Effects of Cigu Xiaozhi Formula on miR-378a-3p Expression and Hh Signaling Pathway in TGF-β1 Induced LX2 Cells

[Objectives]To observe the effects of Cigu Xiaozhi Formula on miR-378a-3p expression and Hh signaling pathway in TGF-β1 induced and activated LX2 cells.[Methods]Cells were divided into control group,induction group,drug-containing serum group,miR-378a-3p inhibitor group,and miR inhibitor NC group.CCK-8 method was used to detect the cell viability of each group,and flow cytometry was used to detect the apoptosis rate of each group.RT-qPCR was used to detect the expression of miR-378a-3p in each group s cells,and RT-qPCR and Western blot were used to detect mRNA and protein expression of Shh,Gli1,Gli2,Col-I,andα-SMA in each group s cells.[Results]Compared with the control group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,andα-SMA mRNA and protein in induction group increased(P<0.01),while the expression of miR-378a-3p decreased(P<0.01).Compared with the induction group,the cell viability and expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA andα-SMA and Gli2 protein decreased in drug-containing serum group(P<0.05),while cell apoptosis rate and miR-378a-3p expression increased(P<0.01).In miR-378a-3p inhibitor group,cell viability and the expression of Shh,Gli1,Gli2,Col-I,α-SMA mRNA and Gli1,Gli2,α-SMA protein increased(P<0.05,P<0.01),while the apoptosis rate and miR-378a-3p expression decreased(P<0.05,P<0.01).[Conclusions]Cigu Xiaozhi Formula containing serum can upregulate miR-378a-3p expression and downregulate the expression of Gli2 andα-SMA in TGF-β1 induced LX2 cells,thereby inhibiting the activation of LX2 cells and exerting the effects of anti liver fibrosis.

Amphioxus Gli knockout disrupts the development of left–right asymmetry but has limited impact on neural patterning

The Gli transcription factors are the primary mediators of Hedgehog(Hh)signaling.Vertebrate genomes contain multiple Gli paralogues with different functions downstream of Hh signal receipt,in part explaining the complexity of cellular responses to Hh that allow concentration-dependent target gene activation.Amphioxus is a chordate that split from the vertebrate lineage early in the evolution of chordates,before the genome duplications that occurred in early vertebrate evolution.It has a single Gli gene whose transcripts can be alternately spliced to yield two protein isoforms called GliS and GliL.We generated two knockout mutations in amphioxus Gli,one that affects the whole gene and a second that only affects GliL.Both knockouts showed major morphological and molecular defects in the development of left–right asymmetry,a phenotype that is similar but not identical to that previously found in Hh mutants.Hh signaling also patterns the amphioxus neural tube.Here,however,knockout of GliL showed no identifiable phenotype,while knockout of the full gene showed only small changes to the expression of one gene family,Olig.Other genes that were prominently affected by Hh knockout were not altered in expression in either knockout.Reasons for the differences between Hh and Gli knockouts in the pharynx and neural tube are discussed in the context of the likely different functions of amphioxus Gli isoforms.