Effect Of Duyun Maojian Tea Extract On LX-2 Extracellular Matrix Through Nrf2/HO-1 Signaling Pathway

Objective:The Lipopolysaccharide(LPS)-induced human hepatic stellate LX-2 cells(LX-2)was used to observe Nrf2/HO-1 signaling pathway related targets and the expression of LX-2 extracellular matrix.To investigate the mechanism of improving hepatic fibrosis by Duyun Maojian tea extract at cell level.Methods:1.Respectively from the Weng ’an,Duyun Spring and Autumn tea of dry tea leaves a total of four tea samples sent to the analysis and test research institute in Guizhou,using national standard method and high performance liquid chromatograph specifically Duyun Maojian tea catechins,caffeine and the content of flavonoids and other primary nutrients and comparison,choose the highest nutrient content of tea samples deionized water and ethanol mixed purification after the production of tea extract.2.The phenotype of LX-2 cells is activated Hepatic stellate cells(HSCs),which is used as the research object in vitro experiments.Different concentrations of Duyun Maojian tea extracts(0ug/ml,5ug/ml,10ug/ml,20ug/ml,40ug/ml,80ug/ml,160ug/ml,320ug/ml and 640ug/ml)were used to intervene LX-2 in logarithmic growth period for 24 hours.Cell activity was detected by CCK8 method to determine the appropriate in vitro Cell intervention concentration.LX-2 in logarithmic growth phase was divided into 6 groups: Blank control group(without any treatment),model group(LPS treatment),tea extract low-concentration,medium-concentration and high-concentration groups and control group(low-concentration silybin 5.0umol/L),except blank control group,other groups were added with 500ng/ m L LPS for 24 hours,tea group was added with the corresponding concentration of tea extract,The control group was treated with silybin for 24 hours.Quantitative real-time PCR(q PCR)was used to detect the m RNA expression of COL I(Type I collagen protein,COL I)and α-SMA(A-smooth muscle actin,α-SMA)in LX-2.The expressions of COL I,Nrf2(Nuclear factor-related factor 2,Nrf2)and HO-1(Heme oxygenase-1,HO-1)proteins in LX-2 were detected by Western-blot.Results:1.Among the four tea samples,the content of catechins in Weng ’an Spring tea was the highest,up to(19.07±0.15)%,which was significantly different from that in Duyun Spring tea(18.56±0.14)%、Autumn tea(15.31±0.11)% and Weng ’an Autumn tea(18.01±0.20)%(P<0.01);The caffeine content of Weng ’an Autumn tea was the highest,followed by Spring tea(3.30±0.15)and(3.29±0.02)%,respectively,and there was no significant statistical difference between the two groups.Weng ’an Autumn tea had the highest flavonoids content,followed by Spring tea,which were(0.75±0.03)% and(0.46±0.06)%,respectively(P<0.001),Weng ’an Spring tea and Duyun Spring tea had significant differences(P<0.001).2.According to the results of IC50,the low,medium and high concentration of tea extract acting on LX-2 were selected as 40ug/ml,80ug/ml and 160ug/ml,respectively.q PCR and Western-blot results showed that α-SMA m RNA,COL I m RNA and protein were expressed in blank control group.After LPS-induced activation of LX-2 cells,the expression levels of α-SMA m RNA and COL I m RNA and protein in model group were significantly higher than those in blank control group,and the expression levels of Nrf2 and HO-1 protein were significantly decreased,the differences were statistically significant(P<0.05).Compared with model group,low silybin concentration significantly decreased COL I and α-SMA m RNA and protein expression,Nrf2 and HO-1 protein expression,and the difference was statistically significant(P<0.05).After the addition of Duyun Maojian tea extract,the expression of α-SMA m RNA and COL I m RNA and protein content of LX-2decreased compared with the model group.With the increase of tea extract concentration,the inhibition effect became more obvious,while Nrf2 and HO-1protein content gradually increased,the difference was statistically significant(P<0.05).Compared with the high concentration tea extract group,the m RNA and protein expressions of COL I and α-SMA in positive control group were higher(P<0.05),and the protein expressions of Nrf2 and HO-1 in positive control group were slightly lower than that in high concentration tea extract group(P<0.05).Conclusions:Duyun Maojian tea extract can improve liver fibrosis and inhibit LPS-induced LX-2 cell proliferation.The mechanism may be that Nrf2/HO-1 signaling pathway is activated to up-regulate Nrf2 and HO-1 expression,and COL I protein expression is down-regulated,thus reducing the synthesis and deposition of extracellular matrix.