| Purpose:Objective to study the effect of shenshuaiyin on TLR4 / MyD88 / NF-?B signaling pathway,Fas / Fas L mediated apoptosis pathway and PI3 K / Akt signaling pathway in rats with chronic renal failure based on image thinking of traditional Chinese Medicine(TCM),and to explore the mechanism of shenshuaiyin in preventing and treating chronic renal failure.Material and method:A total of 84 6-week-old SPF grade healthy male SD rats were selected.12 rats were randomly selected as the normal group,and the other 72 rats were selected as the model group.The model rats were given adenine(200 mg /(kg·d))by gavage for 3 weeks to establish the rat model of chronic renal failure.The rats were randomly divided into model group,shenshuaiyin group,Niaoduqing group,P13 K inhibitor group(ly group) and Shenshuaiyin + ly group.The normal group and model group were given 0.9% Na Cl solution 8 ml /(kg·d)by gavage.The shenshuaiyin group was given compound decoction of traditional Chinese medicine,shenshuaiyin Decoction by gavage at the dosage of 33.3g /(kg·d).Niaoduqing group was given Niaoduqing granules by gavage at the concentration of2.25 g /(kg·d).Ly group was given intraperitoneal injection of P13 K inhibitor,namely LY294002 diluent 50 mg / kg,once a week.Shenshuaiyin + ly group was given shenshuaiyin Decoction by gavage according to the dosage of 33.3g /(kg·d)and intraperitoneal injection of LY294002 diluent 50 mg / kg,once a week.The rats in the above groups were treated for 4weeks.The serum and renal tissue of rats in each group were collected to detect the indexes.Experiment 1: the general state of rats was observed,the appearance of kidney was observed with naked eyes,the pathological changes of kidney tissue were observed under light microscope,and the number of apoptotic cells was detected by TUNEL staining.The levels of serum creatinine(SCR)and urea nitrogen(BUN)were detected by automatic biochemical analyzer.Serum IL-1 was detected by enzyme-linked immunosorbent assay(ELISA)??IL-6?TNF-? Level.Experiment 2: TLR4,MyD88 and NF were detected by Western blot and immunohistochemistry-?B protein expression.Experiment 3: Western blot was used to detect the expression of Fas,Fas L,caspase8 and Caspase3 in kidney tissue.Experiment 4: the expressions of Akt and p-Akt in kidney tissue were detected by Western blot.Results:1Effect of shenshuaiyin on renal morphology and function in rats with chronic renal failure1.1 effect of shenshuaiyin on general state of rats with chronic renal failureNormal group: good mental state,glossy hair color,less depilation,normal activity,flexible reaction,normal daily food intake and water intake,mild odor of padding,normal stool quality,no abnormal defecation.Model group: weight decreased significantly,mental state was depressed,hair color was dry and dark,hair removal occurred,slow reaction,decreased activity,decreased food intake,increased drinking water,smelly and dirty bedding,and loose stool.Shenshuaiyin group: weight loss in different degrees,mild mental depression,dry hair color,light depilation,moderate reaction,moderate activity,moderate food intake,slightly increased water consumption,smelly and filthy padding,normal stool quality,no abnormal defecation.Niaoduqing group: weight decreased in different degrees,mental state was depressed,hair color was dry and fell off,mental state was moderately depressed,reaction was slightly slow,activity was reduced,food consumption was reduced,water consumption was increased,bedding material smell was foul and loose stool.1.2 effect of shenshuaiyin on renal morphology in rats with chronic renal failure1.2.1 effect of shenshuaiyin on the appearance of renal tissue in rats with chronic renal failure Compared with the normal group,the renal volume of the other groups increased in varying degrees.The most obvious change was in the model group,followed by Niaoduqing group,and finally shenshuaiyin group.Compared with the normal group,the surface of renal tissue in the model group showed diffuse fine granular changes,uneven,membrane adhesion,not easy to peel off,and the appearance color was obviously white.The degree of appearance color whiteness was observed by naked eye: model group >Niaoduqing group > shenshuaiyin group > normal group.In addition to the blank group,the renal cortex of each group was thinned to varying degrees,and the boundary of cortex and medulla was not clear.The model group was the most obvious,and there was no significant difference in other groups.1.2.2 effect of shenshuaiyin on body weight and renal index in rats with chronic renal failure There was no significant difference in the body weight of rats in each group before experiment and at the first week of modeling;compared with the normal group,the body weight of rats in the model group decreased significantly at the second and third weeks(P <0.05);during the treatment: at the first week,compared with the normal group,the body weight of rats in the model group,Niaoduqing group and shenshuaiyin group decreased significantly(P < 0.01);at the second and third weeks,compared with the normal group,the body weight of rats in the model group and Niaoduqing group decreased significantly(P <0.01)At the 4th week,compared with the normal group,the body weight of the model group,the Niaoduqing group and the shenshuaiyin group decreased significantly(P < 0.01),and the renal index increased significantly(P < 0.01).Compared with the model group,the body weight of the Niaoduqing group and the shenshuaiyin group decreased significantly(P < 0.01)The body weight of rats in shuaiyin group increased significantly(P < 0.05),while the renal index decreased(P < 0.01 in Niaoduqing group and P < 0.05 in shenshuaiyin group).There was no significant difference in body weight between Niaoduqing group and shenshuaiyin group.1.2.3 effect of shenshuaiyin on renal pathology in rats with chronic renal failure Hematoxylin eosin(HE)staining results: in the normal group,the structure of glomerulus,renal tubule and mesangium was normal,and the interstitium was not widened,and there was no inflammatory cell infiltration.In the model group,there were enlargement of glomerular cysts,atrophy of some renal tubules,uneven thickness of tubular wall,diffuse vacuolar degeneration of renal tubular epithelial cells,increase of fibroblasts,obvious proliferation of mesangial cells,and infiltration of a large number of inflammatory cells around the glomerulus.In Niaoduqing group,vacuolar degeneration of renal tubular epithelial cells was alleviated,mesangial cells were slightly proliferated,and inflammatory cell infiltration was found in some areas.In shenshuaiyin group,fibroblasts decreased,mesangial cells proliferated slightly,and inflammatory cells infiltrated in some areas.The degree of vacuolar degeneration of renal tubular epithelial cells and mesangial cell proliferation in shenshuaiyin group was lower than that in Niaoduqing group.1.3 effect of shenshuaiyin on serum SCr and BUN levels in rats with chronic renal failure Compared with the normal group,the serum levels of SCr and BUN in the model group were significantly increased(P < 0.01);compared with the model group,the serum levels of SCr and BUN in the Niaoduqing group and the shenshuaiyin group were significantly decreased(P < 0.01),but there was no significant difference between the Niaoduqing group and the shenshuaiyin group.2Effect of shenshuaiyin on renal cell apoptosis in rats with chronic renal failure TUNEL staining and microscopic observation: the cells in the kidney tissue of the normal group were normal,and no positive cells were found;the number of apoptosis in the kidney of the model group was significantly increased,and mainly distributed in the medullary area;the apoptosis index in the kidney of the Niaoduqing group and the shenshuaiyin group was lower than that of the model group(P < 0.01);there was no significant difference between the Niaoduqing group and the shenshuaiyin group.3Effect of shenshuaiyin on serum IL-1?,IL-6 and TNF-? levels in rats with chronic renal failureELISA detection of serum IL-1?,IL-6,TNF-? levels: compared with normal group,the expression levels of serum IL-1?,IL-6,TNF-? in model group were significantly increased(P < 0.01);compared with model group,the expression levels of serum IL-1?,IL-6,TNF-? in Niaoduqing group and shenshuaiyin group were significantly decreased(P< 0.01);compared with Niaoduqing group,the expression levels of serum IL-6,TNF-? in shenshuaiyin group were significantly decreased(P < 0.01)The level of serum creatinine was significantly lower than that of control group(P < 0.05).4Effect of shenshuaiyin on the expression of TLR4,MyD88 and NF-k B in renal tissue of rats with chronic renal failureThe protein expression levels of TLR4,MyD88 and NF-k B in kidney tissue were detected by Western blot: compared with the normal group,the protein expression levels of TLR4,MyD88 and NF-k B in kidney tissue of model group were significantly increased(P < 0.01);compared with the model group,the protein expression levels of TLR4,MyD88 and NF-k B in kidney tissue of Niaoduqing group and shenshuaiyin group were significantly decreased(P <0.01),while those of Niaoduqing group and shenshuaiyin group were significantly decreased(P < 0.01)There was no significant difference between the two groups.Immunohistochemistry was used to detect the expression of TLR4,MyD88 and NF-?B protein in renal tissue of rats in each group: under 200 times light microscope,the expression of TLR4,MyD88 and NF-?B protein in normal group was negative,and TLR4,MyD88 and NF-?B protein in model group was strongly positive.Niaoduqingkeli and shenshuaiyin could reduce the expression of TLR4,MyD88 and NF-?B protein in varying degrees after intervention(P < 0.05)There was no significant difference between the two groups.5The effect of shenshuaiyin on the expression of Fas,Fas L,caspase8 and Caspase3 in renal tissue of rats with chronic renal failureThe protein expression of Fas,Fas L,caspase8 and Caspase3 in kidney tissue was detected by Western blot: compared with normal group,the protein expression levels of Fas,Fas L,caspase8 and Caspase3 in kidney tissue of model group were significantly increased(P <0.01);compared with model group,the protein expression levels of Fas,Fas L,caspase8 and Caspase3 in kidney tissue of Niaoduqing group and shenshuaiyin group were significantly decreased(P < 0.01)There was no significant difference between Niaoduqing group and shenshuaiyin group.6Effect of shenshuaiyin on the expression of PI3 K and Akt protein in renal tissue of rats with chronic renal failureThere was no significant difference in the expression of Akt protein in the kidney of each group.Compared with the normal group,the expression level of p-Akt protein and the ratio of p-Akt / Akt in the model group were decreased(P < 0.01);Compared with the model group,the expression level of p-Akt protein and the ratio of p-Akt / Akt in kidney tissue of the ly group were significantly decreased(P < 0.01);Compared with ly group,the expression level of p-Akt protein and the ratio of p-Akt / Akt in kidney tissue of shenshuaiyin + ly group were significantly increased(P < 0.01).Conclusion:1Based on the thinking of TCM image and the pathological image of kidney viscera,it is proposed that spleen and kidney deficiency is the pathological basis of the occurrence and development of chronic renal failure and the source of toxin;Toxin(dampness toxin,phlegm toxin,Blood Stasis Toxin)is an important external cause of aggravating the progress of the disease and prolonging the disease.2It is suggested that "anti toxin,detoxification and detoxification" should be used in one prescription.Shenshuai decoction can strengthen the spleen,benefit the kidney and strengthen the body to resist toxin;Dispel dampness,remove phlegm and remove blood stasis to detoxify;Tongfu Xiezhuo to detoxify,attack and tonic.3According to the above etiology,pathogenesis,treatment principles and methods,experimental studies show that:(1)Shenshuai Yin can significantly improve the general state and renal function of adenine induced chronic renal failure model rats,reduce the pathological damage of renal tissue,inhibit renal inflammatory response and apoptosis,further inhibit renal fibrosis,and achieve the purpose of treating chronic renal failure.(2)Shenshuaiyin can reduce TLR4,MyD88 and NF-?B in renal tissue of rats with adenine induced chronic renal failure,The expression of protein can inhibit the inflammatory reaction and alleviate the inflammatory injury of kidney.(3)Shenshuaiyin can reduce the protein expression levels of Fas,Fas L,caspase8 and Caspase3 in adenine induced chronic renal failure model rats,and inhibit the apoptosis of renal cells.(4)Shenshuaiyin may inhibit renal cell apoptosis in rats with chronic renal failure by activating PI3 K / Akt signaling pathway and further inhibiting its downstream target Fas L. |
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