Research On The Effect And Mechanism Of Citrus Maxima-tea On Inhibiting Liver Cancer Cells Proliferation And Relieving Non-alcoholic Fatty Liver Disease

In order to explore the role of citrus maxima combined with Yinghong NO.9 tea,two characteristic natural products in Guangdong,we explored its effect and mechanisms on inhibiting hepatocellular carcinoma cells(HCC)proliferation.And we also study its role in relieving non-alcoholic fatty liver disease(NAFLD)and its mechanisms.With reference to national standards and literature,the content of active ingredients in citrus-tea was measured and its antioxidant capacity in vitro was determined.The inhibitory effect of Citrus maxima-tea on the proliferation and migration of hepatocellular carcinoma cells was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide(MTT)and wound healing(WH)methods,and the flow cytometry(FL)skill was used to assayed cell cycle and apoptosis of HCC.Furthermore,the molecular mechanism of anti-HCC growth effects was analyzed by western blot(WB)method.Then,the kit was used to detect the changes of physiological indexes in the serum and liver lipids,and hematoxylin-eosin(HE)staining was used to analyze the pathological changes in the liver.Finally,the molecular mechanism of reducing NAFLD effects was explored by WB and immunohistochemistry(IHC).Results:(1)The mainly components in Citrus maxima-tea as follows: tea polyphenols(TP),epigallocatechin gallate(EGCG),Hesperidin,etc.And they have the effect of scavenging various free radicals.(2)Citrus reticulate peel black tea(CRPBT)has a significant inhibitory effect on the proliferation and migration of hepatoma cells,but it has no significant effect on nomal liver cells.CRPBT markedly suppressed the expression of p-phosphoinositide 3-kinase(PI3K)and p-protein kinase B(AKT)without affecting the total PI3 K or AKT in HCC cells.And it significantly down-regulated the expression of matrix metalloproteinase(MMP)-2/9 and other proteins.(3)The inhibition effect of BT-CM on the proliferation of liver cancer cells was significantly lower than that of YT-CM or GT-CM.The cell cycle arrest effect of YT-CM group and GT-CM group on liver cancer cells was stronger than that of BT-CM group,and the apoptosis rate was significantly higher than that of BT-CM group.The tea-Citrus maxima(T-CMs)significantly reduced the protein phosphorylation level of PI3 K,AKT and rapamycin mammalian targets(Mammalian target of rapamycin,mTOR),and significantly regulated the expression level of mitochondrial apoptosis-related proteins.Among them,the regulation effect of YT-CM group and the GT-CM group was stronger.(4)The body weight,weight gain,and Lee's index of mice in T-CMs were significantly reduced.The liver weight and liver index were significantly reduced compared with the model group,and the BT-CM group's effect was the most obvious.The T-CMs significantly regulated various physiological indexes of serum liver in the model group,and the BT-CM group had the most significant regulation.The T-CMs significantly reduced lipid vacuoles and significantly reduced inflammatory infiltration,and the BT-CM group performed best.The regulation of GT-CM group and YT-CM group on AMP activated protein kinase(AMPK)protein phosphorylation level and fatty acid synthase(FAS)protein expression level was better that that of BT-CM group.In addition,the acetyl-CoA carboxylase(Acetyl-CoA carboxylase,ACC)protein phosphorylation level and fatty acid oxidation rate-limiting enzyme-carnitine acyltransferase-1(carnitine palmitoyltransferase 1,CPT1)protein expression level in the BT-CM-treated group was better.Conclusion:(1)Citrus tea contains catechin and hesperidin and other antitumor active ingredients,and has strong antioxidant capacity in vitro.(2)CRPBT inhibited the proliferation,migration and invasion of liver cancer cells by inducing PI3K/AKT and MMPs signaling pathways.(3)T-CMs regulated the expression of mitochondrial apoptosis proteins by activating the PI3K/AKT/mTOR signaling pathway,and induce apoptosis of liver cancer cells.(4)T-CMs promoted fat oxidation and inhibited fat synthesis by activating AMPK/ACC and AMPK/FAS signaling pathways,then reduced fat accumulation and relieved NAFLD.