| Objective: To survey the expression of e NOS,ET-1,IL-1 ?,MCP-1,v WF and TM in the blood of sufferers with chronic renal failure(CRF)and the intervention effect of Qingshen granule.Methods: Sixty-four sufferers with CRF damp heat syndrome(60 cases are expected to increase the number of excluded and dropped cases by less than 15%)were stochastically separated into the therapy group and the contrast group,32 cases in each group.Four cases were excluded in the trial process,and 60 cases were completed in the clinical trial.30 healthy people were selected as the normal contrast group(selected from the health Administration center of our hospital,to test the levels of e NOS,ET-1,IL-1 ?,MCP-1,v WF and TM in serum).The two groups were treated with hypotension,anemia correction,calcium and phosphorus metabolism regulation,retention enema of traditional Chinese medicine and other symptomatic therapy.In the therapy group,Qingshen granules were added,three times a day,one package at a time,for 12 weeks.After 12 weeks,the alterations of serum creatine(SCR),estimated glomerular filtration rate(e GFR),e NOS,ET-1,IL-1 ?,MCP-1,v WF and TM were observed.SPSS 23.0 was used for data analysis.Result: 1.Curative effect of Clinical disease:the total effective rate of the therapy group(80.00%),the contrast group(60.00%),there was striking discrepancy(P < 0.05).2.Curative effect of TCM Syndrome:the total effective rate of the therapy group(86.67%)and the contrast group(53.33%)were prominently different(P < 0.05).3.TCM syndrome integral:before therapy,there was no striking discrepancy between the two groups(P>0.05);after 12 weeks of successive administration,the score of the therapy group diminished prominently(P< 0.01),and the score was lesser than that of the contrast group(P<0.01);there was no prominent alteration in the score of the contrast group(P>0.05).4.Renal function(SCR,e GFR): before therapy,there was no striking discrepancy between the two groups(P>0.05);after 12 weeks of successive administration,SCR in the therapy group diminished prominently(P<0.01),EGFR increased prominently(P<0.01),compared with the contrast group,SCR level diminished,EGFR increased prominently(P<0.01);there was no prominent alteration in renal function in the contrast group between prior-therapy and post-therapy(P>0.05).5.Comparison of e NOS and ET-1 levels: 5.1 pre-therapy: e NOS level of CRF patients with damp heat syndrome was prominently lesser than that of normal group(P<0.01),ET-1 level was prominently higher than that of normal group(P<0.01),there was no striking discrepancy between the therapy group and the contrast group(P>0.05);5.2 after 12 weeks of therapy: the serum e NOS level of the two groups was prominently higher than that of the contrast group(P<0.05 or 0.01);the serum ET-1 level of the therapy group and the contrast group was prominently lesser than that of the contrast group(P<0.05 or 0.01),and the decrease of the therapy group was prominently higher than that of the contrast group(P<0.01).6.Comparison of IL-1 ? and MCP-1 levels: 6.1 pre-therapy: compared with the normal group,the levels of IL-1 ? and MCP-1 in CRF patients with damp heat syndrome were prominently higher(P<0.01).There was no striking discrepancy between the therapy group and the contrast group(P>0.05);6.2 After 12 weeks of therapy,the serum levels of IL-1 ? and MCP-1 in the two groups diminished in various degrees(P<0.05 or 0.01),and the decrease range in the therapy group was prominently greater than that in the contrast group(P<0.01).7.Comparison of TM and v WF levels: 7.1 pre-therapy: compared with the normal group,the TM and v WF levels of CRF patients with damp heat syndrome were prominently higher(P<0.01),and there was no striking discrepancy between the therapy group and the contrast group(P>0.05);7.2 after 12 weeks of therapy: the plasma TM and v WF levels of the two groups were lesser than before(P<0.05 or 0.01),and the decrease of the therapy group was prominently greater than that of the contrast group(P<0.01).8.Safety observation: after 12 weeks of drug intervention,the safety indexes of patients in the therapy group did not alteration prominently.Conclusion: 1.The levels of e NOS and ET-1 were diminished,the levels of IL-1 ?,MCP-1,TM and v WF were increased;2.Qingshen granule can regulate the level of e NOS and ET-1 in serum,reduce the level of IL-1 ?,MCP-1,TM and v WF in plasma,and improve the inflammatory injury of vascular endothelial cells;3.Qingshen granule can improve the quality of life of CRF patients with damp heat syndrome and delay the process of renal failure,One of the mechanisms is to alleviate the inflammatory injury of vascular endothelium;4.Qingshen granule was used in this study,during which there was no adverse drug reaction,which confirmed the safety and good performance of the drug.Objective: to test the expression of e NOS,ET-1 and IL-8,MCP-1 in renal tissue of 5/6 nephrectomy rats and the pathological alterations of kidney,and to observe the Influence of Qingshen granule in various doses on inflammatory injury of renal vascular endothelial cells and renal fibrosis in 5/6 nephrectomy rats.Method:72 male SD rats,2 months old,clean grade,weighing 200 ± 20 g,were selected.After one week of adaptive feeding,the rats were stochastically separated into 6 groups,12 in each group,comprising sham operation group,model group and low,medium and high dose Qingshen granules group,losartan group.Except the sham operation group,the other 60 rats were made 5/6 nephrectomy model.The rats in each group were administrated by gavage on the first day after the success of modeling.Qingshen granules were given corresponding doses of Qingshen granules aqueous solution;losartan group was given losartan potassium tablets suspension;sham operation group and model group were given normal saline gavage.4ml each time,once a day,after 12 weeks of continuous administration,urine was collected in the metabolic cage for 24 hours.Blood from abdominal aorta and left kidney were taken aseptically.Serum creatinine(SCR),blood urea nitrogen(BUN),24-hour urine protein(24-hour protein,24-hour Upro)and creatinine clearance were measured,The m RNA expression levels of e NOS and ET-1 were detected by RT-PCR,The positive expression of IL-8,monocyte chemotactic protein-1(MCP-1),e NOS and ET-1 were detected by immunohistochemistry.The expression of CD31(platelet endothelial cell adhesion molecule)+ IL-8 + MCP-1 and CD31 + e NOS + ET-1 in renal tissue was detected by immunofluorescence triple staining.The pathological changes of renal tissue were observed by HE,Masson and PAS staining under light microscope.Result:1.Renal function: 1.1 blood SCR,bun and 24 h Upro: the levels of SCR,bun and 24 h Upro in model group and drug intervention group were prominently higher than those in sham operation group(P<0.01);the levels of SCR,bun and 24 h Upro in therapy group were prominently higher than those in sham operation group(P<0.01)contrast with the model group,the Upro level diminished(P<0.01 or 0.05);contrast with different dose groups of Qingshen granules,the above indexes diminished most in the high dose group(P<0.01);Comtrast with losartan group,the above indexes in high dose group of Qingshen granules were all diminished(P<0.05),and there was no striking discrepancy between middle and low dose group of Qingshen granules(P>0.05).1.2CCR: contrast with the sham operation group,the CCR of the model group and the drug intervention group diminished prominently(P<0.01);contrast with the model group,the CCR of the drug intervention group ascended prominently(P<0.01);contrast with the dose groups of Qingshen granules,the increase of CCR of the high dose group was the biggest(P<0.01);contrast with losartan group,the CCR of high dose group of Qingshen granules ascended(P<0.05),and there was no striking discrepancy between middle and low dose group of Qingshen granules(P>0.05).2.Plasma TM and v WF: contrast with sham operation group,the levels of TM and v WF in model group and drug intervention group were prominently higher(P<0.01);contrast with the model group,the above indexes in each drug intervention group diminished(P<0.01 or 0.05);contrast with different dose groups of Qingshen granules,the above indexes in the high dose group diminished the most(P<0.01);contrast with losartan group,the plasma TM and v WF levels in the high dose group of Qingshen granules diminished(P<0.05);there was no striking discrepancy between the middle and low dose groups of Qingshen granules(P > 0.05).3.Expression of e NOSm RNA and ET-1 m RNA in renal tissue(RT-PCR): 3.1contrast with sham operation group,the expression of e NOSm RNA in kidney of rats in model group and drug intervention group diminished prominently(P<0.01);the expression of e NOSm RNA in rats in drug intervention group ascended in different degrees contrast with model group(P<0.01);contrast with Different dose groups of Qingshen granules,the increase of e NOSm RNA in high dose group was the biggest(P < 0.01);contrast with losartan group,the expression of e NOSm RNA in kidney of high dose group of Qingshen granules was ascended(P<0.05),but there was no striking discrepancy between middle and low dose group of Qingshen granules(P>0.05).3.2contrast with sham operation group,the expression of ET-1 m RNA in kidney of rats in model group was prominently higher than that in drug intervention group(P<0.01);the expression of ET-1 m RNA in kidney of rats in drug intervention group was prominently lower than that in model group(P<0.01);contrast with Different dose groups of Qingshen granules,the decrease of ET-1 m RNA in high dose group was the biggest(P<0.01);contrast with losartan group,the expression of ET-1 m RNA in kidney of high dose group was diminished(P<0.05).but there was no striking discrepancy between middle and low dose group of Qingshen granules(P>0.05).4.Immunohistochemical staining of e NOS,ET-1,IL-8 and MCP-1 in renal tissue: 4.1 e NOS: in the sham operation group,brown granules were found in glomeruli and some renal tubules;in the model group,the deposition was prominently reduced;contrast with the model group,the deposition of brown granules was ascended in different degrees in each drug intervention group,with the highest deposition in the high dose group of Qingshen granules,followed by that in the Losartan group.4.2 ET-1,IL-8,MCP-1: in the sham operation group,brown granules were found in glomeruli and some renal tubules;in the model group,the deposition was prominently ascended;contrast with the model group,brown granules were diminished in different degrees in each drug intervention group,the deposition in the high dose group of Qingshen granules was the least,followed by that in the Losartan group.5.The average density of e NOS,ET-1,IL-8,MCP-1 in renal tissue: 5.1 e NOS: The mean density of e NOS in renal tissue of rats in model group and drug intervention group was prominently lesser than that in sham operation group(P<0.01);contrast with model group,the average density of e NOS in different dose groups of Qingshen granules and losartan group ascended prominently(P<0.01);.contrast with different dose groups of Qingshen granules,the increase of e NOS in high dose group was the biggest(P<0.01);contrast with losartan group,Qingshen granules ascended prominently(P<0.01)The mean density of e NOS ascended in high dose group(P< 0.05),but there was no striking discrepancy between the middle and low dose group(P >0.05).5.2 ET-1,IL-8 and MCP-1: The average density of ET-1,IL-8 and MCP-1 in renal tissue of model group and drug intervention group were prominently higher than that of sham operation group(P < 0.01);the above indexes of Qingshen granule and losartan group diminished prominently(P< 0.01 or 0.05);contrast with losartan group,the high dose group diminished most prominently(P<0.01);contrast with losartan group,There was no striking discrepancy between the two groups(P> 0.05).6.Expression of CD31+e NOS+ET-1,CD31+IL-8+MCP-1 in renal tissue 6.1 CD31+e NOS+ET-1: CD31 showed bright green fluorescence in the sham operation group,diminished in different degrees in the model group and each drug intervention group;e NOS and ET-1 were expressed in the sham operation group,showed orange and red fluorescence respectively;e NOS was prominently diminished in the model group;e NOS expression in each drug intervention group was enhanced in different degrees contrast with the model group,and the most significant expression was in the high dose group of Qingshen granules The expression of ET-1 in the model group was prominently enhanced,showing bright red fluorescence;the expression of ET-1 in the drug intervention group was weaker than that in the model group,and the expression in the high dose group of Qingshen granules was the weakest.6.2 CD31+IL-8+MCP-1: CD31 showed bright green fluorescence in the sham operation group,diminished in different degrees in the model group and the drug intervention group;IL-8 and MCP-1 were expressed in the sham operation group,showed orange and red fluorescence respectively;the expression of the two in the model group was prominently enhanced,showed bright orange and red fluorescence;the expression of the two drug intervention groups was weakened in different degrees,among which the high dose group of Qingshen granules was used as the table The weakest. 7.Pathological changes of kidney: no obvious pathological changes were found in the renal tissue of rats in the sham operation group;incomplete glomerular structure,partial glomerulosclerosis,dilatation of renal tubule lumen and vacuolar degeneration of epithelial cells were found in the renal tissue of rats in the model group,even atrophy,necrosis and abscission were found,the area of renal interstitium was widened,a large number of inflammatory cell infiltration and distribution of blue stained collagen fibers were found;contrast with the model group,The above indexes of each drug intervention group were alleviated in the drug intervention group,especially in the high dose group and losartan group.Conclusion: 1.5/6 nephrectomy rats had imbalance of endothelial active factor expression and ascended of inflammatory factor expression;2.Qingshen granule can prominently reduce the levels of SCR,bun and 24-hour Upro in the blood of 5 / 6 nephrectomy rats,increase the level of CCR and delay the process of renal function damage;3.Qingshen granule can reduce the pathological damage of 5 / 6 nephrectomy rats;4.Qingshen granule can inhibit the expression of IL-8 and MCP-1 by regulating the balance between e NOS and ET-1,so as to reduce the inflammatory injury of vascular endothelium and play the role of anti renal fibrosis. |
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