Study On The Inhibitory Effect Of Phytic Acid On Liver Fibrosis

ObjectiveTo investigate the inhibitory effect of phytic acid(PA)on liver fibrosis and the effect of PI3K/AKT/NF-κB pathway-related genes by establishing an animal model of carbon tetrachloride(CCl4)-induced liver fibrosis in C57 mice and culturing HSC-T6 cells in vitro,and to investigate the inhibitory effect of PA on liver fibrosis and the potential mechanism.Methods1.Animal experimentsThirty-six C57 mice were randomly divided into control group(CG),model group(MG),phytate group(PG)and silymarin group(SG)according to their body weight.All groups except the control group were given 20% CCl4 olive oil solution(1 ml/kg)intraperitoneally,and the control group was given the same volume of olive oil intraperitoneally twice a week;the phytate intervention group was gavaged with 100 mg/kg phytate,the silymarin group was gavaged with 100 mg/kg silymarin,and the control and model groups were gavaged with saline once a day for 8 weeks,during which the mice were observed for growth The growth and body weight of the mice were observed.After the experiment,the liver index of mice was calculated;the degree of liver tissue damage and fibrosis was observed by hematoxylin-eosin(HE)staining,Masson staining and Sirus Red staining;the contents of ALT,AST,HYP,SOD,GSH and MDA in the serum or liver tissues of mice in each group were measured;the enzyme-linked immunosorbent assay(ELISA)was used to detect IL-1β,GSH and MDA in liver tissues.The expression of m RNA of α-SMA,Col-1,IL-1β,IL-6 and TNF-α in liver tissue was detected by real-time quantitative PCR(RT-PCR);Western blot to detect the effects of PA on the expression ofα-SMA,Col-1 and PI3K/Akt/NF-κB signaling pathway proteins in mouse liver.2.Cellular experimentHSC-T6 cells were divided into control group,model group(given lipopolysaccharide100 ng/m L),phytate low-dose intervention group(100 ng/m L LPS+0.5mmol/L PA),and phytate high-dose intervention group(lipopolysaccharide 100 ng/m L + 1mmol/L PA).The effect of PA on inflammation-related factors IL-1β,TNF-α and IL-6 in HSC-T6 cells was detected by ELISA;the effect of PA on the expression of α-SMA,Col-1 protein and PI3K/Akt/NF-κB signaling pathway protein in HSC-T6 cells was detected by Western blot;the effect of PA on the expression of Col-1,α-SMA and IL-6 in HSC-T6 cells was detected by RT-PCR.The effects of PA on the relative expression of Col-1,α-SMA,IL-1β,IL-6 and TNF-α m RNA in HSC-T6 cells were examined by RT-PCR.Results1.Animal testCompared with the control group,the body weight gain of mice in the model group was significantly lower(P<0.05),and compared with the model group,the body weight gain of mice in the phytate group was higher;compared with the control group,the liver index and serum levels of ALT and AST of mice in the model group were significantly higher(P<0.05),and the liver index and serum levels of ALT and AST of mice in the phytate intervention were significantly lower(P<0.05);compared with the Compared with the control group,the contents of HYP and MDA in liver tissues of the model group were significantly increased(P<0.05)and the contents of SOD and GSH were significantly decreased(P<0.05),and the contents of HYP and MDA in liver tissues were significantly decreased(P<0.05)and the contents of SOD and GSH were significantly increased(P<0.05)after phytate intervention;meanwhile,the results of HE staining and collagen staining showed that The results of HE staining and collagen staining showed that PA significantly improved the necrosis and fragmentation of hepatocytes and reduced the area of collagen fiber deposition;ELISA results showed that the contents of IL-6,IL-1β and TNF-α were significantly increased in the liver of mice in the model group compared with the control group(P<0.05);compared with the model group,PA significantly decreased the contents of IL-6,IL-1β and TNF-α in the liver(P<0.05);RT-PCR results showed that the contents of IL-6,IL-1β and TNF-α were significantly decreased in the liver compared with the control group(P<0.05).PCR results showed that the expression of IL-6,TNF-α,IL-1β,α-SMA,Col-1 m RNA in liver tissues of mice in the model group was significantly increased compared with the control group(P<0.05);PA significantly inhibited the expression of α-SMA,IL-6,TNF-α,IL-1β m RNA in liver tissues of mice compared with the model group(P<0.05);IHC demonstrated that the positive area of CD68 in liver tissue was significantly increased in the model group,while the positive area of CD68 was significantly decreased after phytate intervention(P<0.05);Western blot result analysis showed that compared with the control group,the expression of Col-1,α-SMA,PI3 K,p-Akt,p-NF-κBp65,p-IκBαprotein in liver tissue of mice in the model group The expression of α-SMA,PI3 K,p-Akt,p-NF-κBp65 and p-IκBα in liver tissues of mice in the model group was significantly increased(P<0.05),and compared with the model group,the expression of α-SMA,PI3 K,p-Akt,p-NF-κBp65 and p-IκBα in liver tissues of mice in the phytate intervention group was significantly decreased(P<0.05).2.Cellular experimentsThe IC50(24h)of the effect of PA on HSC-T6 cells was 2.58 mmol/L;CCK-8 assay showed that LPS(100 ng/m L)stimulation promoted the proliferation of HSC-T6 cells,and the proliferation activity of HSC-T6 cells was inhibited after adding phytic acid for intervention(P<0.05),and the inhibition effect was dose-dependent;ELISA assay The results showed that LPS stimulation could promote the release of IL-1β,TNF-α,and IL-6from HSC-T6 cells,and phytic acid could effectively inhibit the release of these inflammatory factors(P<0.05);relative to the control group,the expression of Col-1,α-SMA,IL-1β,TNF-α,and IL-6 m RNA in HSC-T6 cells was significantly increased after LPS stimulation(P<0.05),while Col-1,α-SMA,IL-1β,TNF-α,IL-6 m RNA expression significantly decreased after phytate intervention(P<0.05);Western blot results showed that,compared with the control group,α-SMA,Col-1,PI3 K,p-Akt,p-NF-κ Bp65,p-IκBαprotein expression was significantly higher in the model group compared with the control group(P<0.05);compared with the model group,α-SMA,PI3 K,p-Akt,p-NF-κBp65,pIκBα protein expression was significantly lower in the liver of mice in the phytate intervention group(P<0.05).ConclusionPhytic acid has inhibitory effects on liver fibrosis in both animal and cellular experiments,and the mechanism may be related to the inhibition of PI3K/AKT/NF-κB pathway activity,attenuation of inflammatory response,inhibition of hepatic stellate cell proliferation activation,reduction of extracellular matrix deposition,and improvement of hepatic antioxidant capacity.