| Diabetic nephropathy?DN?is one of chronic complications caused by diabetic mellitus?DM?,which is serious and harmful to human health.DN is characterized by glomerulosclerosis and proteinuria due to microangiopathy.It was reported the prevalence of DM among adults over 20 years old was 9.7%in China,and 30%70%of DM patients may suffer from DN.The occurrence and development of DN is mainly caused by the interaction between genetic susceptibility and environmental factors,such as hyperlipidemia,hypertension and hyperglycemia.Many researchers have carried out a large number of clinical and basic research on the diagnosis and treatment of DN.But there are still many problems can not be fully illuminated,and the progress in the prevention and treatment of DN is very limited.Recent studies have found that injury and apoptosis of podocytes are directly related to proteinuria.Cytotoxic T lymphocyte associated antigen 4immunoglobulin?CTLA-4-Ig?is an immunosuppressive agent generally used in the immune system diseases.Based on the latest research CTLA-4-Ig can be used to deal with podocytes in order to achieve the purpose of treatment of DN.In the first step,the rats were divided into control group,CTLA-4-Ig intervention group and diabetes group,15 in each group.The effects of CTLA-4-Ig on kidneys of diabetic rats were valued through blood biochemical indexes,renal hemodynamics,elasticity of renal parenchyma and renal pathological changes.In the second step,the rats were divided into control group,CTLA-4-Ig intervention DN group and DN group,15 in each group.Besides contents tested in the first step,the expression of CD31,CD34,podocin,nephrin and B7-1 in renal parenchyma were detected.In the third step,the rats were divided into control group,CTLA-4-Ig combined ultrasound microbubbles intervention DN group,CTLA-4-Ig intervention DN group,ultrasound microbubbles intervention DN group and DN group,15in each group.The contents tested in these groups were as same as those in the second step.Part?The role of CTLA-4-Ig in the prevention of DN in ratsObjective:To observe the role of CTLA-4-Ig in the prevention of DN in ratsMethods:Type 2 diabetic animal model were established through high-sugar and high-fat feeding combined with small dose of streptozotocin?STZ?in male Sprague Dawley?SD?rats.Diabetic rats were randomly divided into CTLA-4-Ig group and non-intervention group.The CTLA-4-Ig group were given 0.5mg kg-1 w-1 CTLA-4-Ig 8-10weeks,until the non-intervention group developed into DN.The control group was set,15in each group.Meanwhile,rats in non-intervention group and CTLA-4-Ig group received high-sugar and high-fat diets,and rats in the control group received normal diet.Then the blood biochemical indexes,detection of blood flow parameters of renal main artery through Color Doppler Ultrasound,detection of elasticity of renal parenchymal through ultrasonic elastography,HE staining of renal parenchymal and ultrastructure of podocytes by electron microscopy were all carried out.Results:Compared to the NC group,fasting blood glucose?FBG?,creatinine clearance rate?Ccr?,urine albumin excretion rate?UAER?and KW/BW in the non-intervention and CTLA-4-Ig groups were significantly increased?P<0.05?;Ccr,UAER and KW/BW in the CTLA-4-Ig group were significantly lower compared to the nonintervention group?P<0.05?.There were significant differences in peak systolic velocity?PSV?,end diastolic velocity?EDV?and mean velocity?MV?among these three groups,and these differences were ranked as nonintervention group<CTLA-4-Ig group<control group?P<0.05?;there were significant differences in systolic acceleration?SAC?,pulsatility index?PI?and resistance index?RI?among these three groups,with the differences ranked as nonintervention group>CTLA-4-Ig group>control group?P<0.05?.There were significant differences in the stiffness of kidneys among these three groups,with the differences ranked as nonintervention group>CTLA-4-Ig group>control group?P<0.05?.HE staining and transmission electron microscope observation showed that there were obvious changes in the renal parenchyma of rats in the nonintervention group and the CTLA-4-Ig group,and the changes of kidney in the nointervention group were worese than those in the CTLA-4-Ig group.Conclusion:CTLA-4-Ig could significantly inhibit the damage of renal function,the increase of resistance and decrease of velocity of renal blood flow,the increase of renal parenchyma stiffness,thickening of glomerular basement membrane,reduction of podocytes in diabetic rats.CTLA-4-Ig can significantly delay the renal injury in type 2diabetic rats,and it can surely prevent DN to some extent.Part?The role and mechanism of CTLA-4-Ig in thetreatment of DN in ratsObjective:To observe the role of CTLA-4-Ig in the treatment of DN in rats.Methods:Type 2 DN animal model were established through high-sugar and high-fat feeding combined with small dose of streptozotocin?STZ?in male Sprague Dawley?SD?rats.DN rats were randomly divided into CTLA-4-Ig group and nonintervention group.The CTLA-4-Ig group were given 0.5mg kg-1 w-1 CTLA-4-Ig 8 weeks.The control group was set,15 in each group.Meanwhile,rats in nonintervention group and CTLA-4-Ig group received high-sugar and high-fat diets,and rats in the control group received normal diet.Then the blood biochemical indexes,detection of blood flow parameters of renal main artery through Color Doppler Ultrasound,detection of elasticity of renal parenchymal through ultrasonic elastography,HE staining of renal parenchymal and ultrastructure of podocytes by electron microscopy were all carried out.The expression of CD31,CD34,podocin,nephrin and B7-1 in renal parenchyma were detected.Results:Compared to the NC group,FBG,Ccr,UAER and KW/BW in the nonintervention and CTLA-4-Ig groups were significantly increased?P<0.05?;Ccr,UAER and KW/BW in the CTLA-4-Ig group were significantly lower compared to the nonintervention group?P<0.05?.There were significant differences in PSV,EDV and mean velocity MV among these three groups,and these differences were ranked as nonintervention group<CTLA-4-Ig group<control group?P<0.05?;there were significant differences in SAC,PI and RI among these three groups,with the differences ranked as nonintervention group>CTLA-4-Ig group>control group?P<0.05?.There were significant differences in the stiffness of kidneys among these three groups,with the differences ranked as nonintervention group>CTLA-4-Ig group>control group?P<0.05?.HE staining and transmission electron microscope observation showed that there were obvious changes in the renal parenchyma of rats in the nonintervention group and the CTLA-4-Ig group,and the changes of kidney in the nointervention group were worese than those in the CTLA-4-Ig group.CD31 and CD34 expression in kidney parenchyma of rats in the CTLA-4-Ig group and nonintervention group were higher than those in the control group,the CTLA-4-Ig group and nonintervention group>the control group?P<0.05?,and there were no significant differences between the CTLA-4-Ig group and nonintervention group in CD31 and CD34 expression?P>0.05?.There were significant differences in podocin and nephrin expression in kidney parenchyma of rats among these three groups,and these differences were ranked as the control group>CTLA-4-Ig group>non-intervention group?P<0.05?.There were significant differences in B7-1 expression in kidney parenchyma of rats among these three groups,and these differences were ranked as the control group<CTLA-4-Ig group<non-intervention group?P<0.05?.Conclusion:CTLA-4-Ig could significantly improve the renal function,decrease the resistance and increase the velocity of renal blood flow,decrease the renal parenchyma stiffness,alleviate the thickening of glomerular basement membrane,and inhibit the reduction of podocytes in DN rats.The expression of CD31 and CD34 in renal parenchyma were hardly affected in the CTLA-4-Ig group compared to non-intervention group.The expression of B7-1 in renal parenchyma of the CTLA-4-Ig group was lower than that in non-intervention group.The expression of podocin and nephrin in renal parenchyma of the CTLA-4-Ig group were higher than those in non-intervention group.CTLA-4-Ig can significantly alleviate the renal injury in type 2 DN rats,and it can be used to treat DN.Part?The effects and mechanism of CTLA-4-Ig combined withultrasound microbubbles irradiation in the treatment of DN in ratsObjective:To observe the effects and mechanism of CTLA-4-Ig combined with ultrasound microbubbles irradiation in the treatment of DN in rats.Methods:Type 2 DN animal model were established through high-sugar and high-fat feeding combined with small dose of streptozotocin?STZ?in male Sprague Dawley?SD?rats.DN rats were randomly divided into CTLA-4-Ig combined with ultrasound microbubbles irradiation group,CTLA-4-Ig group,ultrasonic microbubbles radiation group and non-intervention group.The CTLA-4-Ig combined with ultrasonic microbubbles radiation group were given 0.5mg kg-1 w-1 CTLA-4-Ig and 1ml·kg-1·w-1 ultrasonic microbbles at the same time,and the kidneys in which there were microbubbles through injection received ultrasound irradiation at the same time for 8 weeks.The CTLA-4-Ig group were given 0.5mg kg-1 w-1 CTLA-4-Ig for 8 weeks.The ultrasonic microbubbles radiation group were given 1ml·kg-1·w-1 ultrasonic microbbles,and the kidneys in which there were microbubbles through injection received ultrasound irradiation for 8 weeks.The control group was set,15 in each group.Meanwhile,rats in CTLA-4-Ig combined with ultrasonic microbubbles radiation group,CTLA-4-Ig group,ultrasonic microbubbles radiation group and non-intervention group received high-sugar and high-fat diets,and rats in the control group received normal diet.Then the blood biochemical indexes,detection of blood flow parameters of renal main artery through Color Doppler Ultrasound,detection of elasticity of renal parenchymal through ultrasonic elastography,HE staining of renal parenchymal and ultrastructure of podocytes by electron microscopy were all carried out.Then the expression of CD31,CD34,podocin,nephrin and B7-1 in renal parenchyma were detected.Results:FBG of the nonintervention group,ultrasonic microbubbles radiation group,CTLA-4-Ig group and CTLA-4-Ig+ultrasonic microbubbles radiation group has no significant difference?P>0.05?,but all were higher than that of the control group?P<0.05?.Ccr,UAER and KW/BW of the nonintervention group and ultrasonic microbubbles radiation group had no significant difference?P>0.05?,and were higher than the CTLA-4-Ig group,CTLA-4-Ig+ultrasonic microbubbles radiation group and control group?P<0.05?.Ccr,UAER and KW/BW of the CTLA-4-Ig group,CTLA-4-Ig+ultrasonic microbubbles radiation group and control group had significant differences and were ranked as CTLA-4-Ig group>CTLA-4-Ig+ultrasonic microbubbles radiation group>control group?P<0.05?.PSV,EDV and MV of the nonintervention group and ultrasonic microbubbles radiation group had no significant difference?P>0.05?,and were lower than the CTLA-4-Ig group,CTLA-4-Ig+ultrasonic microbubbles radiation group and control group?P<0.05?.PSV,EDV and MV of the CTLA-4-Ig group,CTLA-4-Ig+ultrasonic microbubbles radiation group and control group had significant differences and were ranked as CTLA-4-Ig group<CTLA-4-Ig+ultrasonic microbubbles radiation group<control group?P<0.05?.SAC,PI and RI of the nonintervention group and ultrasonicmicrobubbles radiation group had no significant difference?P>0.05?,and were higher than the CTLA-4-Ig group,CTLA-4-Ig+ultrasonic microbubbles radiation group and control group?P<0.05?.SAC,PI and RI of the CTLA-4-Ig group,CTLA-4-Ig+ultrasonic microbubbles radiation group and control group had significant differences and were ranked as CTLA-4-Ig group>CTLA-4-Ig+ultrasonic microbubbles radiation group>control group?P<0.05?.There were significant differences in the stiffness of kidneys among these five groups,with the differences ranked as nonintervention group,ultrasonic microbubbles radiation group>CTLA-4-Ig group,CTLA-4-Ig+ultrasonic microbubbles radiation group>control group?P<0.05?.HE staining and transmission electron microscope observation showed that changes in the renal parenchyma of rats in the nonintervention group and ultrasonic microbubbles radiation group were worse than the CTLA-4-Ig group and CTLA-4-Ig+ultrasonic microbubbles radiation group.CD31 and CD34 expression in kidney parenchyma of rats in the nonintervention group,ultrasonic microbubbles radiation group,CTLA-4-Ig group and CTLA-4-Ig+ultrasonic microbubbles radiation group has no significant difference?P>0.05?,but all were higher than those of the control group?P<0.05?.There were significant differences in podocin and nephrin expression in kidney parenchyma of rats among these five groups,and these differences were ranked as the controlgroup>CTLA-4-Ig+ultrasonicmicrobubblesradiationgroup>CTLA-4-Ig group>ultrasonic microbubbles radiation group,non-intervention group?P<0.05?.There were significant differences in B7-1 expression in kidney parenchyma of rats among these five groups,and these differences were ranked as the control group<CTLA-4-Ig+ultrasonic microbubbles radiation group<CTLA-4-Ig group<ultrasonic microbubbles radiation group,non-intervention group?P<0.05?.Conclusion:CTLA-4-Ig and CTLA-4-Ig combined with ultrasound microbubbles could both significantly improve the renal function,decrease the resistance and increase the velocity of renal blood flow,decrease the renal parenchyma stiffness,alleviate the thickening of glomerular basement membrane,and inhibit the reduction of podocytes in DN rats.The effects of CTLA-4-Ig combined with ultrasound microbubbles was better than those of CTLA-4-Ig in treatment of DN.The expression of CD31 and CD34 in renal parenchyma were hardly affected in these two groups compared to non-intervention group.The expression of B7-1 in renal parenchyma of these two groups were both lower than that in non-intervention group.The expression of B7-1 in renal parenchyma of CTLA-4-Ig combined with ultrasound microbubbles group was lower than that of CTLA-4-Ig group.The expression of podocin and nephrin in renal parenchyma of these two groups were both higher than that in non-intervention group.The expression of podocin and nephrin in renal parenchyma of CTLA-4-Ig combined with ultrasound microbubbles group was higher than that of CTLA-4-Ig group.CTLA-4-Ig can deal with DN through protecting podocytes,ultrasonic microbubbles irradiation can help CTLA-4-Ig pass through the glomerular basement membrane and reach podocytes.CTLA-4-Ig combined with ultrasonic microbubbles irradiation was significantly better than the simple application of CTLA-4-Ig in the treatment of DN. |
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