Protective Effect And Mechanism Of Cigu Polysaccharide On Liver Injury Induced By Six Heavy Metals

Objective:Through in vivo and in vitro experiments,6 kinds of heavy metals induced liver damage experimental model,on the basis of the successful establishment of liver injury model,to explore the preventive protection effect of Sagittaria sagittifolin polysaccharide on 6 kinds of heavy metals-induced liver injury,and through antioxidant,anti-apoptosis,anti-inflammatory and other aspects from the genetic and protein levels to study the protective mechanism of Sagittaria sagittifolin polysaccharide anti-heavy metal liver injury,with a view to providing experimental basis for the protection of many kinds of heavy metal liver injury prevention and treatment.It also provides new ideas for the development of Sagittaria sagittifolin polysaccharide as a heavy metal detoxifying product.Method:1 Study on the protective effect and mechanism of Sagittaria sagittifolin polysaccharide on 6 kinds of heavy metal-induced liver damage in mice.After one week of adaptive feeding of Kunming mice,five groups were randomly divided into 8 groups,namely,blank control group,model group,positive drug glutathione group,Sagittaria sagittifolin polysaccharide high dose group,Sagittaria sagittifolin polysaccharide low dose group,a total of 40.Each group of mice was gastrically perfused twice a day,including a high dose of Sagittaria sagittifolin polysaccharide(0.8g/kg),a low dose(0.2g/kg),and a positive drug group(0.9 g/kg)4h in advance for gastric irrigation treatment,control group and model The group was given the same volume of physiological saline,after 4 hours of gastric irrigation,molding administration was carried out,in which the model group,the high sugar,low-dose group and the positive drug group were given heavy metal mixture(0.3g/kg)to the stomach,and the blank control group was given the same volume of physiological saline.All mice weighed every 6 days,and the duration of the experiment was 30 days.After the end of the last day of gastric irrigation,fasting 12h after execution,eye blood,dissection to take its liver weighing,fixed.Centrifugal serum,the application of fully automatic biochemical analyzer for AST,ALT,ALP,ALB liver biochemical index detection,the use of HE staining method for pathological observation TNF-α、Bax、Bcl-2、Caspase-3 of liver tissue,to determine whether the establishment of heavy metal model is successful.2 The protective effect and mechanism of Sagittaria sagittifolin polysaccharide on 6 kinds of heavy metal-induced L02 cell damage were studied2.1 The protective effect of merciful Sagittaria sagittifolin polysaccharide on 6 kinds of heavy metals-induced L02 cell damageL02 cells were divided into 8 groups:blank control group,6 heavy metal dyeing groups in different doses(25,50,100,200,400,800,1600ng/ml),and MTT method was used to detect heavy metal mixture LC50 concentration,the L02 cells were divided into 6 groups:blank control group,Sagittaria sagittifolin polysaccharide administration group(0.25,0.5,1,2,4mg/ml),the use of MTT method to detect the non-toxic concentration range of Sagittaria sagittifolin polysaccharide to L02 cells.L02 cells were divided into 6 groups:blank control group,model group(900ng/ml),Sagittaria sagittifolin polysaccharide protection group(900ng/ml heavy metal mixed venom,0.25,0.5,1,2mg/ml of Sagittaria sagittifolin polysaccharide),application MTT method in 24h,48h,72h to detect the protection of the liver damage caused by 6 kinds of heavy metal mixed poisoning,and the microplate method in 24h to detect L02 cell liquid in the glutamate transaminase(ALT),grass transaminase(AST)content.Determine the molding concentration of the heavy metal mixture and the optimal protection time and dose of liver damage caused by Sagittaria sagittifolin polysaccharide to 6 heavy metals.2.2 Effect of merciful Sagittaria sagittifolin polysaccharide on oxidative damage of 6 heavy metals-induced L02 cellsThe cells were divided into 6 groups:blank control group,model group(900ng/ml),Sagittaria sagittifolin polysaccharide protection group(900ng/ml heavy metal mixed venom,0.25,0.5,1,2mg/ml of Sagittaria sagittifolin polysaccharide).The change of reactive oxygen(ROS)content in cells was detected by cell flow method and immunofluorescence method.The cells were divided into 3 groups:blank control group,model group(900ng/ml),Sagittaria sagittifolin polysaccharide optimal protection group(1mg/ml),using protein imprinting method to change the content of Drpl protein in cells;2.3 Effects of merciful Sagittaria sagittifolin polysaccharide on 6 heavy metals-induced L02 apoptosisGrouping and administration of the same 2.2,using immunofluorescence method and cell flow method to detect L02 apoptosis,using Western Blot method to detect TNF-α,Bax,Bcl-2,Caspase-3 protein content changes.Result:1 The protective effect of Sagittaria sagittifolin polysaccharide on liver damage in 6 heavy metals-induced mice and its mechanismThe results showed that compared with the control group mice,the weight of the mice in the model group decreased significantly(P<0.05),and the weight of the mice in the positive drug and the Sagittaria sagittifolin polysaccharide administration group was higher than that of the model group(P<0.05),and the liver index of the model group was higher than that of the control group,and the liver index of the damaged mice was significantly reduced compared with the positive drug and Sagittaria sagittifolin polysaccharide the high-dose group of the model group(P<0.05).Serum biochemical results showed that compared with the control group,the content of ALT,AST,and ALP in the model group were significantly increased,and ALB was significantly decreased.Compared with the model group,the positive drug and Sagittaria sagittifolin polysaccharide administration group could effectively reduce ALT and AST,ALP content,increase ALB content,the difference is statistically significant(P<0.05).HE staining examination of liver tissue pathology showed that the model group showed severe inflammatory immersion in liver tissue compared to the control group,while compared with the model group,the positive drug and the high and low dose group hepatocyte inflammation improved significantly,of which the high dose of Sagittaria sagittifolin polysaccharide improved better than that of the cousin polysaccharide low-dose group.The results of Western Blot showed that the content of TNF-α in the liver tissue of the model group increased significantly(P<0.05),while the content of TNF-α decreased in the Sagittaria sagittifolin polysaccharide group,and compared with the control group,the content of Bax、Caspase-3 in the model group decreased,The expression content of protein increased significantly,the content of Bcl-2 protein decreased,while the expression of apoptosispromoting protein was significantly reduced in the Sagittaria sagittifolin polysaccharide administration group,and the expression of the apoptosis protein Bcl-2 was improved,and the difference was statistically significant(P<0.05).2 The protective effect and mechanism of Sagittaria sagittifolin polysaccharide on 6 kinds of heavy metal-induced L02 cell damage were studiedThe results of MTT experiments show that the cell survival rate of 6 heavy metal mixtures was about 50%at 900ng/ml concentration,so the concentration was chosen as the molding concentration of heavy metal joint induction L02 cells.The experimental results of the nontoxic concentration of Sagittaria sagittifolin polysaccharide showed that the polysaccharides in the range of(0.25,0.5,1,2 mg/ml)had no effect on cell survival,suggesting that the Sagittaria sagittifolin polysaccharide in the concentration range had no cytotoxicity.The results of the injury protection experiment showed that the cell survival rate of the model group decreased significantly at different times compared with the control group(P<0.05),compared with the model group,the polysaccharide administration group of Sagittaria sagittifolin polysaccharide increased the cell survival rate(P<0.05),and the multisaccharide administration of Sagittaria sagittifolin polysaccharide The group can significantly reduce the AST and ALT content in the cell superserfura,suggesting that Sagittaria sagittifolin polysaccharide have a protective effect on heavy metal-induced L02 damage,which,at a concentration of 1mg/ml,has the best liver protection effect after 24h(P<0.05).Therefore,we chose lmg/ml incubation 24h as the concentration and time of administration in the followup experimental mechanism study.2.2 Effect of merciful Sagittaria sagittifolin polysaccharide on oxidative damage of 6 heavy metals-induced L02 cellsIn cell flow and immunofluorescence experiments,compared with the control group,the ROS content in the model group increased significantly(P<0.01)and the fluorescence intensity was highly expressed.Compared with the model group,the Sagittaria sagittifolin polysaccharide administration group significantly reduced the amount of ROS in the cells(P<0.05)and reduced the production of ROS in the cells.In the RT-PCR experiment,compared with the control group,the content of Nrf2,HO-1 mRNA in the cells of the model group increased significantly,and the content of KeaplmRNA decreased significantly,and compared with the model group,the optimal dose group of p Sagittaria sagittifolin polysaccharide reduced the expression of Nrf2,HO-1 mRNA,and elevated Keap1mRNA(P<0.05).Western blotting experiments showed that compared with the control group,the expression of Drpl,Nrf2,HO-1 protein in the model group was significantly increased(P<0.05);compared with the model group,the Sagittaria sagittifolin polysaccharide group inhibited Drpl,Nrf2,and HO-1 in the cells.1 Protein expression(P<0.05).2.3 Effects of merciful Sagittaria sagittifolin polysaccharide on 6 heavy metals-induced L02 apoptosisThe results of cell flow and immunofluorescence experiments showed that,compared with the control group,the apoptosis of the model group was serious,the difference was statistically significant(P<0.01),and the Sagittaria sagittifolin polysaccharide administration group of Mercy significantly improved the apoptosis(P<0.05).In the Western Blot experiment,compared with the model group,the Sagittaria sagittifolin polysaccharide administration group could significantly reduce the expression of the apoptosispromoting protein Bax and Caspase-3 protein,and improve the protein content of Bcl-2(P<0.05),suggesting that the Sagittaria sagittifolin polysaccharide can significantly improve liver apoptosis.Conclusion:Sagittaria sagittifolin polysaccharide has a good protective effect on mice liver injury and L02 cell injury induced by six kinds of heavy metals.Its mechanism may be related to reducing oxidative damage,activating Nrf2 signaling pathway,inhibiting cell apoptosis and reducing inflammation.