| Part one Effects of Yi qi Hua Shi Tong Luo Formula on general condition,renal function and renal his tomorphology in 5 /6 nephrectomy ratsObjective: The improvement of Yi qi Hua Shi Tong Luo Formula with the general condition,renal function and renal disease in 5 /6 nephrectomy rats was observed.Methods: The rat model of chronic renal failure was established by Platt method.32 adult male SD rats were randomly divided into model group(Model),Yi qi Hua Shi Tong Luo recipe group(Yi qi Hua Shi Tong Luo Formula,YHT),benazepril group(Benazepril Hydrochloride,BH)and sham operation group(Sham),(n = 8).The rats in the YHT group were given in tragastric administration of benazepril hydrochloride tablets 0.09mg/100g/d and Sham and Model1ml/100 g deionized water once a day for 12 weeks.24-hour urine of SD rats was collected at the end of the 12 th week for quantitative detection of 24-hour urine protein.The blood was taken from the abdominal aorta of SD rats,and the bleeding serum was separated,which was used to detect the contents of serum creatinine(serum creatinine,S cr)and blood urea nitrogen(urea nitrogen,BUN).The left kidney was extracted,dried with filter paper,weighed and stored in two parts,one in the refrigerator at-80 ℃ and the other in 4% paraformaldehyde for detection.The pathological sections fixed by 4% paraformaldehyde were examined by HE,Masson,Periodic Acid-S chiffstain,PAS and Sirius red staining,to observe the pathomorphological changes of the residual kidney tissue.The above data were shown by mean ±standard deviation.The mean values of each group were compared by single factor analysis of variance(one-way ANOVA),and pairwise comparisons were made by Newman-Keuls test.Results:1.General conditions of rats: the rats in Sham group had normal feeding,drinking and urination,with shiny hair,ruddy ears and ruddy tail,and normal activity reactions.Rats in the Model group,YHT group and BH group all had different degrees of reduction in food intake,increased water intake,gradually increased urine volume,less luster in hair removal,white pinna,white tail,damp and cold,listlessness,and decreased activity.Among them,rats in the Model group had the most significant change,and the treatment group all improved compared with the Model group.Body weight records showed that the weight of rats in Model group was lower than that of rats in Sham group,and the weight increased after the intervention of YHT or BH(P <0.01).There was no statistically significant difference between YHT group and BH group(P > 0.05).2.Rat survival rate: 50 clean grade healthy male SD rats were randomly divided into sham group(10)and model group(40).In the Sham group,10 rats underwent bilateral renal capsule dissection without kidney resection,and2 of them died during the experiment.The rest were modeled by 5/6nephrectomy.During the experiment,7 in the Model group died,4 in the YHT group and 4 in the BH group.The survival rate of this experimental Model was about 60%.3.Left residual kidney weight: compared with Sham group,the residual kidney weight of rats in Model group was significantly reduced.Compared with Model group,residual kidney weight increased in YHT group and BH group(P<0.01).There was no significant difference between the YHT group and the BH group(P > 0.05).4.24-hour urine protein quantification in rats: compared with Sham group,the urine protein level of rats in Model group for 24 hours increased significantly.Compared with the Model group,the 24-hour urinary protein output of the YHT group and BH group decreased(P<0.01).There was no significant difference between the YHT group and the BH group(P > 0.05).5.Rat BUN and S Cr levels: compared with Sham group,rat BUN and S Cr levels in Model group were significantly higher.Compared with the Model group,the BUN and S Cr levels of rats in the YHT group or BH group were significantly reduced(P <0.01),and no significant difference was found between the YHT group and BH group(P > 0.05).6.Histopathology of rat residual kidney6.1.Gross morphological changes of residual kidney in Sham group,the tissue was reddish brown,the surface was smooth and moist,the capsule was easy to peel off,and the boundary of sectional cortex was clear.In Model group,the left residual kidney tissue was grayish brown,small in size,light in weight,hard and brittle in texture,with fine particles on the surface,the capsule was not easy to peel off,and the section cortex became thinner.In YHT group and BH group,the volume and weight of the left residual kidney tissue increased,the texture was soft,the surface was a few fine granules,the capsule was not easy to peel off,and the section cortex was thicker.HE,Masson,PAS and Sirius red staining showed that the glomerular structure of Sham group was clear,no hyperemia,atrophy,neat arrangement of renal tubular epithelial cells,intact basement membrane and no obvious inflammatory cell infiltration in interstitium.In Model group,most of the glomeruli showed fibrosis and hyaline degeneration,glomerulus atrophy,capillary loop collapse,tubular epithelial cell degeneration,necrosis,exfoliation,tubular atrophy and disappearance,renal tubular dilatation,renal interstitial matrix significantly increased,a large number of lymphocytes as the main inflammatory cell infiltration,foam cells appeared.The degree of glomerulosclerosis,tubular atrophy and interstitial fibrosis were alleviated in YHT group and BH group.Conclusion:1.In this study,5/6 nephrectomy was used to successfully replicate the pathological model of chronic renal failure in SD rats,providing a typical stable and reliable experimental basis for the treatment of chronic renal failure and renal fibrosis.2.In order to effectively improve the general condition,renal function and degree of renal fibrosis of CRF rats,the therapeutic effect was comparable to benazepril hydrochloride.3.Yi qi Hua Shi tong Luo prescription is an effective self-designed prescription established under the guidance of traditional Chinese medicine theory,according to the characteristics of clinical etiology and pathogenesis of CRF,combined with the principle of "divide and cure by three focal points",and it has been applied to clinical practice for a long time.This study provides strong experimental data for its clinical application.Part two Effect of Yi qi Hua Shi Tong Luo recipe on Renal Fibrosis-related proteins in 5/6 Nephrectomized RatsObjective: To explore the mechanism of Yi qi Hua Shi Tong Luo recipe in inhibiting renal fibrosis by regulating the expression of renal fibrosis-related proteins in 5/6 nephrectomized rats.Methods: Modeling,grouping and medication as part I.Western blot technology to observe rat kidney TGF –β1(transforming growth factor beta 1)and FN(fiber connection protein),Collagen Ⅰ,Collagen Ⅲ protein expression;Immunohistochemical method to observe the FN,Collagen Ⅲ protein expression;Fluorescence staining was used to observe the expression of TGF-β1 in residual renal tissues.FN m RNA protein expression was detected by real-time fluorescence quantitative PCR.Results:1.Effect of Yi qi Hua Shi Tong Luo recipe on the expression of TGF-β1protein in kidney tissue the expression of TGF-β1 protein in rat kidney was detected by1.1.Western blot technique.The comparison of TGF-β1 protein expression in renal tissue of rats in each group.Compared with Sham group,the expression level of TGF-β1 protein in Model group was significantly higher than that in Model group,and the expression level of TGF-β1 in each treatment group was significantly lower than that in Model group(P < 0.01).There was no significant difference between YHT group and BH group(P>0.05).1.2.The expression of TGF-β1 in renal tissue was observed by fluorescence staining.The staining of TGF-β1protein was green fluorescence,and the nucleus was blue fluorescence by DAPI staining.The immunofluorescence results of TGF-β1,which showed almost no green fluorescence expression in Sham group,high green fluorescence intensity in Model group,suggesting high expression of TGF-β1protein in Model group,and decreased green fluorescence intensity in YHT or BH group compared with Model group.2.Effect of Yi qi Hua Shi Tong Luo recipe on FN,Collagen Ⅰ and Collagen Ⅲ protein in kidney tissue of rats2.1.Westernblot technique was used to detect the expression of FN,Collagen Ⅰ and Collagen III protein in renal tissue of rats.The comparison of protein expression of FN,Collagen Ⅰ and Collagen Ⅲ in renal tissue of rats in each group.Compared with Sham group,the protein expression levels of FN,Collagen Ⅰ and collagen Ⅲ in renal tissue of Model group were significantly increased,and compared with Model group,the expression levels of FN,Collagen Ⅰ and collagen Ⅲ in all treatment groups were significantly lower(P< 0.01).There was no significant difference between YHT group and BH group(P>0.05).2.2.Immunohistochemical method was used to observe the expression of FN and Collagen III protein.The expression of FN and Collagen Ⅲ protein in each group was detected by immunohistochemistry.Compared with Sham group,FN and collagen Ⅲ protein in renal tissue of Model group showed small brown granules,which mainly showed positive reaction in glomeruli,and the expression of FN and collagen Ⅲ protein in each treatment group was significantly lower than that in Model group(P < 0.01).There was no significant difference between YHT group and BH group(P>0.05).2.3.The expression of FN m RNA in renal tissue of rats in each group was detected by qPCR(quantitative real-time PCR,real-time fluorescence quantitative PCR).The comparison of FN m RNA expression in renal tissue of rats in each group.Compared with sham group,the expression of FN m RNA in renal tissue of rats in Model group increased,and compared with Model group,the expression of FN m RNA in each treatment group decreased significantly(P < 0.01).There was no significant difference between YHT group and BH group(P>0.05).Conclusion: Yi qi Hua Shi Tong Luo recipe may inhibit renal fibrosis by regulating the expression of TGF-β 1 and its mediating proteins FN,CollagenⅠ and collagen Ⅲ.This study provides a molecular biological basis for Yi qi Hua Shi Tong Luo recipe in the treatment of CRF and renal fibrosis.Part three Effect of Yi qi Hua Shi Tong Luo Formula on Nrf2-keap1 signal Pathway in Renal tissue of 5/6 Nephrectomized RatsObjective: to investigate the role of nrf2-keap1 signaling pathway in preventing renal fibrosis of 5/6 nephrectomy rats with Yi qi and wet tong Luo prescription,and to further explore the possible mechanism of the therapeutic effect of Yi qi and Hua Shi tong Luo prescription,so as to provide experimental basis for clinical application.Methods: the activity of Superoxide Dismutase(SOD)and the content of Malondialdehyde(MDA)in renal tissue homogenate were determined after the rats were dissected at the end of 12 weeks.The expressions of interleukin-6(IL-6),interleukin-1(IL-1),interleukin-10(IL-10)and Tumor Necrosis factor-receptor(TNF-α)in renal tissues were determined by ELISA.Oxidative stress-related factors in kidney tissues were detected by Western blot.Nuclear factor-e2 related factor 2(Nrf2),kelch-like ech-associated protein-1(Keap1),NADPH oxidase 4(Nox4),Heme oxygenase 1(HO-1),Nuclear factor a p pa b(NF-k B),Intercellular adhesion molecule-1(ICAM-1).The expressions of NF-k B,ICAM-1 and Nox4 were observed by immunohistochemistry.The expression of Nrf2 and TNF-α in the nuclei of renal tissues was observed by fluorescence staining.The expression level of Nrf2 m RNA was detected by real-time fluorescence quantitative PCR.1.gasification dampness t2 dm party effects on kidney tissue SOD activity and MDA content Compared with the Sham group,the Model rat kidney tissues of SOD activity and MDA content increased,compared with Model group,YHT and BH group rats kidney tissue increased SOD activity and MDA content decreased(P < 0.01).YHT and BH group,there was no statistically significant difference(P > 0.05).2.The effect of Yi qi Hua Shi tong Luo prescription on the inflammatory factors IL-6,IL-1,IL-10,TNF-α,NF-κ B and ICAM-1 in renal tissues2.1 ELISA was used to detect the expression of IL-6,IL-1,IL-10 and TNF-αprotein in the renal tissues of rats in each group.Compared with Sham group,the expression levels of IL-6,IL-1 and TNF-α protein in the kidney tissues of rats in the Model group were significantly increased,and the expression levels of IL-10 protein were significantly decreased(P<0.01).Compared with the Model group,the expression levels of IL-6,IL-1 and TNF-α protein in each treatment group were significantly reduced(P<0.01),and the expression levels of IL-10 protein were significantly increased(P<0.01).There was no significant difference between the YHT group and the BH group(P > 0.05)2.2.The protein expression of NF-κ B and ICAM-1 in renal tissue of rats was observed by Western blot technique.The comparison of NF-κ B and ICAM-1 protein expression in renal tissue of rats in each group.Compared with Sham group,the expression levels of NF-κ B and ICAM-1 protein in renal tissue of Model group were significantly increased,and compared with Model group,the expression levels of NF-κ B and ICAM-1 protein decreased significantly after treatment with YHT or BH(P < 0 01).There was no significant difference between YHT group and BH group(P > 0.05).2.3.the expression of NF-κ B and ICAM-1 in each group was observed by immunohistochemistry.The expression of NF-κ B and ICAM-1 in each group was detected by immunohistochemistry.Compared with the Sham group,NF-κ B and ICAM-1 proteins in the Model group presented small brown and yellow granules,and were mainly positive in the glomerulus.Protein expression increased significantly(P<0.01).Compared with the Model group,NF-κ B and ICAM-1 protein expression levels were significantly reduced in each treatment group(P<0.01),and the difference was statistically significant.There was no significant difference between the YHT group and the BH group(P > 0.05).2.4.the expression of NF-κ B in the nucleus was observed by fluorescence staining.The NF-κ B protein was labeled as red fluorescence and the DAPI staining cell nucleus as blue fluorescence.Results:immunofluorescence results of NF-κ B showed that the NF-κ B protein Sham group had almost no red fluorescence expression.The high intensity of red fluorescence in the Model group suggested that NF-κ B protein was highly expressed in the Model group.After the intervention of YHT or BH,the intensity of red fluorescence in the nucleus was lower than that in the Model group.3.The detection of oxidative stress-related factors Nrf2,Keap1,Nox4 and HO-1 in renal tissues by Yi qi Hua Shi prescription3.1.Western blot was used to observe the protein expression of Nrf2,Keap1,Nox4 and HO-1 in the renal tissues of rats.The comparison of the protein expression of Nrf2,Keap1,Nox4 and HO-1 in the renal tissues of rats in each group.Compared with Sham group,the expression levels of Keap1 and Nox4 proteins in the renal tissues of rats in the Model group were significantly increased,and the expression levels of Nrf2 and HO-1 proteins were significantly decreased(P<0.01).Compared with the Model group,the protein expression levels of Keap1 and Nox4 significantly decreased after YHT intervention(P<0.01),and the protein expression levels of Nrf2 and HO-1 significantly increased(P<0.01),with statistically significant differences.There was no significant difference between the YHT group and the BH group(P > 0.05).3.2.Nox4 protein expression was observed by immunohistochemistry.The expression of Nox4 protein in each group was detected by immunohistochemistry.Compared with Sham group,Nox4 protein in the kidney tissues of rats in the Model group presented small brown and yellow granules,mainly presenting positive reaction in the glomerulus.Protein expression increased significantly(P<0.01).Compared with the Model group,Nox4 protein expression was significantly reduced in each treatment group(P<0.01),with statistically significant differences.There was no significant difference between the YHT group and the BH group(P > 0.05).3.3.the expression of nuclear Nrf2 in the nucleus was observed by fluorescence staining.The Nrf2 protein was labeled as red fluorescence,and the nucleus was labeled as blue fluorescence by DAPI staining.The results showed that the Nrf2 immunofluorescence results showed that compared with Sham group,the expression of Nrf2 protein in the kidney tissue nucleus of Model group was significantly decreased,and the expression of Nrf2 in YHT group and BH group was significantly increased.3.4.the expression of Nrf2 m RNA in renal tissues of rats in each group was detected by qPCR.The comparison of the expression of Nrf2 m RNA in renal tissues of rats in each group.Compared with Sham group,the expression of Nrf2 m RNA in kidney tissues of Model group was decreased(P<0.01).Compared with the Model group,the expression of Nrf2 m RNA in each treatment group increased(P<0.01),and the difference was statistically significant.There was no significant difference between the YHT group and the BH group(P > 0.05).Conclusion: 1.The activity of SOD was significantly decreased,the content of MDA was significantly increased,the expression of inflammatory mediators such as IL-1 and IL-6 was significantly increased,and the expression of IL-10 anti-inflammatory cytokines was decreased.This model has decreased antioxidant capacity,enhanced oxidative stress,decreased anti-inflammatory ability and aggravated inflammatory injury;2.The Nrf2/Keap1 signaling pathway in the renal tissue of rats after 5/6nephretomized can affect the expression of oxidative stress and inflammatory related proteins Nox4,HO-1,IL-6,IL-1,IL-10,NF-κ B,TNF-α,ICAM-1.YHT intervened to regulate the Nrf2/Keap1 signaling pathway,which affected the expression of the above proteins,corrected the imbalance between the inflammatory signal and the antioxidant response in the renal tissues of the 5/6 nephrectomy rats,significantly improved the antioxidant capacity of the 5/6 nephrectomy rats,inhibited their oxidative stress,and reduced the inflammatory response.This may be one of the mechanisms by which YHT can treat CRF and inhibit renal fibrosis.conclusion :1.In this study,the pathological injury of chronic renal failure in SD rats was successfully made by 5/6 stroke nephrectomy,which provides a reliable animal model for the treatment of chronic renal failure and renal fibrosis.2.Yi qi Hua Shi Tong Luo recipe can effectively improve the renal function and the process of renal fibrosis in rats with chronic renal failure,and the curative effect is similar to that of benazepril hydrochloride.3.Under the guidance of traditional Chinese medicine theory,Yi qi Hua Shi tong Luo prescription is an effective self-designed prescription which is established according to the characteristics of clinical etiology and pathogenesis of CRF and combined with the principle of "divide and cure by three focal points" and has been applied to clinical practice for a long time.This study provides powerful experimental data for its clinical application.4.Yi qi Hua Shi Tong Luo can inhibit renal fibrosis by affecting the expression of TGF-β1 and its mediated related proteins that promote the progression of renal fibrosis;5.By activating Nrf2-keap1 signaling pathway,Yi qi and Hua Shi regulate the imbalance between the inflammatory signal and the antioxidant response,and affect the expression of pro-fibrotic proteins,so as to achieve the treatment of CRF and the inhibition of renal fibrosis.This may be one of the mechanisms of action of YHT. |
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