Mechanisms Of Liver And Kidney Injuries Induced By Mercury And Protective Effects Of Rutin

【Background】With the continuous development of economy and the growing prosperity of transportation,environmental pollution is harmful to the health which has become one of the major public health problems all over the world.There are many kinds of environmental pollutants;heavy metal is one of the major categories,such as mercury,lead,cadmium etc.It is now believed that oxidative stress,inflammation,mitochondria dysfunction and apoptosis are closely related to the pathogenesis of multiple organ injuries caused by heavy metals.Many studies have found that mercury and its compounds can cause injuries to the nervous system,kidney and liver.But most of the researches are focus on the phenomenon of some organs injuries.It is scarce about the mechanism of injuries induced by HgCl₂.In the past years,a large number of literatures showed that some of the substances with antioxidant function could alleviate the injuries induced by HgCl₂ in liver and kidney,such as vitamin E,selenium,curcumin,etc.Rutin as a classical antioxidant,has many physiological functions such as anticonvulsant,antidepressant,antibacterial,anti-inflammatory and antioxidation.A large number of documents and information indicate that mecury toxicity is related to antioxidative strss,and Rutin may have a protective effect on liver injury induced by HgCl₂.MAPKs and PI3 K /Akt signal pathways are very important in the regulation of cell proliferation and apoptosis process,we hypothesized that these signal pathways may play a key role in the protection of Rutin on injuries of liver and kidney induced by HgCl₂.According to the above research progress,the focus of our study is as followed: First,make clear of molecular mechanism of liver and kidney injuries induced by HgCl₂.Second,prove whether Rutin has some protective effects on liver and kidney injuries induced by HgCl₂.Final,explore the protection mechanism of Rutin in this process.Make clear of the above questions will undoubtedly help people acknowledge the function of Rutin and play a role in human health especially in present serious environmental pollution.【Objective】1.To investigate the characteristics of liver and kidney injuries induced by HgCl₂,and to clarify the mechanism of injuries induced by HgCl₂.2.To investigate the role of ERK and Akt signaling molecules in the liver and kidney injuries induced by mercury and the protective effects of Rutin.3.To explore the dose effect of hepatocyte injury induced by HgCl₂ and the dose dependent protection of Rutin.4.To determine the relationship between phosphorylation of ERK and Akt and apoptosis,oxidative stress during the protective effect of Rutin.【Methods】1.The protection of Rutin on liver injury induced by HgCl₂ in vivo experiment.SD rats were divided into normal group,Rutin group,HgCl₂ group and HgCl_<sup>2+Rutin group,adaptive feeding for 3 days.The forth day,1)Normal group: rats were given physiological saline by intraperitoneal injection,and were administered 5%DMSO saline by intragastric administration for 3 days.2)Rutin group: rats were given physiological saline by intraperitoneal injection,and were administered 150mg/Kg Rutin by intragastric administration for 3 days.3)HgCl₂ group: rats were given 1.5mg/kg HgCl₂ by intraperitoneal injection,and were administered 5%DMSO saline by intragastric administration for 3 days.4)HgCl_<sup>2+Rutin group rats were given 1.5mg/kg HgCl₂ by intraperitoneal injection,and were administered 150mg/Kg Rutin by intragastric administration for 3 days.After the treatment,the body weight,the ratio of liver weight/body weight were detected.ALT,AST,ALB activities in serum were detected dy biochemical instrument.The level of ROS was detected by DHE probe.The levels of hepatic MDA and GSSG were detected by kits to evaluate hepatic oxidative stress.The content of hepatic CAT,GSH,T-SOD and Cu-ZnSOD and the GSH/GSSG were detected by kits,the antioxidant proteins expression were detected by Western Blotting to evaluate the function of hepatic antioxidant defense system.The protein expressions of apoptosis family were detected by Western Blotting to evaluate hepatic apoptosis.The expression of Akt/P-Akt and Erk/P-Erk were detected by Western Blotting.2.The protection of Rutin on kidney induced by HgCl₂ in vivo experiment.Rat groups and treatments are the same to the above.The body weight and the ratio of kidney weight/body weight were detected.UREA、CRE activities in serum were detected dy biochemical instrument.The level of ROS was detected by DHE probe.The levels of nephritic MDA and GSSG were detected by kits to evaluate nephritic oxidative stress.The levels of nephritic CAT,GSH,T-SOD and Cu-ZnSOD and the GSH/GSSG detected by kits,the antioxidant proteins expression were detected by Western Blotting to evaluate the function of hepatic antioxidant defense system.The protein expressions of apoptosis family were detected by Western Blotting to evaluate nephritic apoptosis.The expression of Akt/PAkt and Erk/PErk were detected by Western Blotting.3.The vitro experiment of liver injury induced by HgCl₂.L02 cells were incubated with 1-150μM HgCl₂ for 3-8 h.Then cell viability was detected by MTT.Cell apoptosis was detected by Annexin V-FITC/PI.The level of ROS was detected by DHE probe.4.The vitro experiment about the protection of Rutin on cell injury induced by HgCl₂.L02 cells were divided into Con,Ru,HgCl₂,20μMRu+HgCl₂,40μMRu+HgCl₂,60μMRu+HgCl₂.L02 cell were pre-incubated with Rutin for 12 h and then treated with HgCl₂ for 6h,after this treatment cell viability was detected by MTT,cell apoptosis was detected by Annexin V-FITC/PI.The level of ROS was detected by DHE probe.MMP and MPTP were detected by kits to evaluate mitochondrial function.MDA and GSSG levels were detected by kits to evaluate the improvement of Rutin on oxidative stress induced by HgCl₂.The contens of CAT,GSH,T-SOD,Cu-ZnSOD and GSH/GSSG were detected by kits,the proteins expression of SOD1,SOD2 and CAT were detected by Western Blotting to evaluate the protection of Rutin on antioxidant defense system.The proteins expressions of Bcl2,Bax were detected by Western Blotting to evaluate the protection effect of Rutin on apoptosis.The expression of Akt/P-Akt and Erk/P-Erk were detected by Western Blotting,and use inhibitors of PI3 K and Erk to explicit the protection mechanisms of Rutin on the injuries induced by HgCl₂.【Results】1.Rutin protects liver from injury induced by HgCl₂.Rutin improve the weight decrease and kidney weight/body weight increased,ameliorate liver function and decreased oxidative stress,enhance the expression of antioxidant proteins and the activity of antioxidant enzyme,increase protein expression of Bcl2,decrease that of Bax.Rutin can also activate the phosphorylation of ERK and Akt to some extent.2.Rutin protects kidney from injury induced by HgCl₂.Rutin improve the weight decrease and liver weight/body weight increased,ameliorate kidney function and decreased oxidative stress,enhance the protein expression of antioxidant proteins and the activity of antioxidant enzyme,increase protein expression of Bcl2,decrease that of Bax.Rutin can also activate the phosphorylation of ERK and Akt to a certain extent.3.HgCl₂ can dose dependently decrease cells viability in a certain concentration range.HgCl₂ can also increase cell apoptosis and reactive oxygen species in dose dependently.a good model of liver cell injury was establish.4.Rutin pre-treatment can alleviate the oxidative stress induced by HgCl₂.Rutin can significantly improve the decreased cell viability,inhibit the increased level of ROS,alleviate the reduction level of CAT,GSH and T-SOD induced by HgCl₂.Rutin can also enhance antioxidant defense system,increase the proteins expression of SOD2,CAT,Nrf2.5.Rutin pre-treatment can significantly inhibite the cell apoptosis induced by HgCl₂,enhance the protein expression of Bcl2,decrease that of Bax,decrease the ratio of Bax/Bcl2.6.Rutin pre-treatment can improve mitochondrial dysfunction induced by HgCl₂.Rutin can improve the decreased mitochondrial membrane potential and mitochondrial membrane pore channel activity induced by HgCl₂.7.Rutin pre-treatment improve the low level of the phosphorylation.of ERK and Akt induced by HgCl₂.Rutin can increase the level of ERK phosphorylation significantly and also improve the phosphorylation of Akt,but not significant.8.Inhibitors of ERK（U0126）and Akt（Ly）can eliminate the protect effect of Rutin on decreased cell viability induced by HgCl₂.Using these inhibitors before Rutin,the protein expression of Bcl2 decrease,Bax and casepase3 increase,the the protein expression of SOD1、SOD2 and CAT reduce.Rutin can activate the phosphorylation of ERK and Akt to enhance the antioxidant defense system to play its protective effect on cell injury induced by HgCl₂.【Conclusion】1.HgCl₂ can destroy the antioxidant defense system,and lead to oxidative stress,mitochondrial damage and apoptosis.2.HgCl₂ can inhibit phosphorylation of ERK and Akt to some extent and participate in the tissue and cell injury.3.Rutin can enhance the antioxidant defense system function,and reduce the production of ROS,as well as improve the activity of antioxidant enzymes,and slow down the tissue injuries and apoptosis.4.Rutin may enhance the antioxidant defense system function and alleviate organ injuries and apoptosis by activating phosphorylation of ERK and Akt.