Effects Of Danshen Injection On Renin Angiotensin System In Renal Tubules Of Diabetic Rats

PART 1:The effect of Danshen injection on the renal local renin-angiotensin system in diabetic ratsObjective:Diabetic rats were developed by fed with high-glucose and high-fat diet and intraperitoneal injection of streptozotocin(STZ).The general situation,biochemical indexes,proteinuria,renal function,and renal pathology were investigated after treatment with Danshen injection or metformin,and the expression of angiotensin?type 1 receptor(AT1)and Mas in renal of diabetic rats were detected to investigate the influence of Danshen injection on the renal local renin-angiotensin system in diabetic rats.Methods:40 SD rats(male)were treated with STZ by intraperitoneal(50 mg/kg)and high-glucose and high-fat diet to induce diabetic rats.Then randomly divided into four groups:control group,model group,Danshen group,and metformin group.Treatment with different drugs for 6 weeks,the body weight,and blood glucose were recorded each week.After six weeks of administration,the rats were placed in a clean metabolic cage to collect the urine for detecting 24-hour urinary protein.Blood samples were collected from the abdominal aorta,and the serum creatinine and the urea nitrogen were measured by biochemical instruments.After anesthesia with 10%chloral hydrate,the kidney of rats were weighted,then the samples were dehydrated and embedded in paraffin wax.Each piece of wax was cut into 5?m thick tissue sections for HE staining and immunohistochemical detection.Results:Compared with the normal group,the body weight of diabetic rats were significantly decreased,while the blood glucose,24-hour urinary protein,serum creatinine and urea nitrogen increased significantly at the 6~thh week.Danshen injection could decrease 24-hour urinary protein,serum creatinine and urea nitrogen significantly(P<0.05).HE staining of kidney tissue showed that Danshen injection could reduce the glomerular hypertrophy,the proliferation of mesangial cells,the proliferation of basement membrane and atrophy of renal tubules in diabetic rats.The AT1 and Mas mainly distributed in the renal tubules.Immunohistochemistry results showed that compared with the normal group,the expression of AT1 in the model group significantly increased and the expression of Mas was significantly reduced,Danshen injection group could significantly reduce AT1 expression and increase Mas expression.Conclusions:Danshen injection could improve renal tubular lesions and reduce 24-hour urinary protein levels in diabetic ratsSerum creatinine and urea nitrogen levels also decreased significantly after treatment with Danshen injection.Danshen injection could inhibit the expression of AT1 and increase the expression of Mas protein,indicating that Danshen injection could treat diabetic nephropathy by balancing ACE2-Ang(1-7)-Mas and ACE-AngII-AT1 axis.PART 2: Protective effect of Danshen injection on NRK-52 E cells injured by high glucose and its influence on the expression of AT1 and MasObjective: To investigate the effect of high glucose on the expression of AT1,Mas and PI3 K in NRK-52 E cells and the influence of Danshen injection.Methods:?.The concentration of Danshen injection were determined by pre-experiments,then the experiment was divided into 3 groups: normal control group(NG,5.5 mmol/L glucose);high glucose model group(HG,80 mmol/L glucose);Danshen group(240 ?g crude drug/m L Danshen + 80 mmol/L glucose).The morphology of NRK-52 E cells was observed by inverted microscope.The effect of Danshen injection on the proliferation of high glucose-injured cells was detected by MTT assay at different time(24,48,and 72 h).The expression of AT1,Mas,PI3 K and p-PI3 K proteins was detected by Western Blot or immunofluorescence.?.The experiment was divided into 4 groups: normal control group(NG,5.5 mmol/L glucose);Ang? model group(Ang?,10-5 mol/L);Danshen injection group(240 ?g crude drug /m L Danshen + 10-5 mol/L Ang?);Losartan group(10-5 mol/L Losartan +10-5 mol/L Ang?).MTT assay was used to detect the effects of Danshen injection on the cells proliferation injured by Ang?,Western Blot was used to detect the effect of PI3 K and p-PI3 K protein expression in each group.Results:?.The results of MTT indicated that(1)The proliferation of NRK-52 E cells was significantly inhibited by 80 mmol high glucose culture medium for 24 h,48 h and72h(P<0.01).(2)10-5 mol/L Ang? significantly inhibited the proliferation of NRK-52 E cells for 24 h(P<0.01).The proliferation of NRK-52 E cells inhibited by high glucose or Ang? can be reversed by Danshen injection(P<0.01 or P<0.05).?.The results of immunofluorescence showed that the expression of PI3 K and p-PI3 K protein significantly increased with high glucose treatment for 24 h compared with the normal group.Compared with the model group,Danshen injection could reduce the expression of PI3 K and p-PI3 K proteins.?.The Western blot analysis results show that:(1)High glucose treatment for 24 h could increase the expression of PI3 K,p-PI3 K,AT1 protein in NRK-52 E cells,and reduce the expression of Mas protein(P<0.01 or P<0.05)compared with the normal group.Treatment with Danshen injection significantly inhibited the expression PI3 K and p-PI3 K,and increased the expression of Mas.but has no significant effect on AT1 expression.(2)Compared with the normal group,high glucose treatment for 48 h could decrease the expression of PI3 K,p-PI3 K,Mas protein in NRK-52 E cells(P<0.01 or P<0.05),and increased the expression of AT1 protein slightly,but there was no significant difference.Compared with the model group,the expression of PI3 K,p-PI3 K,Mas protein in the cells of Danshen group increased significantly,and the expression of AT1 protein decreased significantly(P<0.01).(3)10-5 mol/L Ang? could reduce the expression of PI3 K,p-PI3 K protein in NRK-52 E cells after 24 h treatment compared with normal group.The expression of PI3 K,p-PI3 K protein in the cells of Danshen group was significantly increased(P<0.01)compared with the model group.Conclusions: High glucose could activate intracellular renin-angiotensin system by increasing the expression of AT1 and reducing the expression of Mas.Danshen injection can inhibit the expression of AT1 and increase the expression of Mas,suggesting that Danshen injection can balance ACE2-Ang(1-7)-Mas and the ACE-Ang?-AT1 pathway alleviate NRK-52 E cell damage induced by high glucose,and this effect is may related to PI3 K activation.