| Aim1.To explore the role of MST1 in regulating the expression of genes and proteins related to non-alcoholic fatty liver fibrosis;2.To explore the possibility that LBP may regulate the expression of genes and proteins related to non-alcoholic fatty liver fibrosis by targeting MST1,thus providing new strategies and targets for clinical treatment..Materials and Methods1.Select human hepatic stellate cell LX-2 and human hepatocarcinoma cell Hep G2,respectively give 100 μM,200 μM,400 μM palmitic acid(PA)treatment and 2 ng/m L,5 ng/m L,10 ng/m L transforming growth Factor-β1(TGF-β1)treatment,all treated for 24 hours,then MTT cytotoxicity test was used to detect cell viability,oil red O staining was used to detect cell fatty degeneration,and Western blot and q RT-PCR experiments were used to detect cell fibrosis factors The protein and m RNA expression levels of α-smooth muscle actin(α-SMA)and type I collagen(Collagen Ⅰ).2.Use si Mst1 to transfect LX-2 cells in normal culture or TGF-β1 induction for 24 hours and detect cell viability by MTT cytotoxicity test;Western blot test for Mst1,p Smad2/3,Smad2/3,α-Detect the protein expression levels of SMA and Collagen Ⅰ;detect the m RNA expression levels of Mst1,Smad3,Collagen Ⅰ,Smad4,and α-SMA in cells by q RT-PCR experiment.3.The p CDH-Mst1 overexpression plasmid was used to transfect LX-2 cells in normal culture or induced by TGF-β1 for 24 h,and the cell viability was detected by MTT cytotoxicity test;the cells were Mst1,p Smad2/3,and Smad2 by Western blot test./3.The protein expression levels of α-SMA and Collagen Ⅰ were detected;the m RNA expression levels of Mst1,Smad3,Collagen Ⅰ,Smad4,and α-SMA in cells were detected by q RT-PCR experiment.4.Give different doses of LBP to interfere with normal culture or TGF-β1 induced LX-2cells for 24 hours,and detect the cell viability by MTT cytotoxicity test;use Western blot to detect the protein expression of Mst1,Collagen Ⅰ and α-SMA in the cells The level of detection;the m RNA expression level of Mst1,Collagen Ⅰ and α-SMA in the cells was detected by q RTPCR experiment.5.Give the same dose of LBP to interfere with normal culture or TGF-β1-induced LX-2cells,collect cells at different time points for analysis,and detect cell viability by MTT cytotoxicity test;Western blot test for Mst1 and Collagen Ⅰ in cells And the protein expression level of α-SMA was detected;the m RNA expression levels of Mst1,Collagen I and α-SMA in the cells were detected by q RT-PCR experiment.6.LBP was used to intervene si Mst1 to transfect LX-2 cells induced by TGF-β1 for 24 h,and the cell viability was detected by MTT cytotoxicity test;Western blot was used to detect Mst1,p Smad2/3,Smad2/3,and α-The protein expression levels of SMA and Collagen Ⅰ were detected;the m RNA expression levels of Mst1,Smad3,Collagen Ⅰ,Smad4,and α-SMA in cells were detected by q RT-PCR experiment.7.LBP was used to intervene the p CDH-Mst1 overexpression plasmid to transfect LX-2cells induced by TGF-β1 for 24 h,and the cell viability was detected by MTT cytotoxicity test;Western blot was used to detect Mst1,p Smad2/3,and Smad2/ 3.The protein expression levels of α-SMA and Collagen Ⅰ were detected;the m RNA expression levels of Mst1,Smad3,Collagen Ⅰ,Smad4,and α-SMA in cells were detected by q RT-PCR experiment.Results1.Using PA and TGF-β1 to induce LX-2 cells and Hep G2 cells,respectively,and successfully construct a fatty liver fibrosis model100 μM,200 μM,400 μM PA and 2 ng/m L,5 ng/m L,10 ng/m L TGF-β1 were used to induce LX-2 cells and Hep G2 cells for 24 h,respectively.The results of MTT experiment showed that PA and TGF-β1 were induced The cell survival rate of Hep G2 and LX-2 cells decreased significantly with the increase of the inducing factor concentration(P<0.01).The PA induction group inhibited the survival rate of Hep G2 and LX-2 cells more significantly than the TGF-β1 induction group The results of oil red O staining showed that the lipid droplet content in Hep G2 and LX-2 cells in the PA induction group increased significantly with the increase of PA concentration,and the lipid droplet content in LX-2 cells in the TGF-β1 induction group increased with the concentration of TGF-β1 Western blot experiments showed that the protein expression levels of α-SMA and Collagen Ⅰ,in Hep G2 cells were significantly up-regulated after 200μM and 400 μM PA induced Hep G2 cells(P<0.01),5 ng/m L,10 ng/m L TGF-β1induced α-SMA and Collagen Ⅰ,protein expression levels in LX-2 cells were significantly upregulated(P<0.01);q RT-PCR results showed that α-SMA and Collagen Ⅰ m RNA expression levels in Hep G2 cells were significantly increased after 200μM PA induction Increased(P<0.01).After induction of 5 ng/m L and 10 ng/m L TGF-β1 the expression levels of α-SMA and Collagen Ⅰ,m RNA in LX-2 cells were significantly up-regulated(P<0.01).The above results indicate that 200μM and 400 μM PA are more toxic to Hep G2 and LX-2 cells,5 ng/m L,10 ng/m L TGF-β1 induces the formation of intracellular lipid droplets in LX-2 cells,and select10 ng after comparison /m L TGF-β1 induced LX-2 cells for 24 h as a fatty liver fibrosis cell model for follow-up experiments.2.Mst1 can regulate the expression of protein and m RNA of non-alcoholic fatty liver fibrosis related factorsThe p CDH-Mst1 overexpression plasmid was used to transfect liver fibrosis model cells for 24 hours.Western blot and q RT-PCR results showed that compared with the control group,Mst1 overexpression can reduce intracellular liver fibrosis-related factors α-SMA and CollagenⅠ,PSmad 2/3 and Smad 2/3 protein and Mst1,α-SMA,Collagen Ⅰ,Smad 3,Smad 4 m RNA expression levels(P<0.01).The LX-2 cells induced by TGF-β1 were transfected with si Mst1.The results of Western blot and q RT-PCR showed that compared with the control group,the intracellular hepatic fibrosis-related factors α-SMA,Collagen Ⅰ,p Smad 2/3 and Smad 2/3protein expression and α-SMA,Collagen Ⅰ,Smad 3,Smad 4 m RNA expression levels increased to varying degrees(P<0.01).3.LBP increases the expression of Mst1 protein and m RNA in a concentration or time dependent mannerIncubate LX-2 cells with 0 μg/m L,100 μg/m L,300 μg/m L,600 μg/m L,and 900 μg/m L LBP for 6 h,12 h,24 h,and 48 h.The results of the MTT experiment show that LX-2 cell survival rate decreased with the increase of LBP concentration(P<0.01),600 μg/m L LBP was selected to incubate LX-2 cells,cell survival rate decreased with the increase of incubation time(P<0.01);use 0 μg LX-2 cells were incubated for 24 h with LBP,100 μg/m L,300 μg/m L,600μg/m L,and 900 μg/m L.The results of Western blot and q RT-PCR experiments showed that 300μg/m L,600 μg/m L,900 μg/m L LBP can increase the expression of Mst1 protein,and the effect is best at a concentration of 600 μg/m L(P<0.01);600 μg/m L LBP was used to incubate LX-2cells for 6 h,12 h,24 h,48 h.The results of Western blot experiment and q RT-PCR experiment showed that LBP increased the expression of Mst1 protein and m RNA in a time-dependent manner(P<0.01).The above results indicate that LBP can significantly increase the expression of Mst1 protein and m RNA at a concentration of 600 μg/m L,so 600 μg/m L LBP was selected to intervene for 24 h for follow-up experiments.4.LBP regulates the expression of protein and m RNA of non-alcoholic fatty liver fibrosisrelated factors by affecting Mst1600 μg/m L LBP was used to incubate the liver fibrosis cell model that had been transfected with Mst1 si RNA and Mst1 overexpression.The results of Western blot showed that compared with the control group,the intracellular liver fibrosis-related factors α-SMA,Mst1 overexpression were compared with the control group.The protein expression levels of Collagen Ⅰ,p Smad 2/3 and Smad 2/3 were significantly reduced(P<0.01).The results of q RTPCR showed that the m RNA expression levels of α-SMA,Collagen Ⅰ,Smad3 and Smad 4 in cells were significantly reduced(P<0.01);After transfection with si Mst1,the results of Western blot showed that the protein expression levels of α-SMA,Collagen Ⅰ,p Smad 2/3 and Smad 2/3were significantly up-regulated compared with the control group(P<0.01),the results of q RTPCR showed that the m RNA expression levels of α-SMA,Collagen Ⅰ,Smad3 and Smad 4 were significantly up-regulated(P<0.01).We found that LBP can significantly increase the expression of Mst1 and regulate the expression of genes and proteins related to non-alcoholic fatty liver fibrosis.Conclusion1.Mst1 can inhibit the expression of non-alcoholic fatty liver fibrosis related genes and proteins;2.LBP could provide new strategies and targets for clinical treatment by targeting Mst1 to regulate the expression of non-alcoholic fatty liver fibrosis-related genes and proteins. |
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