The Mechanism Of CircAno6/miR-296-3p/TLR4 Signal Axis Promotes The Activation Of HSCs By Mediating Inflammatory Cascade And The Intervention Effect Of Shuganjianpi Formula

1 ObjectiveTo elucidate the mechanism of circRNA Ano6(circAno6)/miR-296-5p/Toll-like receptor 4(TLR4)signal axis mediating inflammatory cascade reaction,inducing hepatic stellate cell(HSCs)activation and the intervention effect of Shuganjianpi formula(SGJPF).2 Methods2.1 Study on the mechanism of circAno6/miR-296-3p/TLR4 signal axis mediating inflammatory cascade reaction and inducing HSCs activation(1)Nucleocytoplasmic separation and fluorescence in situ hybridization(FISH)were used to detect the localization of circAno6 in cells.(2)Circ Ano6 overexpression plasmid and small interference RNA(sh RNA)were constructed and transfected into JS-1.(3)Real-time quantitative PCR(RT-qPCR)was used to detect the expression of circAno6 under different transfection concentration and time,and the best transfection concentration and transfection time were selected.(4)Effects of circAno6 overexpression/sh RNA on proliferation and cell cycle of JS-1cells were detected by cell counting kit 8(CCK8)and flow cytometry.(5)RT-qPCR,enzyme linked immunosorbent assay(ELASA),western blot(WB)and Immunofluorescence(IF)were used to detect the effects of circAno6overexpression/sh RNA on JS-1 activation markers α-SMA,collagen Ⅰ,inflammatory markers IL-1β,IL-18,NLRP3 and circAno6/miR-296-3p/TLR4 signal axis.(6)The database of National Biotechnology Information Center(NCBI)was used to query the gene sequences of circAno6,miR-296-3p and TLR4,and the potential binding sites of circAno6 and miR-296-3p to TLR4 were predicted by RNAhybrid and miRanda online tools,and the targeted regulation relationship among them was verified by double luciferase report experiment.2.2 Study on the mechanism of SGJPF regulating circAno6/miR-296-3p/TLR4 signal Axis,reducing inflammatory cascade reaction and inhibiting HSCs activation(1)Screening the best intervention concentration and time of SGJPF-containing serum.(2)To observe the effect of SGJPF-containing serum on the viability,cycle,α-SMA and collagen Ⅰ of JS-1 cells.(3)To observe the effect of SGJPF-containing serum on inflammatory indexes and circAno6/miR-296-3p/TLR4 signal axis.(4)Through the recovery test,on the basis of overexpression of circAno6,SGJPFcontaining serum was given to observe the effect of SGJPF on the viability,cycle,α-SMA and collagen Ⅰ of JS-1 cells.3 results3.1 Study on the mechanism of circAno6/miR-296-3p/TLR4 signal axis mediating inflammatory cascade reaction and inducing HSCs activation3.1.1 Expression and localization of circAno6Nuclear and cytoplasmic separation and FISH experiments showed that circAno6 was mainly expressed in the cytoplasm.3.1.2 Transfection efficiency of circAno6The interference efficiency of circAno6 was the best when the small interference RNA of circAno6 was sh RNA3,the concentration was 100 pmol and the time was 48 h,and the overexpression of circAno6 was the best when the concentration of circAno6 overexpression plasmid was 3 μg and the time was 48 h.3.1.3 Effect of circAno6 overexpression/interference on the function of JS-1 cellsOverexpression of circAno6 can promote the proliferation of JS-1,decrease the number of cells in G1 phase,and increase the expression of JS-1 activation index α-smooth muscle actin(α-SMA)and collagen type Ⅰ(collagen Ⅰ).After circAno6 interference,the effect is opposite.3.1.4 Effect of circAno6 overexpression/interference on inflammatory indexesCompared with model group,the expressions of Interleukin-1β(IL-1β),Interleukin-18(Interleukin-18)and NOD-like receptor 3(NLRP3)were increased after circAno6 overexpression,while IL-1β,IL-18 and NLRP3 levels decreased after circAno6 interference.3.1.5 Effect of circAno6 overexpression/interference state on circAno6/miR-296-3p/TLR4 signal axisCompared with model group,miR-26b-5p expression was significantly decreased and Toll-like receptor 4(TLR4)expression was significantly increased after circAno6 overexpression,while miR-26b-5p expression was significantly increased and TLR4 expression was significantly decreased after circAno6 interference.3.1.6 Verification of the regulatory relationship among circAno6,miR-296-3p and TLR4Bioinformatics analysis showed that there were binding sites between circAno6 and miR-296-3p and between miR-296-3p and TLR4.Double luciferase report experiment confirmed that miR-296-3p significantly inhibited luciferase activity in circAno6-WT and TLR4-WT groups.The results suggest that circAno6 could regulate the expression of TLR4 through competitive binding to miR-296-3p.3.2 Study on the mechanism of SGJPF regulating circAno6/miR-296-3p/TLR4 signal axis,reducing inflammatory cascade reaction and inhibiting HSCs activation3.2.1 Screening of the optimal drug-containing serum for SGJPFThe concentration of serum containing SGJPF was 20% and the action time was 48 h,which was the best condition for SGJPF to interfere with JS-1 cells.3.2.2 Effect of SGJPF optimal drug-containing serum on the function of JS-1 cellsCompared with model group,SGJPF-containing serum significantly decreased the viability of JS-1 cells,increased the number of cells in G1 phase,and inhibited the expression of JS-1 cell activation indexes α-SMA and Collagen Ⅰ.3.2.3 Effect of the optimal drug-containing serum of SGJPF on inflammatory indexesCompared with model group,the expression of downstream inflammatory indexes IL-1 β,IL-18 and NLRP3 decreased significantly after intervention with SGJPF-containing serum.3.2.4 Effect of SGJPF optimal drug-containing serum on circAno6/miR-296-3p/TLR4 signal axisCompared with the model group,under the action of SGJPF drug-containing serum,the expression of circAno6 and TLR4 decreased significantly,while the expression of miR-296-3p increased significantly.3.2.5 Recovery experimentThe optimal drug-containing serum of SGJPF could significantly inhibit the viability of JS-1 cells under the condition of circAno6 overexpression,increase the number of G1 cells,and significantly reduce the expression of α-SMA and Collagen Ⅰ.4 Conclusion(1)circAno6 can act as ce RNA to competitively bind miR-296-3p,promote TLR4 expression,mediate inflammatory cascade reaction,induce overactivation of HSCs,and participate in the pathogenesis of LF.(2)SGJPF can regulate the circAno6/miR-296-3p/TLR4 signal axis,reduce the inflammatory cascade reaction and inhibit the activation level of HSCs,so as to play a therapeutic effect on LF.