RESUMO
Schwann cells play a critical role in peripheral nerve regeneration through dedifferentiation and proliferation. In a previous study, we performed microarray analysis of the sciatic nerve after injury. Accordingly, we predicted that long non-coding RNA NONMMUG014387 may promote Schwann cell proliferation after peripheral nerve injury, as bioinformatic analysis revealed that the target gene of NONMMUG014387 was collagen triple helix repeat containing 1 (Cthrc1). Cthrc1 may promote cell proliferation in a variety of cells by activating Wnt/PCP signaling. Nonetheless, bioinformatic analysis still needs to be verified by biological experiment. In this study, the candidate long non-coding RNA, NONMMUG014387, was overexpressed in mouse Schwann cells by recombinant adenovirus transfection. Plasmid pHBAd-MCMV-GFP-NONMMUG014387 and pHBAd-MCMV-GFP were transfected into Schwann cells. Schwann cells were divided into three groups: control (Schwann cells without intervention), Ad-GFP (Schwann cells with GFP overexpression), and Ad-NONMMUGO148387 (Schwann cells with GFP and NONMMUGO148387 overexpression). Cell Counting Kit-8 assay was used to evaluate proliferative capability of mouse Schwann cells after NONMMUG014387 overexpression. Polymerase chain reaction and western blot assay were performed to investigate target genes and downstream pathways of NONMMUG014387. Cell proliferation was significantly increased in Schwann cells overexpressing lncRNA NONMMUG014387 compared with the other two groups. Further, compared with the control group, mRNA and protein levels of Cthrc1, Wnt5a, ROR2, RhoA, Rac1, JNK, and ROCK were visibly up-regulated in the Ad-NONMMUGO148387 group. Our findings confirm that long non-coding RNA NONMMUG014387 can promote proliferation of Schwann cells surrounding the injury site through targeting Cthrc1 and activating the Wnt/PCP pathway.
RESUMO
ETHNOPHARMACOLOGY RELEVANCE: The decoction of the flowers of Abelmoschus manihot (L.) Medic was traditionally used for the treatment of jaundice and various types of chronic and acute hepatitis in Anhui and Jiangsu Provinces of China for hundreds of years. Phytochemical studies have indicated that total flavonoids extracted from flowers of A. manihot (L.) Medic (TFA) were the major constituents of the flowers. Our previous studies have investigated the hepatoprotective effects of the TFA against carbon tetrachloride (CCl4) induced hepatocyte damage in vitro and liver injury in vivo. This study aimed to investigate the protective effects and mechanisms of TFA on α-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury in rats. MATERIAL AND METHODS: The hepatoprotective activities of TFA (125, 250 and 500mg/kg) were investigated on ANIT-induced cholestatic liver injury in rats. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were used as indices of hepatic cell damage and measured. Meanwhile, the serum levels of alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), total bilirubin (TBIL), direct bilirubin (DBIL), and total bile acid (TBA) were used as indices of biliary cell damage and cholestasis and evaluated. Hepatic malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), glutathione transferase (GST), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) were measured in the liver homogenates. The bile flow in 4h was estimated and the histopathology of the liver tissue was evaluated. Furthermore, the expression of transporters, bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), and Na(+)-taurocholate cotransporting polypeptide (NTCP) were studied by western blot and reverse transcription-quantitative real-time polymerase chain reaction (RT-PCR) to elucidate the protective mechanisms of TFA against ANIT-induced cholestasis. RESULTS: The oral administration of TFA to ANIT-treated rats could reduce the increases in serum levels of ALT, AST, LDH, ALP, GGT, TBIL, DBIL and TBA. Decreased bile flow by ANIT was restored with TFA treatment. Concurrent administration of TFA reduced the severity of polymorphonuclear neutrophil infiltration and other histological damages, which were consistent with the serological tests. Hepatic MDA and GSH contents in liver tissue were reduced, while SOD and GST activities, which had been suppressed by ANIT, were elevated in the groups pretreated with TFA. With TFA intervention, levels of TNF-α and NO in liver were decreased. Additionally, TFA was found to increase the expression of liver BSEP, MRP2, and NTCP in both protein and mRNA levels in ANIT-induced liver injury with cholestasis. CONCLUSION: TFA exerted protective effects against ANIT-induced liver injury. The possible mechanisms could be related to anti-oxidative damage, anti-inflammation and regulating the expression of hepatic transporters. It layed the foundation for the further research on the mechanisms of cholestasis as well as the therapeutic effects of A. manihot (L.) Medic for the treatment of jaundice.