| Objective1 To observe the intervention effect of Fushen granule on the injury of human colonic epithelial cells induced by urea.2 To study the effect of Fushen granule on renal function and intestinal barrier injury in CKD model rats.3 To explore the mechanism of Fushen granule in interfering with intestinal barrier injury induced by urotoxin.Methods 1 Establishment of an in vitro experimental model of human colonic epithelial T84 cells treated with urea(1)MTT method was used to detect the effects of urea and Fushen granule drugcontaining serum on the proliferation of human colon epithelial T84 cells.(2)Human colonic epithelial T84 cells were cultured in vitro.T84 cells were treated with 0mg/dl,72mg/dl and 144mg/dl urea for 24 hours and 48 hours to simulate intestinal epithelial barrier injury.The relative expression levels of tight junction proteins zonula occluden1(ZO1)and Claudin-1 were detected by Western Blot method,so as to evaluate the degree of intestinal epithelial injury and select the best concentration.The experiment was divided into control group、the model group、low dose Fushen granule containing serum group、high dose Fushen granule containing serum group and NF-κ B inhibitor group.(3)Immunofluorescence staining was used to detect the expression of tight junction protein Claudin-1 in intestinal epithelium before and after treatment.(4)Western Blot test was used to detect the expression of ZO1、Claudin-1 、nuclear transcription factor-κB P65 、 phosphorylated P65(p-P65)、 IκBα and phosphorylated IκBα(p-IκBα)and the expression levels of tumor necrosis factor-α(TNFα)、malondialdehyde(MDA)、intercellular adhesion molecule-1(ICAM-1)、chemokine ligand-2(CXCL2)、L-selectin(CD62L)and cyclooxygenase(COX2).(5)Quantitative real-time polymerase chain reaction(q RT-PCR)was used to observe the expression levels of ZO1、Claudin-1、nuclear factor E2-related factor 2(Nrf2)、interleukin-6(IL6)、ICAM-1、CD62L and CXCL2 m RNA.2 The in vivo CKD rat model was established by 5/6 nephrectomy.(1)60 healthy male SD rats were randomly divided into sham operation group(group C,n = 12)and model group(n = 48).CKD rats in the model group were made by 5 /6 nephrectomy.After 7 days of nephrectomy,the rats in the model group were randomly divided into model group、low dose Fushen granule intervention group、high dose Fushen granule intervention group and NF-κB inhibitor group.There were 15 rats in each group,and each treatment group was given corresponding drug intervention,continuous intragastric administration for 6 weeks.During the whole experiment,the rats were fed with ordinary feed and freely fed with water.At the end of the 6th week,blood plasma were collected by abdominal aortic method and the intestines and kidneys were collected for follow-up examination.(2)Biochemical method was used to detect the contents of Scr 、BUN.High efficiency liquid chromatography was used to detect the contents of IS、PCS.(3)The pathological changes of kidney and colon were observed by HE staining.(4)Immunohistochemical(IHC)was used to observe the expression of ZO1、Claudin-1 in colon.(5)Western Blot test was used to detect the expressions of ZO1、Claudin-1、COX2、TNF α、CXCL2 and CD62 L.Results 1 Results of cell experiment:(1)There was no significant difference in absorbance after intervention with different concentrations of urea and Fushen granule drug-containing serum(P<0.05).(2)The results of Western blot showed that different concentrations of urea decreased the expression of tight junction protein ZO1 and Claudin-1 in different degrees(P<0.01 or P<0.05),and the model of T84 cells treated with 144mg/dl urea for 48 hours was the best.(3)Immunofluorescence detection showed that compared with the control group,the fluorescent expression of Claudin-1 decreased,and compared with the model group,the expression of Claudin-1 increased in each treatment group.(4)The results of Western blot showed that compared with the control group,the protein expression levels of ZO1 and Claudin-1 in the model group decreased significantly(P<0.01),the protein expression levels of p-P65/P65 and p-IκBα/IκBα increased significantly(P<0.01),the protein expression levels of TNFα、CXCL2、ICAM-1、CD62L、COX2 and MDA increased significantly(P<0.01);Compared with the model group,the protein expression of ZO1 and Claudin-1 in each treatment group increased in different degrees(P<0.01),while the protein expression levels of pIκBα/IκBα、p-P65/P65 、TNFα、CXCL2、ICAM-1、CD62L、COX2 and MDA decreased in different degrees(P<0.01 or P<0.05).(5)QRT-PCR test showed that compared with the control group,the expression levels of ZO1、Claudin-1 and Nrf2 m RNA decreased significantly(P<0.01),while the expression levels of IL6、ICAM-1、CD62L and CXCL2 m RNA increased in different degrees(P<0.01);Compared with the model group,the expression levels of ZO1、Claudin-1 and Nrf2 m RNA in each treatment group increased in different degrees(P<0.01);The expression levels of IL6、ICAM-1、CD62L and CXCL2 m RNA decreased in different degrees(P<0.01 or P<0.05).2 Results of animal experiment:(1)Biochemical indexes: Compared with the sham operation group,the contents of serum creatinine and blood urea nitrogen increased significantly(P<0.01),IS and PCS increased in different degrees(P<0.01 or P<0.05);Compared with the model group,the contents of Scr、BUN decreased in different degrees(P<0.01),and the contents of IS and PCS decreased in different degrees(P<0.01 or P<0.05).(2)Pathological observation: Compared with the sham operation group,the rats in the model group showed glomerular necrosis,atrophy,renal tubular epithelial edema and degeneration,and inflammatory cell infiltration in the renal interstitium;there were different degrees of submucosal edema and a large number of inflammatory cell infiltration in the colon of rats.Intestinal gland structure destruction and exfoliation;compared with the model group,the pathological damage of kidney and colon in each treatment group was alleviated in varying degrees.(3)Immunohistochemical results showed that compared with the sham operation group,the expression of ZO1 and Claudin-1 decreased,and compared with the model group,the expression of ZO1 and Claudin-1 increased in different degrees in each treatment group.(4)Western blot showed that compared with sham operation group,the protein expression of ZO1 and Claudin-1 decreased significantly(P<0.01),and the protein expression levels of COX2 、 TNFα 、 CXCL2 and CD62 L increased significantly(P<0.01);Compared with the model group,the protein expression levels of ZO1 and Claudin-1 in each treatment group increased in different degrees(P<0.01 or P<0.05),while the protein expression levels of COX2、TNFα、CXCL2 and CD62 L decreased in different degrees(P<0.01 or P<0.05).Conclusion(1)The drug-containing serum of Fushen granule can increase the level of tight junction protein and gene expression in intestinal epithelium,reduce the level of oxidative stress and inflammation,reduce the damage of urea to T84 cells,and protect intestinal epithelial barrier.(2)Fushen granule can reduce the contents of Scr、 BUN、PCS and IS in CKD rats,reduce the pathological damage of kidney and colon in CKD rats,protect the structure and function of kidney and colon,enhance the intestinal tight junction of CKD rats,reduce the level of oxidative stress,reduce intestinal inflammation in CKD rats,promote the repair of intestinal barrier function,and protect intestinal barrier in CKD rats.(3)Fushen granule can alleviate the intestinal barrier injury in CKD,and its mechanism is closely related to the regulation of IκBα/NF-κB signaling pathway by Fushen granule. |
