| Objective:To observe the expression of p38,p-p38,extracellular signal-regulated kinase 5(BMK1/ERK5)and p-ERK5 in peripheral blood mononuclear cells of patients with chronic renal failure(CRF)with damp-heat syndrome and the intervention effect of Qingshen Granule on these indexes.Methods:70 patients with chronic renal failure were randomly divided into treatment group and control group,35 cases in each group,and 30 cases in normal group(p38,ERK5 and their phosphorylated protein expression in peripheral blood mononuclear cells were detected).At the end of the course of treatment,32 patients in the treatment group(excluding 3 cases,adding Jinshuibao 1 case,Bailing capsule 1 case,stopping medication without reason)and 32 patients in the control group(excluding 3 cases,adding Bailing capsule 1 case,urea Duqing granule 2 cases).The patients in the treatment group were given Qingshen Granule orally three times a day,one pack at a time,for 12 weeks.Serum creatinine(Scr),p38,p-p38,ERK5 and p-ERK5 were observed before and after treatment,and the changes of estimated glomerular filtration rate(eGFR)were estimated.Results:1.Clinical disease curative effect: The total effective rate of the treatment group was 81.25%,and that of the control group was 53.13%.There was significant difference between the two groups(P < 0.05).2.Therapeutic effect of TCM syndromes: the total effective rate of treatment group was 87.5%,and that of control group was 75%.There was significant difference between the two groups(P < 0.05).3.Serum creatinine: There was no significant difference in serum creatinine between the two groups before treatment(P > 0.05);after 12 weeks,serum creatinine in the treatment group was significantly lower than that before treatment(P < 0.01),and also significantly lower than that in the control group(P < 0.01);there was no significant difference in serum creatinine in the control group after 12 weeks compared with that before treatment(P > 0.05).4.eGFR: There was no significant difference between the two groups before treatment(P > 0.05);after 12 weeks,eGFR in the treatment group was significantly higher than that before treatment(P < 0.01),and significantly higher than that in the control group(P < 0.01);after 12 weeks,there was no significant difference in eGFR between the control group and that before treatment(P > 0.05).5.Before treatment,the serum levels of p38,p-p38,ERK5 and p-ERK5 in the two groups were significantly higher than those in the normal group(P < 0.01).There was no significant difference in the levels of p38,p-p38,ERK5 and p-ERK5 between the treatment group and the control group(P > 0.05).After treatment,the levels of p38,p-p38,ERK5 and p-ERK5 in the treatment group were significantly lower than those in the control group(P < 0.01).(The levels of p38,p-p38,ERK5 and p-ERK5 in the control group had no significant difference before and after treatment(P > 0.05).6.Safety analysis: After 12 weeks,the blood,urine,feces,liver function and electrocardiogram of the patients in the treatment group had no significant changes compared with those before treatment.Conclusion:1.The levels of p38,p-p38,ERK5 and p-ERK5 in peripheral blood mononuclear cells of patients with Damp-heat Syndrome of chronic renal failure increased.2.Qingshen Granule can alleviate the clinical manifestation of damp-heat syndrome of chronic renal failure and improve renal function.3.Qingshen Granule can reduce the expression of p38 and ERK5 in patients with Damp-heat Syndrome of chronic renal failure.4.There were no adverse reactions during the treatment of Qingshen Granule in the treatment group,which indicated that Qingshen Granule was safe and effective.Objective: The expression of P38,p-p38,ERK5 and p-ERK5 proteins in renal tissues of rats with renal fibrosis after 5/6 nephrectomy was detected,and the relationship between the above indexes and renal fibrosis was explored.To observe whether different doses of Qingshen granules can play an important role in Anti-renal fibrosis by reducing the activity of p38 MAPK and ERK5 MAPK signaling pathways.Methods: Seventy-two male SD rats,2 months old,clean grade,weighing 200 + 20 g,were purchased from the Experimental Animal Center of Anhui Medical University.They were managed in a closed manner and fed for one week.One week later,they were randomly divided into six groups: normal group,model group,high,medium and low dose group of Qingshen granule and losartan group.Except 12 rats in the normal group,60 rats in the model group were given 5/6 nephrectomy models according to the standard experimental method.The rats in each group were given 2 ml once a day on the first day after the establishment of the model.The high-dose group,middle-dose group and low-dose group of Qingshen Granule were given Qingshen Granule water solution by 4.0 g/kg.d,8.0 g/kg.d and 16.0 g/kg.d respectively.Losartan potassium tablets(50 mg/kg.d)were given to the rats in the Losartan group./ The suspension was orally administered at 13mg/kg.d.The normal group and the model group were given the same amount of saline by gavage.Each group was administered continuously for 12 weeks.Serum creatinine(Scr),endogenous creatinine clearance(Ccr),blood nitrogen(BUN),24-hour urine protein(24-hoururine protein,24 h Upro)and urine creatinine(Ucr)were measured.The protein expression of P38,p-p38,ERK5 and p-ERK5 in kidney tissue was detected by Western blot.HE staining,Masson staining and PAS staining were performed.The pathological changes of kidney tissues in six groups were observed by color,and the corresponding proteins were double labeled by immunofluorescence,and then the expression of the proteins was observed by confocal laser scanning microscopy.Results:1.Scr,BUN,24 h Upro: There was significant difference between model group and normal group(P < 0.01);there was significant difference between Qingshen Granule dose group,losartan group and model group(P < 0.01);there was significant difference among Qingshen Granule dose groups(P < 0.01),among which high dose group was the lowest;Qingshen Granule high dose group was significantly lower than losartan group(P < 0.01);There was no significant difference between the middle and low dose group and losartan group(P > 0.05).2.Ccr: Compared with the normal group,the model group decreased significantly(P < 0.01);the Qingshen granule dose group,losartan group and the model group increased(P < 0.01);the Qingshen granule dose group was significantly different(P < 0.01),and the high dose group had the highest level;the middle and low dose group of Qingshen granule had no significant difference compared with losartan group(P > 0.05),and the high dose group of Qingshen granule had the highest level of Ccr in rats(P > 0.05).Compared with losartan group,it was significantly higher(P < 0.01).3.Expressions of P38,p-p38,ERK5 and p-ERK5 proteins(Western blot method):(1)P38 and ERK5: The expression of P38 and ERK5 in model group was significantly higher than that in normal group(P < 0.01);the expression of P38 and ERK5 in each dose group of Qingshen granule was significantly lower than that in model group(P < 0.01);the difference between each dose group of Qingshen granule and losartan group was not significant(P > 0.05).(2)p-p38 and p-ERK 5: The expression of p-p38 and p-ERK 5 in model group was significantly higher than that in normal group(P < 0.01);the expression of p-p38 and p-ERK 5 in each dose group of Qingshen granule and losartan group was significantly lower than that in model group(P < 0.01);the expression of p-p38 and p-ERK 5 in each dose group of Qingshen granule was the lowest;the expression of p-ERK 5 and p-p38 in high dose group was lower than that in losartan group(P < 0.01).The expression of p-ERK-5 and p-p38 in middle and low dose groups of Qingshen Granule was not significantly different from that in losartan group(P > 0.05).4.Immunofluorescence double staining was used to detect the expression of CD31 and p-p38,CD31 and p-ERK5(laser confocal microscopy scanning imaging):(1)CD31+p-p38:p-p38 was not expressed in the normal group,and the expression of CD31+p-p38 was significantly decreased in the model group.The expression of CD31+p-p38 in the high-dose group was the lowest,and the expression of CD31+p-p38:p-p38 in the middle-dose group and the low-dose group was not significantly different from that in the Losartan group.(2)CD31+p-ERK5:p-ERK5 was not expressed in the normal group,and the expression of CD31+p-ERK5 was significantly decreased in the model group.The expression of CD31+p-ERK5 in the high-dose group was the lowest,and there was no significant difference between the low-dose group and the middle-dose group.5.Pathological changes of the kidneys of rats in each group: There was no abnormal pathological structure of the kidneys in the normal group;in the model group,the epithelial cells of the renal tubules were exfoliated,vacuolated,the lumen was dilated,a large number of inflammatory cells were infiltrated in the renal interstitium,a large amount of blue-stained collagen fibers were accumulated,the size and shape of the glomeruli were different,mesangial cell proliferation and matrix increase were observed,and partial focal glomerulosclerosis was observed.Compared with the model group,the above pathological changes of rats in each treatment group were significantly alleviated,especially in the high dose group of Qingshen Granule.Conclusion: 1.Activation of p38 MAPK and ERK5 MAPK signaling pathways is involved in the formation of renal interstitial fibrosis.2.Qingshen Granule can reduce the levels of SCr and BUN in rats with renal fibrosis,reduce the amount of 24 H urinary protein,and increase Ccr.3.Qingshen Granule can alleviate the degree of pathological damage of renal tissue in rats with renal fibrosis.4.Qingshen Granule can reduce the expression levels of p38,p-p38,ERK5 and p-ERK5 in renal tissue of rats with renal fibrosis,and delay RIF by inhibiting the activation of p38 and ERK5 signaling pathways. |
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