Based On NF-κB Signal Transduction Pathway To Research Clinical And Experimental Study On The Effect Of Clearing Away Heat,eliminating Dampness,removing Blood Stasis On Renal EMT

Objective:To observe the effect of Qingshen Granule on expression of NF-κp65 and p-IκBα in peripheral blood NF-κB signal transduction pathway of chronic renal failure(CRF) patients with damp heat syndrome.To investigate whether Qingshen granule could inhibit the incidence of epithelial-mesenchymal transition(EMT) and improving renal fibrosis by inhibiting the NF-κB signal pathway in patients with damp heat syndrome of chronic renal failure.Methods:68 CRF patients with damp heat syndrome,according to the random number table,were divided into treatment group and control group with 34 cases each,and 20 healthy persons were selected as normal group(for detecting NF-κBp65 and p-IκBα in peripheral blood).5 cases had been eliminated during treating,including 2 cases in treatment group,3 cases in control group. As the result,63 cases completed their treatment,including 32 cases in treatment group and 31 cases in control group.There were 18 male and 14 female patients in treatment group,who were (50.50+10.67) years old,and there were 17 male and 14 female patients in control group who were (47.86 +12.75) years old.Both two groups were given the basic treatment of Western medicine and retention enema of traditional Chinese medicine,what’s more,the treatment group was treated with Qingshen granule,3 times a day with 1 bag for 8 weeks.1.To evaluate the clinical effect,the TCM syndromes and the integral of TCM syndrome in the two groups before and after the treatment.2.To estimate glomerular filtration rate(eGFR) by detecting serum creatinine(Scr) in both two groups before and after treatment.3.To detect the level of NF-κBp65 and p-IκBα in peripheral blood mononuclear cells(PBMC) in both two groups.Results:1.Comparison of efficacy:The total effective rate of the treatment group was 84.38%,and that in control group was 54.84%.The difference was significant between two groups (P<0.01).2.Comparison of TCM syndromes:The total effective rate of TCM syndromes in the treatment group was 81.25%,but that in control group was 51.61%, and the difference was significant between the two groups (P<0.01).3.Comparison of TCM syndrome score:symptom scores in two groups showed no significant difference before treatment (P>0.05).The TCM syndrome score of the treatment group was significant difference between that before and after treatment (P<0.01).The TCM syndrome score of the control group was no significant difference between that before and after treatment (P>0.05).There was significant difference between treatment group and control group in TCM syndrome score after treatment (P<0.01).4.Comparison of Scr and eGFR:(1)pre-treatment differences between treatment and control groups was not statistically significant Scr level(P>0.05),The Scr level significantly descended in treatment group after treatment(P<0.01).There was no statistical difference between pre-treatment and post-treatment in Scr in control group (P>0.05).Scr level in treatment was lower than that in control group (P<0.05).(2)There was no statistical difference between pre-treatment and post-treatment in eGFR in treatment group(P>0.05).The eGFR ascended after treatment in treatment group (P<0.01).There was no statistical difference between pre-treatment and post-treatment in control group (P>0.05).The eGFR in treatment group was higher than that of control group (P<0.05).5.Comparison of NF-κBp65 in PBMC:Compared with the normal group,NF-κBp65 contents of the treatment group and the control group pre-treatment in PBMC were higher normal group (P<0.01).There was no statistical difference in NF-κBp65 contents between treatment group and treatment group before performance(P>0.05). The NF-κBp65 content was descended in treatment group after performance (P<0.01), but it was no statistical difference in control group (P>0.05).There was statistical difference in NF-κBp65 content in PBMC from treatment group and control group after intervention (P<0.01).6.Comparison of p-IκBα in PBMC:p-IκBα content in PBMC from treatment group and control group pre-treatment were higher than that from normal group (P<0.01). There was no statistical difference in p-IκBα content in PBMC from treatment group and control group(P>0.05).p-IκBα content in PBMC from treatment group was significantly descended after performance (P<0.01).The difference of p-IκBα content in PBMC in the control group was significant after treatment(P>0.05).On the contrary, that made sense in treatment group after performance (P<0.01).Conclusion:1.Qingshen granule could improve clinical symptoms of CRF patients with damp heat by decreasing Scr,increasing eGFR and protecting renal function.2. The fact that Qingshen granule could reduce NF-κBp65,p-IκBα level in PBMC from patients of CRF with damp heat syndrome manifested this medicine could inhibit signal transduction pathway of NF-κB.3.No patient showed side effects during treatment and safety indications were normal.All above manifested this medicine was reliable in health.Objective:To observe the effect of Qingshen Granule on expression of nuclear factors-κBp65 (NF-KBp65), p-IκBα,IKKα,Monocytechemotatic protein-1 (MCP-1), Intercellular cell adhesion molecule-1 (ICAM-1), alphasmooth muscle actin (α-SMA), E-cadherin in rat renal fibrosis.To investigate the Qingshen granule could suppress the unilateral ureteral obstruction(UUO)NF-κB signal channel,reduce renaltubular EMT, play anti renal fibrosis(RF) function or not.Methods:48 male SD rats were randomly divided into 4 groups, including sham oper ation group, model group, Qingshen granule group, pyrrolidine and dithiocarbamate (PDTC) group. The experimental rats was detected before the blood urea nitrogen (BUN), serum creatinine(Scr),no significant difference (P>0.05).Excepted for 12 rats from sham operation group received ureteral separation without ligation,the other 36 rats were established as UUO model by the normal method. Qingshen granule group: the Qingshen granule dissolved in warm water with 0.4g Qingshen/ml and gavage at a dose of lml/100g. PDTC group:PDTC was dissolved in warm water with 15mg/ml and gavage at a dose of lml/100g; Rats in sham operation group and model group were given a gavage with warm water once a day, at a dose of lml/100g for 4 weeks.1. To detect Scr, BUN and 24 hour urinary protein (24h Upro) by biochemical assay.2. To detect the expression of α-SMA and E-cadherin in rat renal tissue.3.To detect content of protein of NF-κB p65,p-IκBα,IKKα, MCP-1 and ICAM-1 in renal tissue by Western blot.Results:1.Comparison of BUN and Scr:(1)There was no difference among groups in BUN before treatment (P>0.05),while BUN was higher in Qingshen granule group, PDTC group, model group than sham operation group after treatment (P<0.01). BUN in Qingshen granule group and PDTC group were lower than that in model group (P<0.01),and BUN in Qingshen granule group was lower than that in PDTC group (P<0.05).(2) Scr among different groups was not significant before treatment (P>0.05). Scr in Qingshen granule group, PDTC group and model group was higher than that in sham operation group after treatment (P<0.01).Scr in Qingshen granule group and PDTC group was lower than that in model group (P<0.01).Scr in Qingshen granule group was lower than that in PDTC group (P<0.05).2.comparison of 24 hour urinary protein:24hUpro in PDTC group and model group was higher than that in sham operation group (P<0.01).24hUpro in Qingshen granule group was higher than that in sham operation group (P<0.05).24hUpro in Qingshen granule group and PDTC group was higher than that in model group (P<0.01). 24hUpro in Qingshen granule group was lower than that in PDTC group (P<0.05).3.Comparison of expression of ICAM-1 protein in rat renal tissue from all groups (Western blot method):Expression of ICAM-1 protein in renal tissue from PDTC group and model group was higher than that from sham operation group(P<0.01). Expression of ICAM-1 protein in renal tissue from Qingshen granule group was higher than that from sham operation group(P<0.05).Expression of ICAM-1 protein in renal tissue from Qingshen granule group and PDTC group was higher than that from model group (P<0.01).Expression of ICAM-1 protein in renal tissue from Qingshen granule group and PDTC group was higher than that from model group(P<0.01),but expression of ICAM-1 protein in renal tissue from Qingshen granule group was lower than that from PDTC group(P<0.05).4.Comparison of expression of IKKa protein in renal tissue from all groups (Western blot method):Expression of IKKa protein in renal tissue from PDTC group and model group was higher than that from sham operation group(P<0.01).Expression of IKKa protein in renal tissue from Qingshen granule group was higher than that from sham operation group(P<0.05),but expression of IKKa protein in renal tissue from Qingshen granule group and PDTC group was lower than that from model group(P<0.01).Expression of IKKa protein in renal tissue from Qingshen granule group was lower than that from PDTC group,but the difference was not statistically.5.Comparison of expression of MCP-1 protein in renal tissue from all groups (Western blot):Expression of MCP-1 protein in renal tissue from PDTC group and model group was higher than that from operation group(P<0.01).Expression of MCP-1 protein in renal tissue from Qingshen granule group was higher than that from sham operation group(P<0.05).Expression of MCP-lprotein in renal tissue from granule group and PDTC group was lower than that from model group(P<0.01),what’s more, this protein from Qingshen granule group was lower than that from model group(P<0.05).6.Comparison of expression of NF-κBp65 protein in all groups(Western blot method): Expression of NF-κBp65 protein in renal tissue from Qingshen granule group, PDTC group and model group was higher than that from sham operation group (P<0.01). Expression of NF-κBp65 in renal tissue from PDTC group and Qingshen granule group was higher than that from that from model group(P<0.01).Expression of NF-κBp65 protein in kidney tissue from Qingshen granule group was lower than that from PDTC group(P<0.05).7.Comparison of expression of p-IκBα protein in renal tissue from all groups(Western blot method):Expression of p-IκBα protein in kidney tissue from Qingshen granule group,PDTC group and model group was higher than that from sham operation group (P<0.05).Expression of p-IκBα protein in renal tissue from Qingshen granule and PDTC group was lower than that from model group (P<0.05). Expression of p-IκBα protein in renal tissue from Qingshen granule group was lower than that from PDTC group (P<0.05).8.Comparison of immunohistochemical α-SMA staining:Semi-quantitative scores of a-SMA from Qingshen granule group, PDTC group and model group were higher than that from sham operation group (P<0.01). Semi-quantitative scores of α-SMA from Qingshen granule group,and PDTC group were lower than that from model group (P< 0.01).Semi-quantitative scores of α-SMA from Qingshen granule group, and model group were lower than that from PDTC group (P<0.05).9.Comparison of immunohistochemical E-cadherin staining:Semi-quantitative E-cadherin scores in Qingshen granule group,PDTC group and model group were lower than that from sham operation group (P<0.01).Semi-quantitative E-cadherin scores in Qingshen granule group and PDTC group were higher than that in model group (P<0.01). Semi quantitative E-cadherin score in Qingshen granule group was higher than that in PDTC group(P<0.05).Conclusion:1.NF-κB signal channel activation was involved in the pathogenesis of UUO rats.2.Treatment of reducing fever,dampness and stasis could descend serum creatinine,urea nitrogen and 24h urine protein.3.Treatment of reducing fever, dampness and stasis could improve renal tubular EMT and play a role in anti kidney fibrosis by suppressing NF-κB signal channel and reducing inflammation.