Effect And Preliminary Study Of Mechanism Of Total Flavonoids From Equisetum On Liverinjury Induced By LPS

As a folk herb,Equisetum arvense L.(Equ.)contains a variety of flavonoids.However,up to now few studies about the pharmacological effects of the flavonoids from Equ.have been reported.In this study,the total flavonoids were isolated and purified from Equisetum.The protective effects of the total flavonoids of Equisetum(ETF)on lipopolysaccharides(LPS)-induced oxidative stress,inflammation,and endoplasmic reticulum stress in mice liver were investigated.Meanwhile,we explored the potencial hepatoprotective molecular mechanism of ETF from CYP450 s and related nuclear receptors pathways.And the main results are as follows:1.Extraction and purification of total flavonoids of EquisetumThe optimum conditions for total flavonoids of Equisetum yield were: enzyme concentration 0.52%,enzyme hydrolysis time 51 min,and temperature 49℃,ratio of liquid to solid25:1,and ethanol concentration 80%.The total flavonoids of Equisetum were purified to reach 51.76% by D-101 macroporous resin.2.The protective effect of ETF in LPS-induced mice.(1)The influences of ETF on liver: The acute liver model of this study was manufactured by intraperitoneal injection of LPS.The effects of ETF on the liver were evaluated by detecting the liver index,liver function and tissue pathology.The results showed that: ETF could significantly reduce the hepatic index;significantly reduce AST,ALT and ALP activity in the serum;and effectively alleviate LPS-induced liver lesions in mice.(2)The influences of ETF on liver oxidative stress and inflammatory factors:The levels of various factors were detected in different groups by the kit methods.The results showed that: ETF could significantly increase the activities of antioxidant enzymes such as superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px),et al..And ETF could obviously decrease the content of free radicals such as malondialdehyde(MDA),nitric oxide(NO),and inducible nitric oxide synthase(i NOS),et al..At the same time,ETF could significantly down-regulate the gene and protein expressions of pro-inflammatory factors,including interleukin(IL)-1 beta and tumor necrosis factor(TNF)-alpha,while up-regulate the protein expression of anti-inflammatory factor IL-10.(2)The influences of ETF on liver endoplasmic reticulum stress factors: Detected the gene and protein expressions of ETF on factors of liver in LPS-induced mice by RT-PCR and Western-blot.The results were as follows: ETF could significantly down-regulated the gene and protein expressions of related factors: cysteine aspartate protein hydrolase(Caspase)-3,CCAAT/enhancer binding protein homologous protein(CHOP)and glucose regulated protein(GRP)-78 gene and protein expression.And ETF could significantly inhibit endoplasmic reticulum stress.3.The influences of ETF on the activities and expressions of CYP450 s and related nuclear receptors in mice liver.(1)The effects on CYP450s: The CYP450 s activities in vitro were detected by "cocktail" method,and the CYP450 s gene and protein expression levels were detected of liver by RT-PCR and Western-blot,respectively.The results showed that: ETF could inhibit expressions of gene and protein of CYP1A2 and 2E1,and inhibit enzyme activity in vitro.Meanwhile,ETF could induce the expressions of gene and protein of CYP2D22 and 3A11,and enhance enzyme activity.(2)The effects on nuclear receptors: The gene and protein expressions of nuclear receptors CAR,PXR and Ah R in liver were detected by RT-PCR and Western-blot.ETF could induce expressions of gene and protein of CAR and PXR,and significantly inhibit the protein expression of Ah R.Above all,the results of this study showed that there was a good protective effect of total flavonoids of Equisetum on LPS-induced liver injury in mice.And its hepatoprotective mechanism may be related to the expression and enzyme activity of CYP450 s.