Effects Of Exogenous Lactic Acid Treatment After Exercise On The Inflammatory Response In DEN Liver Injury Mice

Background: Chronic inflammation is closely related to the development of liver cancer.As an energy substance and signal molecule,lactic acid is widely involved in tumorigenesis and immune regulation.On the one hand,lactic acid can provide energy for tumorigenesis and tumor development.On the other hand,it can regulate the function of immune cells to be conducive to the direction of disease progression,thus aggravating the pathological process.Diethylnirtosamine(DEN)is a class of substance containing nitrosamines,which can cause toxicity to the liver.The pathogenesis of DEN leads to hepatotoxic injury,liver fibrosis,cirrhosis and finally liver cancer.Our research group's previous studies in DEN-induced liver injury mice suggested that moderate intensity exercise but not high intensity interval exercise had obvious anti-tumor effect.The reason may be related to the increase of resting blood lactic acid content after high intensity interval exercise.The accumulation of lactic acid and the inability to remove it quickly may be the key to the failure of the anti-tumor effect of high intensity interval exercise.On this basis,this study intends to construct a model of lactic acid stress after moderate intensity exercise,to delay the recovery of lactic acid after exercise in mice with DEN-induced liver injury,in order to explore the effects of the generation and recovery of lactic acid on exercise benefits.Objective: A moderate-intensity exercise model of DEN-induced liver injury mice was constructed to investigate whether delayed recovery of lactic acid after exercise has adverse effects on the anti-inflammatory effect of exercise,and to further clarify the key role of lactic acid in the anti-tumor effect of exercise. Methods: Forty male C57 BL / 6 mice were 3 weeks old.Randomly divided into4 groups: negative control group(NC group),DEN model group(DEN group),DEN plus moderate intensity exercise group(DET group)and DEN plus moderateintensity exercise plus exogenous lactic acid treatment group(DTL group).After adapting to the environment,the mice were injected with DEN.6 weeks after DEN treatment,exercise intervention was performed and training method was treadmill exercise.The exercise program as follows: training from Monday to Friday,and rest on Saturday and Sunday.The exercise intensity is 65-75% of the maximum oxygen uptake,12m/min,and exercise for 40 min per day for 17 weeks.After exercise training,lactic acid treatment was performed in the DTL group for 14 weeks.Lactate gluconeogenesis experiments was performed at 17 weeks of mice age in every group.The body composition of the animals was analyzed 1 day before the sacrifice.At the end of all the interventions,blood was taken from the heart cavity after anesthesia,and the liver of the mice were harvested.Hematoxylin-eosin was used to stain the morphology of liver cells.Immunohistochemistry for cell labeling of CD3,CD4,CD8 a,CD11c and F480.ELISA was used to determine the serum concentration of IL-1?,TNF-? and IL-10 in mice.RT-q PCR was used to determine the transcription level of liver inflammatory factors,and western blotting was used to determine the expression level of liver inflammation-related proteins.Results :(1)Inflammatory response characteristics of DEN liver injury: Compared with the NC group,the body weight,body composition,and gluconeogenesis levels of the DEN group mice did not show significant changes,but all indicators were reduced;immune cell aggregation occurred in liver tissue;IHC results suggested that the percentage of CD8 a stained area was significantly reduced(p <0.05),and the percentage of F480 stained area was significantly increased(p <0.05);in terms of serum factors,the concentration of IL-1? was significantly increased(p <0.0001),the concentration of TNF-? was significantly increased(p <0.01),and the concentration of IL-10 was significantly increased(p <0.0001);the liver m RNAresults demonstrated that the expression of IL-1? was significantly increased(p<0.0001),the expression of IL-6 was significantly increased(p <0.01),the expression of MCP-1 was significantly increased(p <0.001).(2)Effects of exercise on inflammation in DEN liver injured mice: Compared with the DEN group,the body composition of the DET group showed no significant changes;The percentage of area stained by CD3 and CD11 c showed a decreasing trend;the concentration of serum TNF-? decreased significantly(p<0.001);m RNA expression of liver IL-6 decreased significantly(p <0.05),m RNA expression of liver MCP-1 decreased significantly(p <0.01).(3)Effects of exogenous lactic acid treatment after exercise: Compared with DET group,there was no significant difference in body weight of DTL group in the early stage of lactic acid stress,but the body weight of DTL group decreased significantly in the later stage;the agglutination of immune cells in liver tissue showed a increasing trend;the percentage of CD4 and F480 staining area increased;the serum cytokine results suggested that the concentration of IL-1? was significantly increased(p <0.01),the concentration of TNF-? was significantly increased(p <0.05),and the concentration of IL-10 was significant increased(p<0.01);the liver m RNA results demonstrated that IL-1? expression was significantly reduced(p <0.05);liver western blot results suggested that the expression of IL-6,NLRP3 and p-STAT3 had an increasing trend.Conclusion:(1)DEN leads to obvious inflammatory cell infiltration in liver tissues,that is,increase expression of T cells and macrophages;increase the serum IL-1? and TNF-? inflammatory cytokine concentration;at the transcriptional level,the expression of hepatic inflammatory factors was increased.(2)Exercise,to some extent,can reduce the infiltration of inflammatory cells in the liver caused by DEN,avoid more serious tissue damage and reduce the expression of serum inflammatory factors and m RNA of liver inflammatory factors.(3)Exogenous lactic acid treatment after exercise can reduce the weight of mice,may aggravate the infiltration of inflammatory cells,increase the concentration of serum inflammatory factors,to a lesser extent,increase the expression level of inflammatory proteins,suggesting that the delayed clearance of lactic acid after exercise may have a negative effect on the anti-inflammatory benefits of exercise.