Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Wildlife Diseases > Animals used for experiments

Protective Effect of Beta-Casomorphin-7on Renal Injury of Rats with Diabetic Nephropathy Induced by Stz and Its Mechanism Study

Author ZhangWei
Tutor ZhangYuanShu
School Nanjing Agricultural College
Course Basic Veterinary
Keywords β-casomorphins-7 Diabetic nephropathy Oxidative stress Tubularepithelial-myofibroblast transdifferentiation RAS SD rat NRK-52E
CLC S858.91
Type PhD thesis
Year 2013
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In this study, effects of beta-casomorphin-7on renal funtion, morphological changes, oxidative stress, tubular epithelial-myofibroblast transdifferentiation (TEMT) and renin-angiotensin system (RAS) were first evaluated on the rats with diabetic nephropathy which were induced by STZ. The damage responses of AngⅡ on renal tubular epithelial cells were detected in vitro and studied the mechanism of beta-casomorphin-7on renal injury of rats. The research included four parts:1The protective effect of β-casomorphin-7on renal injury on rats with diabetic nephropathyThis study was designed to investigate the protective effect of P-casomorphin-7on renal injury of streptozotocin-induced diabetic rats. Eight SD rats were selected as the control group in40rats,32rats were induced by intraperitoneal injection of STZ (60mg/kg body weight) to make the diabetic model. The diabetic rats were divided into model group which were given intragastric administration of saline and β-casomorphins-7group (β-CM-7group) which were given intragastric administration of7.5×10-6mol/kg body weight β-casomorphin-7. The food intake, water intake and the body weight were measured every day. The fasting blood glucose was measured every5days. All rats were sacrificed after30days of feeding with or without β-casomorphin-7, the serum and the kidney were collected and8rats were selected from every group for the following experiments:1) The urine glucose, urine protein, renal function, serum insulin and glucogan were detected.2) The pathological and fibrosis of kidney were detected by HE, MASSON and Sirius red.3) The mRNA and protein expression of type Ⅰ and IVcollagen in the kidney of rats were detected. Results:1)27rats were successfully induced into diabetic model in32rats and successful rate is84%. During the experiment, the water intake and food intake were increased significantly, the body weight was decreased significantly, the fasting blood glucose had been higher than16.7mmol/L during the experiment, the content of insulin was decreased (P<0.05) and the content of glucagon (P<0.05) was increased in the rats of model group. Compared with the rats of model group, β-CM-7decreased the food intake, water intake and fasting blood glucose, increased the body weight.2) After30days treatment, the urine glucose, urine protein, serum creatinine, blood urea nitrogen (P<0.05) and renal index (P<0.01) in the rats of model group were significantly increased and the kidney had obvious histopathological changes, β-casomorphin-7decreased the above index and renal histopathological changes.3) There were excessive deposition of collagen fibers in glomerulus and tubulointerstitial in the rats of model group, the mRNA and protein of type Ⅰ and Ⅳ collagen were significantly increased in the kidney of model group (P<0.05), β-casomorphin-7reduced deposition of collagen fibers in kidney of diabetic rats. Conclusions:Intraperitoneal injection of STZ could been used for inducing the diabetic nephropathy which first performed that more water intake, more food intake and loss of weight, and after30days the rats in model group performed glycosuria, proteinuria, renal hypertrophy, renal dysfuction and renal fibrosis. β-casomorphin-7had a protective effect on the kidney of rats with diabetic nephropathy induced by STZ.2The possible mechanism of β-CM-7on renal injury of rats with diabetic nephropathyWe had found that β-CM-7had the protective effect on renal injury of rats with diabetic nephropathy in the previous experiment and this study explored the possible mechanism from the oxidative stress and renal tubular epithelial-myofibroblast transdifferentiation (TEMT). All rats were treated the same as the first chapter and the kidney were collected for the following experiments:1) The contents of MDA and H2O2, the SOD, GPx and T-AOC were determined by biochemical assay.2) Expression of α-smooth muscle actin (α-SMA), E-cadherin, vimentin and cytokeratinl9(CK19) mRNA in the rats were detected by real-time PCR. The protein expression and distribution of α-SMA and E-cadherin were detected by Western-blot and immunohistochemistry. Results:1) The activity of SOD and GPx (P<0.01) were significant lower and the contents of MDA (P<0.01) were significant higher than the control group in the kidney. β-CM-7increased the SOD (P<0.01), GPx (P<0.05), and decreased the MDA (P<0.05) of rats with diabetic nephropathy. The contents of T-AOC and H2O2in β-CM-7group were between control group and model group, but there is no statistics significance (P>0.05).2) Compared with the control group, the mRNA expression of α-SMA (P<0.01) and vimentin (P<0.05) increased significantly and the expression of E-cadherin (P<0.05) and CK19(P<0.01) mRNA were reduced in model group. β-CM-7reduced the mRNA expression of α-SMA and vimentin (P<0.05), increase the mRNA expression of E-cadherin (P<0.05) and CK19(P<0.01).3) The distribution of E-cadherin was decreased significantly and the α-SMA was increased significantly, renal tubular epithelial cell appeared tubular epithelial-myofibroblast transdifferentiation and β-casomorphin-7antagonized all these changes. The protein expression of E-cadherin and α-SMA were consistent with the expression of mRNA. Conclusion:β-CM-7reduced renal oxidative stress and renal tubular epithelial-myofibroblast transdifferentiation which may be one of the protective mechanisms of β-CM-7on renal injury3The effect of β-casomorphin-7on RAS of kidney in the rats with diabetic nephropathyWe had found that β-CM-7had the protective effect on renal injury of rats with diabetic nephropathy which was associated with enhancing the antioxidant capacity and inhibiting the renal tubular epithelial-myofibroblast transdifferentiation (TEMT) in the previous experiment. To further explore the new mechanism of beta-casomorphin-7on the renal injury of rats with diabetic nephropathy from the renin-angiotensin system (RAS) in this experiment. All rats were treated the same as the first chapter and the kidney were collected for the following experiments:The content of AngⅡin the kidney of rats were detected by radioimmunoassay. Expression of ACE, AT1, ACE2and MAS mRNA in the kidney of rats were detected by real-time PCR. The protein expression of ACE and ACE2were detected by Western-blqt. Results:1) Compared with control group, content of AngⅡ (P<0.01) was significantly increased in the kidney of rats in model group (P<0.01). β-casomorphin-7decreased content of AngⅡ (P=0.035), but it was also higher than the control group.2) Compared with control group, the expression of ACE2mRNA and protein (P<0.05) was decreased significantly, the expression of ACE protein and mRNA, the AT1mRNA were significantly increased (P<0.05), the expression of MAS mRNA was decreased (P=0.09) which did not have statistical significance in the kidney of model group. β-CM-7could increased the expression of ACE2mRNA and protein(P<0.05), decrease the expression of ACE mRNA and protein, AT1mRNA (P<0.05) and MAS mRNA (P=0.07) in the kidney significantly. The ratio of ACE/ACE2mRNA and ACE/ACE2protein was higher in the kidney of the model group than the control group (P<0.01), β-CM-7decreased the ratio of ACE/ACE2(P<0.05). Conclusion:The RAS were activated in the kidney of rats with diabetic nephropathy and the axis of ACE-AngⅡ-AT1was dominant. The high contents of Angll involved in the renal injury and fibrosis. β-casomorphin-7protected the renal injury and fibrosis of rats with diabetic nephropathy through increasing the expression of ACE2, decreasing the content of AngⅡ and the expression of AT1. AngⅡ was a key factor in renal injury of rats with diabetic nephropathy and it may be a key pathway which β-CM-7protected the renal injury of rats with diabetic nephropathy.4The effect of β-casomorphin-7on the oxidative stress and TEMT of cells treated with AngⅡ in vitroThe research is further to study the effect and mechanism of β-casomorphin-7on the oxidative stress and tubular epithelial-myofibroblast transdifferentiation (TEMT) of NRK-52E cells treated with AngⅡ. NRK-52E cells were cultured in the DMEM medium and the cells were divided into four group:control group, AngⅡ group, β-CM-7group and AT1receptor blocker group (ARB group). The cells of the AngⅡ group were treated with lnM AngⅡ in DMEM medium, the cells of β-CM-7group were treated with lnM AngⅡ and10-5M β-casomorphin-7in DMEM medium, the cells of ARB group were treated with lnM AngⅡ and10-5M losartan in DMEM medium and the cells of control group were cultured in DMEM medium. All cells were treated for72h and the cells and the culture medium were collected. The index of oxidative stress and antioxidant were detected by biochemical assay. The mRNA expression about TEMT, RAS and TGF-β1were detected by real-time PCR. The protein expression about TEMT and RAS were detected by western-blot and immunofluorescence. The contents of Angll or TGF-β1were detected by radioimmunoassay or ELISA. Result:1) The contents of ROS and MDA were increased (P<0.01) and the SOD and GPx were decreased (P<0.01) in the cells treated with Angll, which increased the oxidative stress of cells. P-CM-7significantly reduced the contents of ROS and MDA, increase the activity of SOD and GPx in the NRK-52E cells treated with Angll, which alleviated the oxidative stress of cells.2) The mRNA and protein expression of α-SMA (P<0.01) was increased and E-cadherin (P<0.01) was decreased in the NRK-52E cells treated with Angll, AT1receptor blocker (ARB) antagonized this changes of α-SMA and E-cadherin and β-CM-7also significantly alleviated the changes of α-SMA and E-cadherin of cells.3) The mRNA and protein expression of ACE was increased (P<0.05), ACE2was decreased (P<0.01&P<0.05)) and the content of TGF-β1was increased in the cells treated with AngⅡ. β-CM-7significantly increase the mRNA and protein expression of ACE2, decrease the mRNA and protein expression of ACE and the contents of AngⅡ and TGF-β1. Conclusion:The oxidative stress and TEMT of NRK-52E cells could be induced by AngⅡ through AT1receptor and TGF-β1. β-CM-7alleviated the oxidative stress and TEMT of NRK-52E cell treated with AngⅡ which is associated with increasing the expression of ACE2, decreasing the expression of ACE and the contents of Ang Ⅱ and TGF-β1.

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