diet was fed to 40 rats to establish the NASH model. Then model rats were intragastrically administrated with 40, 80, 160 mg/(kg•day) TFHL, respectively. The pathological changes of liver tissues in NASH rats were detected by oil red O and hematoxylin-eosin (HE) stainings. The expression of Nrf2 in rat liver was examined through immunohistochemistry. The level of 8-iso-prostaglandin F2α in serum was detected through enzyme linked immunosorbent assay (ELISA). The mRNA and protein levels of Nrf2 and other related factors in liver tissue were measured by real-time reverse transcriptionpolymerase chain reaction and western blot. RESULTS: Lipid deposition, hepatic steatosis, focal necrosis in lobular inflammation and ballooning degeneration were emerged in livers of NASH rats. The 8-iso-prostaglandin F2α in the serum of NASH rats increased significantly compared with the control group (P<0.05). The mRNA of Nrf2, hemeoxyenase1 (HO-1) and the mRNA and protein levels of quinine oxidoreductase (NQO1) in NASH rats liver tissue showed a striking increase, while the mRNA levels of Keap1, r-glutamylcysteine synthethase (rGCS) and glutathione S-transferase (GST) were significantly decreased compared with the control group (P<0.05). After TFHL treatment, 8-iso-prostaglandin F2α level in serum significantly decreased, and Nrf2 mRNA and protein levels in hepatocytes nucleus enhanced compared with the model group (P<0.05 or 0.01). Meanwhile the Keap1 mRNA, the mRNA and protein levels of HO-1, NQO1 antibody, rGCS antibody, GST increased after TFHL treatment (P<0.05 or 0.01). CONCLUSIONS: Nrf2 and other related factors were involved in development of NASH, and they also served as an important part in its occurrence. By regulating expression of Nrf2 and other related factors, TFHL may play a role in antioxidative stress and prevention of NASH.

Differential expression of microRNA related to irritable bowel syndrome in a rabbit model.

OBJECTIVE: This study aimed to evaluate the differential expressions of microRNAs (miRNAs) in white hair black eye (WHBE) rabbits of irritable bowel syndrome (IBS). METHODS: WHBE and Japanese white (JW) rabbits were divided into the control and IBS groups. The IBS groups were exposed to moist heat, stress and low-dose laxatives. Their intestinal movement rate was measured. Blood samples were taken to detect serum 5-hydroxytryptamine (5-HT) and dopamine levels and colonic tissues were obtained to detect c-Fos expression by immunohistochemistry. Deep sequencing technology was used to obtain miRNA sequences in the intestinal tissues of WHBE and JW control groups. Expressions of 14 miRNAs were measured by real-time polymerase chain reaction in both the control and the IBS model groups. RESULTS: Serum 5-HT and dopamine levels, intestinal movement rate and c-Fos expressions in the WHBE rabbits were significantly increased compared with the control group. MiR-29a-3p, miR-24-3p, miR-221-3p, let-7f-5p, let-7g-5p, let-7i-5p, miR-192-5p, miR-126-3p and miR-130b-3p expressions in WHBE IBS rabbits at day 14 were significantly higher than those in the control group while miR-324-3p and miR-132 were downregulated. MiR-29a-3p, let-7i-5p, miR-192-5p and miR-126-3p were significantly upregulated only in JW IBS rabbits at day 14 and miR-324-3p, miR-223-3p and miR-132 were significantly downregulated in JW IBS group. MiR-24-3p, miR-221-3p, let-7f-5p, miR-126-3p and miR-130b-3p expressions in WHBE IBS rabbits were higher than that in JW IBS rabbits. CONCLUSIONS: Twelve miRNAs were differentially expressed in IBS rabbits. Five are specific in WHBE IBS rabbits, suggesting that they play a role in increased sensitivity to IBS.

[The influence of miR-21 on HK-2 EMT cells induced by TGF-beta 1].

OBJECTIVE: To investigate the effect of the miR-21 and its target mRNA in renal tubular epithelial mesenchymal transformation (EMT) model induced by transformation growth factor-ß1(TGF-ß1) in human renal tubular epithelial (HK-2) cells. METHODS: HK-2 cells were divided into 6 groups:normal control group, TGF-ß1 group, miR-21 mimic negative group, miR-21 mimic group, miR-21 inhibitor negative group and miR-21 inhibitor group. EMT model was established in HK-2 cells induced by 4 ng/ml TGF-ß1. The level of miR-21, the mRNA and protein expression of EMT related factors were detected. MiR-21 mimic plasmid and miR-21 inhibitor plasmid were transfected into HK-2 cells that treated with TGF-ß1 respectively using liposome transfection technique. Observe the impact of overexpression or inhibition expression of miR-21 on the mRNA and protein expression of EMT related factors and PTEN. RESULTS: ①Compared with the normal group, the level of miR-21 was significantly increased in model group (P<0.05), the mRNA and protein expression levels of epithelial cells marker E-cadherin was significantly decreased (P<0.01), while the mRNA and protein levels of mesenchymal cells marker α-SMA was significantly increased (P<0.05,P<0.01). ②Compared with the miR-21 mimic negative group, the level of miR-21 in miR-21 mimic group increased significantly (P<0.01), the mRNA and protein expression levels of PTEN and E-cadherin decreased significantly (P<0.05,P<0.01), the mRNA and protein levels of α-SMA increased significantly (P<0.05,P<0.01). Compared with the miR-21 inhibitor negative control group, the level of miR-21 in miR-21 inhibitor group decreased significantly (P<0.01), the mRNA and protein expression levels of PTEN and E-cadherin increased significantly (P<0.05,P<0.01), the mRNA and protein levels of α-SMA decreased significantly (P<0.05,P<0.01). CONCLUSIONS: MiR-21 may play an important role in EMT induced by TGF-ß1 in HK-2 cells and regulate the expression of EMT related factors its target gene PTEN.

[Protective effect of astragalus saponin extracts on kidneys of diabetic rats].

To study the protective effect of astragalus saponin extracts (AS) on kidneys of diabetic rats. Totally 32 diabetic rats induced by streptozotocin (STZ) were divided into AS high and low dose groups, the positive control group and the model group (DM group) and orally administered with 50 mg x- kg(-1) x d(-1) AS 200, 25 mg x kg(-1) x d(-1) valsartan, 10 mL x kg(-1) x d(1) physiological saline, respectively. Another 8 healthy rats were collected in the normal control group (NC group, physiological saline 10 mL x kg(-1). d(-1)). All rats were treated for consecutively 6 weeks. After the administration, the body weight was measured every week, the concentration of blood glucose was monitored on week 2, 4 and 6. The total urine and total urinary protein (U-TP) in 24 h were measured by the metabolic cage method on week 6; At the end of week 6, blood samples were collected from hearts to detect blood urea nitrogen (BUN), serum creatinine (Scr), uric acid (UA) , total cholesterol (CH) triglyceride (TG) by biochemical methods. Kidneys were collect to calculate the kidney hypertrophy index and observe the pathological sections. The laboratory results show that in the DM group, the blood glucose, metabolic cost in 24 h, kidney hypertrophy index, U-TP, BUN, Scr, UA, TG were significantly higher than that in the NC group (P < 0.01, P < 0.05) , with significant pathological changes; After the intervention with AS, the metabolic value in 24 h, kidney hypertrophy index, U-TP, BUN, Scr, UA, TG were significantly lower in the high dose group (P < 0.01, P < 0.05), and the kidney hypertrophy index, BUN, Scr, UA, TG in the low dose group were also significantly lower (P < 0.05), with slight reduction in renal pathological changes in both groups. In conclusion, Astragalus saponin extracts have a certain protective effect on kidneys of diabetic rats.

[Effect of Nrf2 and related factors on the progression of nonalcoholic steatohepatitis].

OBJECTIVE: To explore the role of NF-E2-related factor 2(Nrf2) and its related factors in the progression of nonalcoholi steatohepatitis (NASH) by investigating the alterations of lipid metabolism and liver histopathology as well as the changes of mRNA and protein expression levels of Nrf2 and its related factors in rats during NASH progression. METHODS: Male SD rats were randomly divided into normal group and model group, which were administrated with high fat diet to establish nonalcoholic steatohepatitis model. The rats from both groups were randomly killed at the end of 4, 12 weeks respectively. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) were detected in the serum and liver tissue; Changes in fat deposition in liver tissue were determined by oil red O staining. HE staining were used to observe the pathological changes of liver tissue and to calculate nonalcoholic fatty liver disease (NAFLD) activity score (hepatic steatosis, inflammation and ballooning degeneration of liver cells). The expression of Nrf2 in liver was detected by immunohistochemical staining. The mRNA and protein levels of Nrf2 and related factors in liver were determined by Realtime PCR and Western blot, respectively. RESULTS: After 4 weeks of high fat diet, the levels of ALT, AST, TC in rat serum and TC, TG, LDL-C in liver were significantly increased compared with that of the normal group (P < 0.01, P < 0.05). After 4 weeks of high fat diet, the levels of ALT, AST, TC, TG in serum and TC, TG, LDL- C in liver increased further (P < 0.01, P < 0.05). Until the 12th week, the content of HDL-C in liver was significantly lower than that of the normal group (P < 0.05). At the end of the 4th or the 12th week, lipid droplets in the model rat liver cells were heavily dyed red and hepatic steatosis increased severely, with ballooning degeneration of liver cells. With the extension of high fat diet feeding time, fat deposition in the liver tissue, hepatic steatosis, NAFLD score, Nrl2 expression were significantly increased (P < 0.01). Expression levels of mRNA and protein of Nrf2, heme oxyenase 1(HO1), NADPH quinone oxidoreductase 1(NQO1), γ-glutamylcysteine synthethase (γ-GCS), glutathione S-transferase (GST) in the model rats increased or decreased at the end of the 4th or the 12th week differentially, (P < 0.01, P < 0.05) with the more significant changes at the end of the 4th week than the 12th week. CONCLUSION: Nrf2 and its related factors may be involved in the occurrence and development of nonalcoholic fatty liver disease, which may play an important role in the process of NASH formation.

[Study on treatment effect and mechanism of Hirsutella sinensis mycelium in idiopathic pulmonary fibrosis in rats].

OBJECTIVE: To study and discuss the effect and mechanism of Hirsutella sinensis mycelium (HSM) on idiopathic pulmonary fibrosis in rats. METHOD: Forty Wistar rats were divided into five groups: the normal control group, the model control group, the high-dose group (1.0 g x kg(-1) HSM), the low-dose group (0.5 g x kg(-1) HSM), and the positive control group (10 mg x kg(-1) hydrocortisone). In addition to rats in the normal control group, the pulmonary fibrosis model was established by injecting 5 mg x kg(-1) bleomycin into rat tracheas for consecutively 28 days, in order to observe their lung function, lung tissue hydroxyproline, cytokines and pathology. RESULT: After rats were administered with HSM, 0.5 g x kg(-1) and 1.0 g x kg(-1) HSM could significantly decrease lung index and hydroxyproline content (P<0.01), while notably improving pulmonary function, alveolus inflammation and fibrosis degree (P<0.05, P<0.01); 1.0 g x kg(-1) HSM could decrease significantly protein expressions of TNF-alpha, IL-1beta and TGF-beta1 in lung tissues, while increasing significantly protein expressions of IFN-gamma (P<0.05). CONCLUSION: HSM have better effect in treating idiopathic pulmonary fibrosis in rats. Its treatment effect and mechanism are related to the regulation of TNF-alpha, IL-1beta and TGF-beta1 and IFN-gamma imbalance.

Silica-supported sulfonic acid-functionalized ionic liquid coated with [bmim][PF6] as a scavenger for the synthesis of amides.

The methodology of using a silica gel-supported functionalized ionic liquid as a scavenger in the purification of parallel synthesis products was demonstrated. Silica-supported sulfonic acid-functional ionic liquid was synthesized by etherification, aminate, and quaternary aminate from activated silica gel and 3-chloropropyl trimethoxysilane, imidazole, and 1,4-butanesultone, which was followed by acidification using trifluoromethanesulfonic acid and anion exchange with potassium hexafluorophosphate. A conventional ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate was then used to coat the surface of the silica gel. The silica-supported functionalized ionic liquid was used as a scavenger in the removal of excess amine in the parallel synthesis of amides. Desired products were obtained in excellent yields and purity with a sequestration time of less than 100 min at room temperature. After scavenging, the scavenger was easily filtered out and regenerated.

[Monitoring inbreeding of WHBE rabbits using microsatellites].

Microsatellite polymorphisms were analyzed in F5, F6 and F7 WHBE rabbits (Oryctolagus cuniculus) to monitor inbreeding. Out of 21 microsatellite loci, 11 were successfully amplified and showed polymorphic. For the F5 WHBE rabbits, the number of alleles per locus (Na) ranged from 3 to 9 and the mean effective number of alleles (Ne) was 1.81. The mean value of observed heterozygosity (Ho) and polymorphism information content (PIC) of the 11 polymorphic loci were 0.381 and 0.524, respectively. The power of cumulative discrimination (CDP) was 1.0. The value of cumulative exclusion probability of the 11 polymorphic loci in the absence (CPE-1) or in the presence of genetic information on the first parent (CPE-2) was 0.926 and 0.993, respectively. For the F6 WHBE rabbits, Na per locus ranged from 3 to 8 and the mean value of Ne was 1.68. The mean value of Ho, PIC, CDP, CPE-1, CPE-2 of the 11 polymorphic loci were 0.356, 0.548, 1.0, 0.931, 0.994 respectively. For the F7 WHBE rabbits, Na per locus ranged from 2 to 6 and the mean value of Ne was 1.51. The mean value of Ho, PIC, CDP, CPE-1, CPE-2 of the 11 polymorphic loci were 0.287, 0.498, 1.0, 0.891, 0.986 respectively. The average number of effective alleles and the average observed heterozygosity were decreasing continuously in F5, F6 and F7, suggesting that the purity of WHBE rabbits was increasing continuously.