| Objective:This study was to investigate the mechanism of action of lipopolysaccharide(LPS)/toll-like receptor-4(TLR4)signaling pathway on the activation and proliferation of hepatic stellate cells(HSC).As a target to provide new ideas for the treatment of nonalcoholic fatty liver fibrosis.Method:HSC cells were divided into 4 groups:1 blank group;2 control group;3 sample group 1(100 ng/ml LPS)4 sample group 2(200 ng/ml LPS),5 replicate wells in each group,continue to culture for 24h,48h,72h,At 96 h,the survival rate of HSC was determined by MTT assay.In addition,there were 3 groups:1 control group;2100 ng/ml LPS group;3200 ng/ml LPS group,5 culture dishes in each group,and then continued to culture for 12h,24h,36h,48h,respectively,and the cells were taken to extract total RNA,using Real.-Time PCR method was used to determine TLR4,typelcollagen,MMP-13,2,TIMP-1,2,BAMBI mRNA levels at 12h,24h,36h in 100ng/ml LPS group;protein was extracted from cells at 48h,using Western Blot method The expression levels of a-SMA and TLR4 were determined.The other three groups were:1 control group;2 LPS group and 3 anti-TLR4 neutralizing antibody group;MTT assay was used to detect the proliferation of each group,and the typeIcollagen of each group was compared by Real-Time PCR.And BAMBI mRNA levels.Results:1.After LPS is added,the growth of HSC cells is very strong,star-shaped or polygonal,the volume is significantly increased,the cell processes are extended outward,the lipid droplets in the cytoplasm are significantly reduced or even disappeared,and the cells are fused to each other in a focal or monolayer.It is more significant in the 200 ng/ml LPS group.2.The results of MTT assay showed that after adding LPS at different concentrations,the cell proliferation rate increased significantly,which was time-dependent,and the difference was statistically significant(P<0.01).Compared with the control group,the proliferation rate after LPS treatment.Significantly increased,the difference was statistically significant(P<0.01).After adding 1 mg/ml of TLR4 antibody and 100 ng/ml of LPS,the proliferation rate of LPS group was significantly higher than that of the control group,which was time-dependent,and the difference was statistically significant(P<0.01),The anti-TLR4 neutralizing antibody group was not significantly increased(P>0,05),3.The results of Real-Time PCR showed that the expression of TLR4,typelcollagen,MMP-13,2 and TIMP-1 mRNA in HSC cells increased significantly at 24h and 36h after treatment with 100ng/ml LPS,Statistically significant(P<0.01),compared with LPS treatment for 0 hours,24h,36h significantly increased,the difference was statistically significant(P<0.01),while TIMP-2,BAMBI mRNA expression compared with the control group,24h,36h significantly decreased,the difference was statistically significant(P<0.01).After adding anti-TLR4 neutralizing antibody(lmg/mL)and LPS(100ng/ml)in HSC,compared with the control group,the expression of type Icollagen mRNA was significantly increased and the expression of BAMBI mRNA was significantly decreased in the LPS group.The difference was statistically significant.Significance(p<0.01),while there was no significant difference between the anti-TLR4 neutralizing antibody group(p>0.05).4.The results of Western Blot assay showed that the protein expression levels of TLR4 and a-SMA in the two groups were significantly higher than those in the control group,and the difference was statistically significant(P<0.01).Conclusions:1.LPS stimulates the activation of HSC and increases the expression of TLR4.2.LPS promotes the proliferation and activation of HSC through the LPS/TLR4 signaling pathway.3.Activated HSC expresses a-SMA,MMP-13,2,TIMP-1,2,type Icollagen.4.LPS down-regulates the expression of BAMBI through the TLR4 signaling pathway and promotes HSC activation. |
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