Research On The Protective Effect And Mechanism Of Electroacupuncture Pretreatment On Kidney In Hyperglycemic Mice

ObjectiveThe kidney is the most sensitive to hyperglycemia and the earliest organ involved.Hyperglycemia can directly cause renal tissue damage,and further lead to irreversible impairment of renal function.In this study,following the academic thought of"treating non-disease"in traditional Chinese medicine,the acupoints"Zusanli"(ST36)and"Shenshu"(BL23)were selected to intervene the hyperglycemia mice induced by streptozotocin(Streptozotocin,STZ)by electroacupuncture.The effect and mechanism of electroacupuncture preconditioning on renal injury in hyperglycemic mice were explored with the instantaneous receptor potential cation channel 6(transient receptor potential cation channel 6(TRPC6)as the breakthrough point.Methods125 male C57BL/6 mice aged 4-6 weeks,weighing 18±2g,were fed adaptively for one week.The disposal of the animals in this experiment was strictly in accordance with the requirements of the document"guidance on being kind to Experimental Animals"issued by the Ministry of Science and Technology[2006].It has been approved by the Experimental Animal Ethics Committee of Hubei University of traditional Chinese Medicine(examination and approval No.:HUCMS201809011).125 mice were randomly divided into 6 groups according to the method of random number table.(1)Control group(C,N=20):normal feeding,no other intervention.(2)Model group(M,N=20):the mice were fixed in the same way as the electroacupuncture pretreatment group on the 7th day of experiment 1,and 1%STZ solution(50 mg/kg body weight/day)was injected intraperitoneally on the 8th and 12th day of the experiment.(3)Electroacupuncture pretreatment group(E,n=25):on the 7th day of experiment,electroacupuncture was performed at"ST36"and"BL23"points.0.18 mm×13 mm sterile acupuncture needle was used for acupuncture,"ST36"straight needling,depth 3 mm,"BL23"oblique needling 4 mm,ipsilateral"BL23"and"ST36"connected with a pair of electrodes of HANS-200 electroacupuncture therapeutic apparatus,using sparse and dense waves with a frequency of 2 Hz/15 Hz,gradient current.0.3 m A lasted for 5 min,0.4 m A,lasted for 5 min,0.5 m A,lasted for 20min,and lasted for 30 min,once a day for 7 days.Among them,5 rats extracted acupuncture serum for cell culture,only electroacupuncture pretreatment,no other detection.The remaining 20 mice were intraperitoneally injected with STZ solution on the 8th and 12th day of the experiment,in the same way as the model group.(4)Sham electroacupuncture group(S,n=20):in experiment 1,the fixed method and acupoint selection for 7 days were the same as electroacupuncture pretreatment group,acupuncture points were subcutaneously treated with no electricity,and the needles were retained for 30 min,once a day for 7 days.On the 8th and 12th day of the experiment,STZ solution was injected intraperitoneally in the same way as the model group.(5)Electroacupuncture preconditioning+TRPC6 agonist group(ET,n=20):Hyperforin dicyclohexylammonium salt(HP)solution(5mg/kg)was injected intraperitoneally on the 1st and 4th day of the experiment,and the other operations were the same as those in the electroacupuncture pretreatment group.(6)Model+TRPC6 agonist group(MT,n=20):HP solution was used in the same way as above,and the other operations were the same as the model group.In vitro cell experiment:HK-2 cell line was divided into three groups.(1)Normal group:cells cultured with 5mmol/L glucose.(2)High glucose group:cells cultured with 25mmol/L glucose.(3)Acupuncture serum group:cells were cultured with acupuncture serum+25mmol/L glucose.The cells of the above three groups were detected after 72 hours of culture.Detection method.The main results were as follows:(1)on the 0th and 15th day of the experiment,the body weight of mice was weighed,the blood glucose was measured,and the food intake and excretion on the same day were measured.(2)after the detection of blood glucose,part of the right kidney was examined by transmission electron microscope,and the rest of the kidney tissue protein was extracted;after the left kidney was fixed,it was embedded in paraffin and sectioned along the sagittal plane of the kidney.(3)the morphology of renal tissue was observed by HE staining,the sectional area of glomeruli and the proportion of damaged renal tubules were counted,the proliferation of Mesangial matrix in glomeruli was observed and evaluated by Masson and PAS staining,and the changes of foot process of podocytes in each group were observed by transmission electron microscope.(4)Immunohistochemistry and Western-Blot,WB were used to detect the expression of podocyte related proteins Neprin,WT1,Desmin,apoptosis related proteins Caspase 3,Bcl-2/Bax,TRPC6 protein and ERK1/2phosphorylation level in renal tissue.(5)apoptosis of renal tubular epithelial cells was detected by Tunel method.(6)the intracellular Ca²⁺level of HK-2 cells was detected by calcium imaging.Results1.Effects of electroacupuncture pretreatment of"ST36"and"BL23"on blood glucose level in hyperglycemic mice.Compared with the control group(6.83±0.43 mmol/L),the blood glucose of the model group(13.17±0.46 mmol/L)was significantly increased(P<0.0001),and the blood glucose of the electroacupuncture pretreatment group was significantly lower than that of the model group(P<0.0001).Compared with the electroacupuncture pretreatment group,the blood glucose of the electroacupuncture pretreatment+TRPC6agonist group increased(12.32±1.05mmol/L),and compared with the model group,the blood glucose of the model+TRPC6 agonist group increased(P<0.05).Compared with the control group,the food intake of the model group was significantly increased(P<0.0001),and that of the electroacupuncture pretreatment group was significantly lower than that of the model group(P<0.0001),and that of the electroacupuncture pretreatment+TRPC6agonist group was significantly higher than that of the electroacupuncture group(P<0.0l),and that of the model+TRPC6 agonist group was significantly higher than that of the model group(P<0.05).Compared with the control group,the excretion of mice in the model group increased significantly(P<0.0001).Compared with the model group,the excretion of mice in the electroacupuncture pretreatment group decreased significantly(P<0.05).The ratio of Bcl-2/Bax in the model+TRPC6 agonist group was significantly lower than that in the model group(P<0.01).5.Effects of electroacupuncture Pretreatment of"ST36"and"BL23"on TRPC6 expression and ERK Phosphorylation in Kidney of hyperglycemic mice.(1)Immunohistochemical method was used to detect the expression of TRPC6 in the kidney of mice in each group.The results showed that compared with the control group,the expression level of TRPC6 in the model group was significantly up-regulated(P<0.001),and that in the electroacupuncture pretreatment group was down-regulated compared with the model group(P<0.001).Compared with the electroacupuncture pretreatment group,the glomerular TRPC6 expression level in the electroacupuncture pretreatment+TRPC6 agonist group was up-regulated(P<0.05).Compared with the model group,the expression of TRPC6 in the glomeruli of the model+TRPC6 agonist group was up-regulated.The results of WB showed that compared with the control group,the expression of TRPC6 in the model group was significantly up-regulated,compared with the model group,the expression of TRPC6in the electroacupuncture pretreatment group was down-regulated,compared with the electroacupuncture pretreatment group,the expression of TRPC6 in the electroacupuncture pretreatment+TRPC6 agonist group was significantly up-regulated,and compared with the model group,the expression of TRPC6 in the model+TRPC6 agonist group was significantly up-regulated.The level of ERK phosphorylation in kidney tissue of mice in each group was detected by immunohistochemical WB.The results showed that compared with the control group,the phosphorylation level of ERK in the model group was significantly up-regulated,and that in the electroacupuncture pretreatment group was significantly lower than that in the model group,and that in the electroacupuncture pretreatment+TRPC6 agonist group was significantly higher than that in the electroacupuncture pretreatment group(P<0.05).Compared with the electroacupuncture pretreatment group,the ERK phosphorylation level in the electroacupuncture pretreatment+TRPC6 agonist group was significantly up-regulated(P<0.01).Compared with the model group,the level of ERK phosphorylation in the model+TRPC6 agonist group was significantly up-regulated(P<0.01).There was no significant difference in the level of JNK and P38 phosphorylation among the groups.6.Effect of acupuncture serum on the concentration of Ca²⁺in HK-2cells.The intracellular Ca²⁺concentration of HK-2 cells in each group was detected by calcium imaging.The results showed that the intracellular Ca²⁺concentration in HK-2 cells in the high glucose group was significantly higher than that in the normal group(P<0.001),and that in the HK-2 cells cultured with acupuncture serum was significantly lower than that in the high glucose group(P<0.001).ConclusionThe main results are as follows:(1)electroacupuncture pretreatment of"ST36"and"BL23"can significantly inhibit the increase of blood glucose and improve the general condition of mice induced by STZ.(2)electroacupuncture pretreatment of"ST36"and"BL23"can exert renal protective effect by reducing podocyte injury and inhibiting apoptosis of renal tubular epithelial cells.(3)the renal protective effect of electroacupuncture pretreatment of"ST36"and"BL23"may be achieved by down-regulating the expression of TRPC6 and reducing the phosphorylation of ERK.