| Objective: Diabetic Nephropathy (DN) has been the second cause ofdeath in patients with diabetes. It is also the main cause of end-stage renalfailure(ESRF), but the mechanism of DN is still not known clearly by usnowadays. So how to prevent and treat it effectively in the early stage of DN isan extremely urgent subject concerned by studies at home and abroad. Recentstudies demonstrated that mitochondria damage, especially mitochondrialoxidative stress and cell apoptosis maybe involved in the mechanism of DN.Drp1,mitochondrial division related protein, has been proven to beupregulated in a variety of cell types in high sugar environment,which wasalso accompanied by mitochondrial ROS synthesis increasing and cellapoptosis. So we constructed diabetic animal model by STZ intraperitonealinjection to observe the expression change of mitochondrial division relatedprotein Drp1, mitochondrial oxidative stress index Mn-SOD, GSH andapoptosis related indicators BAX, BCL2in diabetic mice kidney tissue. Wealso gave resveratrol(RSV)intervention treatment,which is a new kind ofnatural antioxidant,to explore whether RSV has beneficial effect on diabeticmice kidney and try to explain its mechanism. Through this experiment weprovided ideas and experimental basis for seeking new treatment target andeffective drugs of DN on clinical.Methods:Choose90healthy male CD-1mice.Execute right nephrectomyunder chloral hydrate anesthesia after one week adaptability feed. Then dividethem into two groups randomly after two weeks:Control group(n=40) andModel group(n=48). Mice in model group are established a singleintraperitoneal injection of STZ(140mg/kg) and mice in control group areestablished by a single intraperitoneal injection of Citric acid buffer equally.The DM model was considered to be successful when blood glucose≥16.7 mmol/L2times continuously after72hours. Divide Control mice into twogroups randomly: Normal control group(group A)(n=20)and Normal+RSV(group B)(n=20); Divide successful model mice into two groups randomly:Diabetes group(group C)(n=24)and Diabetes+RSV treatment group(groupD)(n=24). Group B and D are given RSV carboxymethyl cellulose solution5mg/kg/d through lavage intervention and group A, C are given equivalentcarboxymethyl cellulose solution everyday. Mice eat and drink freely and donot use any other drugs during the experiment. Choose6mice from eachgroup randomly at the4th,8th,12th weekend. Collect24h urine, blood andrenal tissue samples. Test body weight(BW), kidney weight(KW), bloodglucose(BG),24h urinary protein(24hUP), serum cholesterol(TC),triglyceride(TG), serum creatinine(Scr), urea, high density lipoprotein(HDL),low density lipoprotein(LDL), GSH content and renal cortical Mn-SODactivity. Renal pathology change is observed by HE and PAS staining. Theexpressions of Drp1, BAX, BCL2in kidney are detected byimmunohistochemistry and Western-Blot.All the data are analyzed bySPSS13.0statistics software, P<0.05was considered to be statisticalsignificant different.Results:14-5days After STZ injection, mice in Group C, D appeared obviouspolydipsia, polyphagia, polyuria, weight loss, listlessness,and appearedabdominal obesity gradually.The situations above in group D recoveredpatially after receiving RSV.(Fig.1.2)2BW in Group C, D was significantly lower than groupA. BW in GroupD was higher than Group C after receiving8weeks RSV therapy(P <0.05),but it was still lower than groupA (P <0.05). During the whole experimentperiod, KW/BW in Group C was the highest, group D followed, groupA, Bwas the lowest(Fig.3.4;Table1).3There was no statistic difference in BG,24hUP, TC, TG, HDL,LDL,Scr, Urea between Group A and B during the whole experiment period.BG in Group C, D was significantly higher than group A (P <0.05). There wes no statistics difference between Group C and D during the wholeexperiment period (P>0.05);24hUP in Group C raised obviously (P<0.05),24hUP in Group D recovered patially after receiving RSV therapy, butit was still significantly higher than group A during the experiment period (P <0.05); TC,TG in GroupC, D began to raise significantly until the8th weekendand the level in Group C was much higher(P <0.05). LDL in GroupC, D alsobegan to raise significantly at the end of8th week, but LDL level in groupDdid not decreased significantly than Group C until the12th weekend (P <0.05); HDL level in Group C decreased significantly at the12th weekend (P <0.05) and that in group D was still on a normal level (P>0.05);Scr, Urea levelin Group C, Dwas significantly higher than group A after DM model wasmade successfully (P <0.05),Scr in Group D decreased significantly at theend of the4th,8th,12th week (P <0.05) and Urea in Group D recovered tonormal at the end of the8th,12th week.(Fig.5-12;Table2-5)4Compared with group A, serum GSH content, renal cortical Mn-SODvigor in Group B were higher at the same period, but there was no statisticallysignificant difference (P>0.05). And that in Group C decreased significantly(P <0.05). After application of RSV, serum GSH content and renal Mn-SOD vigor in Group D recovered in different degrees at the4th,8th,12thweekend (P <0.05)(Fig.13.14;Table6).5HE and PAS staining showed that glomerular volumn and glomerularmesangial area was much more broaden in Group C, basement membrane wasthicker, matrix increasing and mesangial cell proliferation were alsoobvious.Renal tubular lumen in Group C expansed and part of the brushborder falled off. All the changes above was less severe in GroupD(Fig.15-18).6Immunohistochemistry and Western-Blot showed that the expression ofDrp1, BAX was significant increased and BCL2was significant decreased inGroup C at the4th,8th and12th weekend (P<0.05).The changes above wasrecovered obviously in Group D at the same time(P<0.05).The expression ofBAX in Group B was lower than group A at the end of8th,12th week(P<0.05).(Fig.19-27;Table7.8)Conclusion:1The Drp1expression was upregulated in renal tissue from diabeticmice.The renal cortical Mn-SOD activity and serum GSH content decreased.At the same time, renal BAX expression was upregulated and BCL2expression was downregulated.All the changes above were accompanied by24h UP increasing, renal pathological change aggravating,kidney functiondeclining and lipid metabolism disorder. All above indicating thatmitochondrial division process, mitochondrial oxidative stress and cellapoptosis may be involved in the occurrence and development of diabeticnephropathy and Drp1may be a new target of DN treatment.2The general condition of diabetes mice can be improved by RSV andthe changes of24hours urinary protein quantity, renal pathology, renalfunction, lipid metabolism disorders can also be recovered in different degrees.At the same time, renal cortical Mn-SOD activity, serum GSH content andBCL2expression level increased and Drp1,BAX expression decreased. ButRSV had no effect on BG.All above illustrate RSV may play renal beneficialeffect by relieving mitochondrial injury, improving mitochondrial antioxidantstress ability and reducing renal cell apoptosis. |
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