MicroRNA133b Attenuates Schistosomiasis Liver Fibrosis By Inhibiting The Expression Of CTGF

[Objective]This study explored the role and mechanism of miR-133b in hepatic fibrosis of Schistosoma japonicum by up-regulating the expression of microRNA 133b(miR-133b)in vitro and in vivo.[Method]Hepatic stellate cells(LX-2)were cultured,and LX-2 cells were stimulated at different time points using different concentrations of TGF-?1.LX-2 cells were treated with optimal concentration and time of TGF-?1,miR-133b mimics,miR-133b mimics NC,miR-133b inhibitor mimics and miR-133b inhibitor NC plasmid were transfected into LX-2cells by transient transfection technique;qRT-PCR detected the transcription levels of miR-133b,TGF-?1,Smad3,CTGF,Colla I and?-SMA in LX-2 cells,respectively.A lentiviral vector-based miR-133b virus plasmid(Lenti-miR-133b)and a negative control plasmid(Lenti-NC)were constructed.KM mice were randomly divided into 4 groups,and the mice were challenged by cercariae abdomen application.The mice not attacked by the cercariae were uninfected,and the other mice were infected.One week after the cercariae challenged the mice,PBS,Lenti-NC(1×10~9TU/mL)and Lenti-miR-133b(1×10~9 TU/mL)lentiviral plasmids were injected into the tail vein of the mice,respectively.After the 4w,6w and8w,the anesthesia was performed,and the liver tissue was removed and the blood was taken through the eyeball to prepare for the subsequent experiment.The transcription levels of miR-133b,fibrosis-related molecules CTGF,Colla I and?-SMA,and inflammation-related factors IL-4 and IFN-?in liver tissues of each group were detected by qRT-PCR;the levels of IL-4 and IFN-?in serum were determined by ELISA;the protein expression level of CTGF in liver tissue was detected by western-blot method;mouse liver tissue granuloma and fibrosis changes were assessed by HE staining and Masson trichrome staining;the kit analyzed the content of hydroxyproline and serum ALT in mouse liver tissue.[Results]The results of cell experiments showed that the expression of miR-133b in LX-2 cells treated with TGF-?1 was continuously down-regulated and was time-dependent.The expression of miR-133b was significantly increased after transfection of miR-133b mimics in LX-2 cells.Real-time PCR showed that transfection of miR-133b mimics significantly inhibited the expression of CTGF,Colla I and?-SMA induced by TGF-?1(P<0.05),but had no effect on the expression of TGF-?1 and Smad3.The results of animal experiments showed that the expression of miR-133b was decreased in the liver tissues of mice 4w,6w and 8w after infection with Schistosoma japonicum,after treatment with Lenti-miR-133b,the expression of miR-133b was significantly up-regulated(P<0.05).Real-time PCR showed that the expression of fibrosis-related factors CTGF,Colla I and?-SMA in the liver of the infected group were significantly higher than those in the uninfected group(P<0.05);the expression levels of CTGF,Colla I and?-SMA in liver tissues of Lenti-miR-133b treated mice were significantly lower than those in PBS group and lentivirus-negative control group(P<0.05).The results of HE staining and Masson's trichrome staining showed that the egg granuloma and fibrosis changed significantly after 6 weeks of infection in mice,compared with the PBS group and the lentivirus-negative control group,the degree of liver lesions in the miR-133b treatment group was significantly reduced,the analysis of hydroxyproline content in liver tissue showed that the hydroxyproline content in the liver tissue of the infected group was significantly higher than that in the uninfected group(P<0.01);the mice were 6w and 8w after the cercaria attack,the hydroxyproline content of the miR-133b treated group was significantly lower than that of the PBS group and the lentivirus negative control group(P<0.05);the serum ALT expression level analysis showed that the serum ALT level of the infected group was significantly higher than that of the uninfected group(P<0.05),compared with PBS and lentivirus negative control group,the serum levels of ALT in the miR-133b treatment group were decreased(P<0.05).Quantitative real-time PCR and ELISA analysis showed that the IL-4 secretion level in the liver tissue and serum of the infected group was gradually increased after the cercaria attack compared with the uninfected group(P<0.05);IFN-?was 4w after the cercaria attack significantly elevated(P<0.05),6w and 8w decreased;however,there was no significant difference in the expression of IL-4 and IFN-?in each infected group.[Conclusion]miR-133b may inhibit the activation of HSC and the deposition of collagen by targeting silencing of CTGF to alleviate liver fibrosis in mice with schistosomiasis.