Amilioration Of CCl₄-Induced Liver Injury In Rats By Selenizing Astragalus Polysacchrides And Combination Of Selenium Yeast With Gum Arabic

Liver is an extremely important organ responsible for the metabolism of drugs and toxic chemicals.Liver diseasesare considered one of the global health problemsfound in human and animals,which are more frequently in dogs and cats.Many drugs and chemicalsmay cause severe liver damage.Carbon tetrachloride(CCl4)is a classic chemical liver injury toxicant,and its mechanism of liver injury induced on rats is closely related to the increase of free radicals with the degree of lipid peroxidation andthe activation of Kupffer cells.Selenium(Se),an essential trace element,is widely distributed in various tissues and organs of the body and has been proved to play a significant role in the antioxidant defense system and theprevention of liver diseases and conditions.The antioxidant and anti-inflammatory effects of Astragalus polysaccharides(APS)and Gum Arabic(GA)have been reported.Different sources of selenium,when linked with APS or combined with GA,could improve the activity compared to selenium and these Polysaccharides.Therefore,this study was designed to investigate the effects of selenizing Astragalus polysaccharides(sAPS)and Se-enriched yeast(SY)with Gum Arabic(GA)combination on CCl4-induced liver injury in rats and to provide theoretical basis and data support for the promotion and application of organic selenium polysaccharides in human or animal liver disease prevention.The experiments are described as followings:Experiment 1 Inactivation of Kupffer cells by Selenizing Astragalus polysaccharides prevents CCl4-induced hepatocellular necrosis in the Male Wistar RatSelenizing astragalus polysaccharides-3(SAPS3)was prepared by nitric acid-sodium selenite method and identfied by infrared spectroscopy.The effec ts of sAP S3 on carbon tetrachloride(CCl4)induced hepatocellular necrosis,and its underlying mechanisms were studied in male Wistar rats.Hepatic damage was induced by intraperitoneal injection of CCl4 twice a week,for 3 weeks.Meanwhile,the rats in addition to CCl4 were also exposed to sodium selenite(SS),astragalus polysaccharides(APS),SS+ APS or SAPS3,in parallel by oral gavage once a day for 3 weeks.At the end of 3 weeks,blood and liver tissue were taken.Serum was collected to test the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and antioxidant status parameters.Liver tissue was collected for histopathological examination and determination of messenger RNA(mRNA)expression levels of CD68,tumor necrosis factor-?(TNF-?),interleukin-1 beta(IL-1?)and autophagy related gene7(ATG7)followed by the measurements of CD68,IL-1? andmicrotubule-associated protein light chain 3(LC3)by immunohistochemistry assay(IHC),or TNF-? by immunofluorescence assay(IFA).The results showed that sAPS3 effectively ameliorated CC14 induced hepatocellular necrosis and inflammation and significantly decreased the levels of AST,ALT,malondialdehyde(MDA)and the expression levels of Kupffer cells(KCs)-specific biomarker CD68 and proinflammatory cytokines produced by activated KCs such as IL-1? and TNF-?(P<0.01).While increasing the levels of total antioxidant capacity(T-AOC),glutathione(GSH),glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)(P<0.05)and reduced the expression levels of a key regulator of autophagy in KCs ATG7 or LC3(P<0.05).These findings indicate that sAPS3 could ameliorate CC14-induced hepatocellular necrosis by inactivation of Kupffer cells and its activity may be superior to the application of selenium,APS or combination of selenium with APS.Experiment 2 Amelioration of CCl4-induced chronic liver injury in rats by selenizing Astragalus polysaccharides:Role of proinflammatory cytokines,oxidative stress and hepatic stellate cellsEffect of sAPS on CCl4-induced chronic liver injury and the underlying mechanisms were investigated in the rat.Forty male Wistar rats were randomly divided into five equal groups as follows:control group;CCl4 group;CCl4+APS group;CCl4+SS group and CCl4+sAPS3 group for 7 weeks.The results showed that sAPS3 significantly decreased the levels of ALT,AST,alkaline phosphatase(ALP),lactate dehydrogenase(LDH)in the serum,MDA and hydroxyproline(Hrp)content in liver(P<0.01),and increased the levels of total protein(TP),T-AOC,GSH-Px,and SOD in liver of rats induced by CCl4.In addition,expression levels of antioxidant-related genes(glutathione peroxidase 1(GPX1),superoxide dismutase 1(SOD 1),and nuclear factor E2 related factor2(Nrf2))were significantly increased following supplementation of the sAPS3(P<0.01).Furthermore,sAPS3 effectively ameliorated CCl4 induced hepatic necrosis and inflammation,and it also reduced the expression levels of proinflammatory cytokines including TNF-?,interleukin-6(IL-6),cyclooxygenase-2(COX-2)and nuclear factor-kappa(NF?B)(P<0.01).Moreover,SAPS3 significantly decreased the expression levels of ?-smooth muscle actin(?-SMA),collagen 1,transforming growth factor-beta1(TGF-?1),but increased the Bcl-2/Bax mRNA ratio in rats administered CC14(P<0.01).Taken together,it could be concluded that sAPS3 could increase the activities of Astragalus polysaccharides and sodium selenite to protect the liver from chronic damage by attenuating hepatic inflammation,oxidative stress,fibrogenesis,and induces apoptosis and cell cycle arrest in hepatic stellate cells.Experiment 3 The Potential combined effect of Se-enriched yeast(SY)with Gum Arabic(GA)on the oxidative damage induced by CCl4 and its underlying mechanismsin rats.The antioxidant and anti-inflammatory effects of SY and GA have been reported.The SY was obtained in our lab.However,the combined effects of GA and SY have not been investigated.This study was conducted to estimate the potential combined effects of SY and GA on the oxidative damage induced by CC14 in rats.Forty male Wistar rats were randomly divided into 5 groups with 8 animals per group:(A)worked as control,(B)was administered CC14,(C-E)were fed daily by GA,SY,and GA+SY respectively after mixing with basal diet,following CC14-intoxication for 3 weeks.The results showed that GA+SY supplementation demonstrated protective and antioxidative effects than the administration of either GA or SY,as evident by lesser(P<0.05)activities of serum ALT,AST,ALP and MDA,and increased(P<0.05)the hepatic activities of GSH,GSH-Px and SOD,consistent with up-regulation of antioxidant-related genes including GPX1,SOD1,and heat shock protein 70(HSP70)in CCl4-intoxicated rats.Furthermore,GA+SY supplementation revealed mild pathological damage and effectively attenuated(P<0.05)the expression levels of the Proinflammatory gene including TNF-?,IL-1?,and IL-6.Moreover,GA+SY supplementation alleviated CCl4-induced hepatocytes apoptosis via suppression of caspase-3 expression.We concluded that GA+SY combination displayed strong protective and antioxidative effects against CCl4-induced oxidative damage in rats via its ability to suppress the oxidative stress,inhibit the caspase-3 and pro-inflammatory gene expression.Experiment 4 the Hepatoprotective Effect of SY and GA Combination on CCl4-Induced Chronic Liver Injury in RatsThis study aimed to determine the hepatoprotective effect of SY and GA combination on CC14-induced chronic liver injury in rats and to explore their synergistic mechanisms of action.Forty male Wistar rats were randomly allotted to 5 groups:(A)worked as control,(B)was administered CCl4,(C-E)were fed daily by GA,SY,and GA+SY respectively after mixing with basal diet,following CC14-intoxication for 7 weeks.Compared with GA or SY,GA and SY combination significantly ameliorated CC14-induced reduction in serum TP with elevation in AST and ALT in addition to restoring the histopathological changes and hepatic content of Hrp.GA and SY combination was also effective in reducing lipid peroxidation(MDA),consistent with an increase in T-AOC,GSH,SOD activities,indicating the suppression of liver oxidative stress.FUrthermore,liver inflammation was ameliorated by GA and SY combination through inhibition of NF-?B,TNF-?,COX-2,monocyte chemotactic protein-1(MCP-1),and toll-like receptor 4(TLR-4)over expression in the liver.Moreover,the up-regulation of proliferating cell nuclear antigen(PCNA)expression by GA and SY combination enhanced the regeneration of liver tissue after CCl4-administration.The expression of Collagenl,?-SMA,and TGF?1,was obviously ameliorated by GA and SY combination,suggesting the amelioration of profibrotic response of the liver.Taken together,our current study suggests that GA and SY eombination exhibit a significant hepatoprotective activity,which more efficient than GA or SY alone.They actobviously to ameliorate chronic liver damage,by suppressing inflammation and inhibiting the profibrotic response as well as support the liver regeneration.