| ObjectiveBased on the literature of clinical therapy related on acupuncture and moxibustion for Diabetic Nephropathy(DN),the method of data mining was used to systematically summarize the distribution characteristics,compatibility rules and core acupoints of acupuncture and moxibustion in the treatment of DN,and visualized them in a visual form to provide the basis for further optimizing and standardizing the acupuncture treatment plan for DN.After the core prescription was clarified,Electroacupuncture(EA)was used to intervene in DN model rats,and the effects of EA on DN were investigated by observing the general indexes,renal function indexes,pathological structures of kidneys and podocytes,autophagy expression and insulin resistance,and explore its underlying mechanisms.Method1.Literature research: Retrieve the literature records of acupuncture and its related therapies in the treatment of DN in major Chinese and English databases,screen out the effective literature of clinical research types through software such as End Note and CNKI Research,and extract the main information such as treatment methods,selected acupoints and observations indicators in the literature.and Microsoft Office Excel 2011 was used to record the information and establish a database,thedescriptive analysis was carried out on the frequency,meridian,division of acupoints and the attributes of specific acupoints in the prescription.SPSSModeler18.0 and SPSS22.0statistical software were used to conduct association rule analysis,cluster analysis and factor analysis to deeply explore the core prescriptions and internal laws of acupuncture and treatment of DN.At the same time,the efficacy indicators were also descriptivelyanalyzed to understand the current evaluation methods and efficacy of acupuncture in the treatment of DN,objectively recognize its deficiencies and make improvements.2.Experimental study: 70 SPF healthy male Wistar rats with a body weight of about(180±20)g were fed adaptively for 1 week,10 of them were randomly selected as normal group and fed with ordinary diet,and the remaining rats were fed with high-fat diet for 4 weeks later,high-fat fed rats were given intraperitoneal injection of streptozotocin(STZ)to establish DN rat model.Rats with successful modeling were randomly divided into model group,electroacupuncture(EA)group,autophagy inhibitor(3-MA)??group,electroacupuncture plus autophagy inhibitor(EA+3-MA)??group,10 rats in each group.After the grouping,all rats continued the original feeding,Rats in EA group received electroacupuncture treatmentat acupoint BL23 and ST36,the electrodes were connected on the same side: that is,each pair of electrode leads was connected to BL23 and ST36 on the same side,and both sides were stimulated at the same time.Parametersettings:Equal-amplitude sparse-dense wave,frequency 2/15Hz(sparse wave: 2Hz,dense wave: 15Hz),current intensity 1m A,each treatment for 20 minonce a day,5 days a week for6 consecutive weeks;The autophagy inhibitor group was given intraperitoneal injection of 3-MA at a concentration of 1 m M and a dose of 15 mg/kg,5 times a week for 6 consecutive weeks;the EA+3-MA group was given the above two methods at the same time;the normal group and the model group were only given the same Time capture and fixation without any other intervention.At 0,1,3,and 6 weeks after the intervention,the body weight,24h-UV,24h-UP,and FBG levels of the rats in the normal group,model group,and electroacupuncture group were detected.After the intervention,all rats were fasted for 12 hours,and blood was collected from the heart to determine the serum BUN and Scr levels of the three groups of rats.After the rats in each group were sacrificed,the kidney tissue was quickly taken out for sample collection and processing for testing.PAS staining was used to observe the pathological manifestations of kidney tissue,and transmission electron microscope was used to observe the ultrastructure and mitochondrial morphology of podocytes.Western blotting was used to observe the expression of renal autophagy proteins(Beclin-1,LC3-II/I,p62),the phosphorylation levels of AKT/m TORC1/ULK1 signaling molecules,the podocyte proteins(Nephrin,Podocin)and IRS-1,GLUT1 and PINK1protein;the Immunofluorescence method was used to detect the co-localization expression intensity of Nephrin,LC3 II and GLUT1 proteins in podocytes.Results1.Literature research: A total of 118 literatures were included in this study,with a total of 112 prescriptions,of which a total of 40 acupuncture prescriptions were at most.A total of 69 acupoints were used,and the total usage frequency was 618.Among them,the most frequently used acupoint was BL23(87 times in total,accounting for 87/618),the bladder meridian was used most frequently(179 times in total,accounting for 179/618),the lower extremities were most widely used(263 times in total,accounting for263/618),and the classification of specific acupoints showed that the Back-Shu points were most frequently used(146 times in total,accounting for 146/618);The analysis of association rules showed that: among all the prescriptions,the two-points have the highest association support for ST36-BL23(82.86%);Clustering and factor analysis showed that5 effective clusters were formedfrom 17 high-frequency acupoints,and 6 greatest common factors were extracted.2.Experimental research:1.Effects of electroacupuncture on body weight,urine volume and urine protein,fasting blood glucose and renal function indexes of DN rats.(1)Results of body weight of rats in each group: Before intervention,there was no significant difference in body weight between normal group,model group and EA group(P>0.05).After the intervention,the body weight of the normal rats increased gradually,while the rats in the model group decreased gradually.The body weight of the rats in the model group was significantly lower than that of the normal group in each measurement period(P<0.01);after 1 week of intervention,the body weight of the rats in the EA group was significantly higher than the model group(P<0.01);after 3 weeks of intervention,the body weight of the rats in the EA group was still higher than that in the model group,but there was no statistical significance(P>0.05).(2)Results of 24-hour urine volume and urine protein of rats in each group:Before intervention,the urine volume and urine protein content of rats in the model group were significantly higher than those in the normal group(P<0.01),but no significant differencebetween EAgroup and model group(P>0.05).After the intervention,the urine volume and urine protein content of the rats in the model group were significantly higher than those in the normal group at each detection time(P<0.01);1 week and 3 weeks after the intervention,the urine volume of the rats in the electroacupuncture group was significantly lower than that in the model group(P<0.01),but no significant difference in the urine protein content(P>0.05);6 weeks after the intervention,the urinary protein content of the rats in the EA group was significantly lower than that in the model group(P<0.01),but no significant difference in the urine volume(P>0.05).(3)Results of fasting blood glucose of rats in each group: Before intervention,the fasting blood glucose level of rats in model group was significantly higher than that in normal group(P<0.01),and there was no significant difference between rats in EA group and model group(P>0.05).After the intervention,the fasting blood glucose of the rats in the model group was significantly higher than that of the normal group at each detection time(P<0.01);1 week and 3 weeks after the intervention,there was no significant difference in the blood glucose level between the EA group and the model group(P>0.05).),but 6 weeks after the intervention,the fasting blood glucose of the rats in the EA group was lower than that in the model group(P<0.05).(4)Results of renal function indexes of rats in each group: After intervention,the blood BUN and Scr levels of rats in the model group were significantly higher than those in the normal group(P<0.01),and the levels of BUN and Scr in the EAgroup were significantly lower than those in the model group(P<0.01,P<0.05)2.The effect of electroacupuncture on the renal pathological damage and autophagy expression in DN rats.(1)The morphology of the kidneys of the rats in each group:PAS staining showed that the structure of the glomerulus in the normal group was clear,the shape of the renal tubules was normal,the cells were closely connected,the cytoplasm was uniform and abundant,and there was no cell degeneration and inflammatory cell infiltration.In the model group,the glomerular volume of the rats increased,the extracellular matrix deposition in the glomerulus increased,and the cells proliferated slightly.The glomerular volume of the rats in the electroacupuncture group was decreased,and the cell proliferation was alleviated.Compared with the model group,the glomerular volume of the rats in the 3-MA group became larger,the deposition of extracellular matrix increased significantly,and inflammatory cell infiltration was seen in the interstitial area.Compared with the 3-MA group,the glomerular volume of the rats in the electroacupuncture+3-MA group was reduced,and the deposition of extracellular matrix was reduced.(2)The expression levels of Beclin-1,LC3 II,LC3-II/LC3-I and p62 in each group: Compared with the normal group,the expression levels of Beclin-1,LC3-II and the ratio of LC3-II/LC3-I in the kidneys of the model group rats was significantly decreased(P<0.01),and the expression of p62 protein was significantly increased(P<0.01).The expression of Beclin-1,LC3-II and the ratio of LC3-II/LC3-I in the EA group were significantly higher than those in the model group and 3-MA group(P<0.05,P<0.01),the expression of p62 protein was lower than that in the model group and 3-MA group(P<0.01).the expression levels of Beclin-1,LC3-II and The ratio of LC3-II/LC3-I in EA+3-MA group was significantly higher than that in the 3-MA group(P<0.01),and the expression of p62 protein was lower than that in the 3-MA group(P<0.01).The expression of LC3-II and the ratio of LC3-II/LC3-I in EA+3-MA group was significantly lower than that in the EA group(P<0.01=.(3)The phosphorylation levels of p-AKT,p-mTOR and p-ULK1 in each group: Compared with the normal group,the expression levels of p-AKT and p-m TOR in the kidneys of the model group were significantly increased(P <0.01),and the expression of p-ULK1 was significantly decreased(P < 0.01).The expressions of p-AKT and p-m TOR in the EA group were significantly lower than those in the model group and 3-MA group(P<0.01,P<0.05),and the expression of p-ULK1 was higher than that in the model group and 3-MA group(P<0.01).The expressions of p-AKT and p-m TOR in the EA+3-MA group were significantly lower than those in the 3-MA group(P<0.01),and the expression of p-ULK1 was higher than that in the 3-MA group(P<0.01,P<0.05).The expression of p-AKT in the EA+3-MA group was significantly higher than that in the EA group(P<0.05),and the expression of p-ULK1 was lower than that in the EA group(P<0.01).3.Effects of electroacupuncture on podocyte injury and insulin resistance in DN rats(1)The ultrastructural changes of rat podocytes in each group:the number and morphological structure of podocytes in the normal group were normal,the basement membrane and mitochondrial structure in the podocytes was clearly visible,and no change such as swelling and wrinkling was seen;The number of foot processes of the rats in the model group decreased,and the foot processes were partially fused or detached.Part of the basement membrane showed localized thickening,mitochondrial swelling increased,the structure of the inner crest was blurred,and the particles increased;The basement membrane was relatively reduced,the number of foot processes was relatively increased,the swelling of mitochondria was reduced,and the morphological structure was clearer than that in the model group;the foot process shedding was further aggravated in the 3-MA group,the basement membrane was localized and thickened,the swelling of mitochondria was more obvious,and the internal structure was unclear;The damage of podocytes and mitochondria in the EA+3-MA group was less than that in the 3-MA group.(2)The effect of electroacupuncture on the protein expression levels of Nephrin and Podocin in podocytes of DN rats: Compared with the normal group,the expression levels of Nephrin and Podocin in the model group were significantly decreased(P<0.01).The expressions of Nephrin and Podocin in the EA group were significantly higher than in the model group and 3-MA group(P<0.01,P<0.05).The expression of Nephrin in the EA+3-MA group was significantly higher than that in the 3-MA group(P<0.01),and the expression of Podocin was also significantly higher than that in the 3-MA group(P<0.01)but lower than that in the EA group(P<0.05).(3)Effects of electroacupuncture on the expression of IRS-1,GLUT4 and PINK1 proteins in the kidneys of DN rats: Compared with the normal group,the expressions of IRS-1 and GLUT4 proteins in the model group were significantly decreased(P<0.01),the expression of PINK1 protein was significantly increased(P<0.01).The expression of IRS-1 and GLUT4 in the EA group was significantly higher than that in the model group and 3-MA group(P<0.01),and the expression of PINK1 was significantly lower than that in the model group and 3-MA group(P<0.01).The expression of IRS-1 in the EA+3-MA group was significantly higher than that in the 3-MA group(P<0.01)but lower than that in the EA group(P<0.01),and the expression of GLUT4 was also significantly higher than that in the 3-MA group(P<0.01),The expression of PINK1 was significantly lower than that in the 3-MA group(P<0.01).(4)Co-localized expression of Nephrin,LC3 II and GLUT4 proteins in renal podocytes of rats in each group: high magnification(400×)field of view showed that Nephrin protein was localized and expressed in glomerular podocytes and correlated with the results of WB detection.Consistently,co-localization showed that LC3 II and GLUT4 proteins were also expressed to varying degrees in podocytes.Statistical analysis of the mean immunofluorescence intensity showed that the expression intensity of LC3 II and GLUT4 podocytes in the model group was significantly lower than that in the normal group(P<0.01),and the expression in the EA group was significantly higher than that in the model group(P<0.01,P<0.05),The expression of LC3 II and GLUT4 in the 3-MA group was lower than that in the model group(P<0.05),and the expressions of LC3 II and GLUT4 were significantly increased after further electroacupuncture intervention compared with the 3-MA group(P<0.01).Conclusion1.The core acupoints used in acupuncture treatment for DN are ST36 and BL23,The overall acupoint selection rule reflects the importance of Zang-fuand meridian dialectical thought in the treatment of DN.2.Electroacupuncture can delay the weight loss of DN rats,reduce urine volume and urinary protein excretion,reduce the fasting blood sugar,and improve renal function.3.Electroacupuncture can up-regulate the expression of autophagy in kidney of DN through the AKT/m TOR/ULK1 signaling pathway,and reduce the pathological damage of the kidneys.4.Electroacupuncture can promote the clearance of damaged mitochondria by activating autophagy in DN podocytes,thereby reducing insulin resistance and improving podocyte damage. |
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