Effects Of Mangiferin On Kidney Inflammation Injury And MCP-1/CCR2 Signal Pathway In Spontaneously Hypertensive Rats

Objective:In this study herein,we firstly investigated the effect of mangiferin in the treatment of spontaneously hypertensive rats（SHRs）on kidney damage.By investigating the effect of mangiferin on kidney inflammatory damage and MCP-1/CCR2 signal pathway in SHRs.To explore the protective effect of Mangiferin and the role of MCP-1/CCR2 signaling pathway in inflammatory damage of kidney.Furthermore,we explored the mechanism of the Mangiferin protects SHRs target organs from inflammatory damage,to provide experimental foundation for research in exploring new drugs to prevent and treat hypertension.Methods:1.The Effect of Mangiferin on Blood Pressure in SHRsTaking the 10-week-old male Wistay-Kyoto（WKY）rats and spontaneously hypertensive rats as the object of study.40 male spontaneously hypertensive rats were fed 1 week after adaptation,were randomly divided into Model group,high dose Mangiferin group(100mg·kg^-1·d^-1),medium dose Mangiferin group(50mg·kg^-1·d^-1),low dose Mangiferin group(25mg·kg^-1·d^-1)and Benazepril group(10mg·kg^-1·d^-1);Another 8 male WKY rats were served as control group.According to the experimental group,the rats were administered intragastrically with Mangiferin(100mg·kg^-1·d^-1,500mg·kg^-1·d^-1or 25mg·kg^-1·d^-1)or Benazepril for 8 weeks,respectively.Model group and Control group were given equal volume of saline,gavage administration.Tail systolic pressure（SBP）of the rats was measured by tail culf method once two weeks.2.Effect of Mangiferin on Renal Inflammatory Injury in SHRsRats in each group after the last administration,collected 24-hour urine and deprived of food for 12 hours.Anaesthetize the rats by intraperitoneal injection 10%chloral hydrate,and then set them on the operating table.Renal tissue should be stripped off rapidly,dry them off by filter paper,Processing and conserving accordingly accord to each experiment.The pathomorphological changes of the renal tissue were observed with HE,masson staining and transmission electron microscope.The contents of IL-6and TNF-αwere detected by ELISA.The contents ofβ2-MG、m Alb、BUN and SCr in urine were measured by automatic biochemical analyzer.3.Effect of Mangiferin on MCP-1/CCR2 Signaling Pathway in Kidney of SHRsRats in each group after the last administration in the fasted 24h,can not help but water.Anaesthetize the rats by intraperitoneal injection 10%chloral hydrate,and then set them on the operating table.Renal tissue should be stripped off rapidly,dry them off by filter paper,Processing and conserving accordingly accord to each experiment.The protein expression of MCP-1 and CCR2 were measured by Immunohistochemical staining and Western blot.The m RNA expression of MCP-1 and CCR2 were tested by Real-Time PCR.Results:1.The Change of Systolic Blood Pressure in each group8 weeks after intragastric administration,compared with Model group,the systolic blood pressure of low,medium and high dose Mangiferin group were decreased,the difference are statistically significant（P<0.01）.In comparison with before administration in each group,the systolic blood pressure of medium dose Mangiferin group has a lower trend,but there is no obvious biological significance.The experimental results show that mangiferin can reduce the blood pressure of SHRs.Mangiferin has the effect of lowering the blood pressure.2.Renal Pathological ChangesHE staining of kidney tissue:In the Model group,the renal tubular basement membrane was thickened,inflammatory cell infiltration,and glomerular pyknosis.Compared with Control group,the lesions improved in different degrees after treatment with mangiferin.Masson staining observation renal organization fibrosis degree:The Control group without renal fibrosis.The rats in the Model group were all occurred in renal fibrosis,having a large amount of blue-stained collagen deposits,the structure of kidney tubules are unclear.After treatment with mangiferin,the renal fibrosis are alleviated in different degrees.Transmission electron microscope results show that:Rats in model group have serious injury of kidney,glomerular basement membrane thickening,the foot process fusion or the membrane structure of podocytes are dissolved.We also observe that the foot process are fusional,Non-uniform thickness in a amount of basement membrane of low and medium dose Mangiferin group,but the degree and range are slightly reduced compared with Model group.In the high dose Mangiferin group,the kidney tissue damage are significantly improved,glomerular basement membrane are continuous and uniform,the swelling and fusion of podocytes are greatly alleviated.3.The Effect of Mangiferin on the Expression of IL-6 and TNF-αin KidneyThe expression levels of IL-6 and TNF-αin kidney tissue are significantly increased in the Model group compared with the Control group（P<0.01or P<0.05）,suggest that the modeling is successful.Compared with the Model group,the expression level of IL-6 in medium dose Mangiferin group are significantly decreased（P<0.01）;the expression level of IL-6 in Benazepril and low dose Mangiferin group and the expression level of TNF-αin low and medium dose Mangiferin group and Benazepril group are significantly decreased（P<0.05）;the expression level of IL-6 and TNF-αhave the tendency to lower in high dose Mangiferin group.These results suggest that mangiferin can down-regulate abnormally elevated levels of IL-6 and TNF-αin kidney.4.Effect of Mangiferin on Renal Function in SHRsThe expression levels ofβ2-MG,m Alb and BUN in kidney tissue are significantly increased in the Model group compared with the Control group（P<0.01 or P<0.05）.Compared with the Model group,the expression level ofβ2-MG in Benazepril and high dose Mangiferin group are significantly decreased（P<0.01）;the expression level ofβ2-MG and BUN in medium dose Mangiferin group are significantly decreased（P<0.05）;the expression level ofβ2-MG in low dose Mangiferin group and the expression level of m Alb are significantly decreased（P<0.05）;the expression level of m Alb in medium and high dose Mangiferin group and the expression level of BUN in low and medium dose Mangiferin group have the tendency to lower.Mangiferin had no significant effect on SCr in urine of each group（P>0.05）.5.The Effect of MCP-1/CCR2 Signal Pathway on KidneyWestern blot results showed that the expression levels of MCP-1 and CCR2 in kidney tissue are significantly increased in the Model group compared with the Control group（P<0.01）,suggest that the modeling is successful.Compared with the Model group,the expression level of MCP-1、CCR2 are significantly decreased in Benazepril and high dose Mangiferin group（P<0.01）;the expression level of MCP-1 is significantly decreased in Medium dose Mangiferin group（P<0.05）;the expression level of CCR2 has the tendency to lower in low and medium dose Mangiferin group.These results suggest that mangiferin can down-regulate abnormally elevated levels of MCP-1 and CCR2 in kidney.Immunohistochemistry results showed that the expression levels of MCP-1 and CCR2 in kidney tissue are significantly increased in the Model group compared with the Control group（P<0.01or P<0.05）,suggest that the modeling is successful.Compared with the Model group,the expression level of MCP-1 in Benazepril and low dose Mangiferin group and the expression level of CCR2 in low dose Mangiferin group are significantly decreased（P<0.01）;the expression level of MCP-1 in high and medium dose Mangiferin group and the expression level of CCR2 in Benazepril and medium dose Mangiferin group are significantly decreased（P<0.05）;the expression level of CCR2 has the tendency to lower in high dose Mangiferin group.These results suggest that mangiferin can down-regulate abnormally elevated levels of MCP-1 and CCR2 in kidney.Real-Time PCR results showed that the expression of MCP-1 and CCR2m RNA in kidney tissue are significantly increased in the Model group compared with the Control group（P<0.01or P<0.05）,suggest that the modeling is successful.Compared with the Model group,the expression of MCP-1m RNA in Benazepril group and the expression of CCR2 m RNA in high dose Mangiferin group are significantly decreased（P<0.01）;the expression of MCP-1m RNA in low dose Mangiferin group and the expression of CCR2m RNA in Benazepril and low dose Mangiferin group are significantly decreased（P<0.05）;the expression of MCP-1m RNA in high and medium dose Mangiferin group and the expression of CCR2 m RNA in medium dose Mangiferin group have the tendency to lower.The experimental results show that mangiferin can down-regulate abnormally elevated levels of MCP-1 and CCR2 in kidney.Conclusion:Mangiferin may lower blood pressure,and effectively improve the renal damage in SHRs.The mechanism might be relate to regulate the MCP-1/CCR2 signaling pathway and inhibit immune inflammation.