| Purpose:Based on the theory of "kidney deficiency and toxic evil stasis",this research focuses on the functions of Shenshuaiyin in strengthening spleen and stomach,removing stasis and toxin.It investigates the influence of Shenshuaiyin on chronic renal failure rats via the MAPK signal pathway,endoplasmic reticulum stress,apoptosis and inflammatory factors.This thesis aims to discuss the mechanism of Shenshuaiyin in treating and preventing chronic renal failure.Materials and Methods:52 SD male SPF rats(weight 190±20g)were randomly divided into two groups:normal group(n=10)and experimental group(n=42).The experimental group were induced by adenine gavage,with dose of 200 mg /(kg·d)once a day.The total dose should be within 1.5g per rat.The normal group was treated with 0.9% NaCl 8ml/(kg·d)once a day.After 3 weeks,the experimental group were randomly classified into model group,Shenshuayin group and Niaoduqing group.The normal and model groups were fed with the same volume of 0.9% NaCl solution.Shenshuaiyin group were fed with raw herbs with dose of 33.3g/(kg·d)once a day.Niaoduqing group by feeding niaoduqing particle 2.25g/(kg·d)once a day.Rats in each group were given intragastric administration for 4 weeks continuously.Serum and renal tissues were collected and the indexes were measured.Experiment 1: Serum creatinine(Scr)and blood urea nitrogen(BUN)were tested by the automatic biochemical analyzer.The morphological changes of the kidneys were observed by naked eyes.The pathological changes of Masson dyeing and HE dyeing in each group were observed.Experiment 2: Western blot was used to detect the expression of p38,p-JNK and p-ERK of the renal tissue in each group.Experiment 3:The expression of GRP78,ATF6,CHOP,Bax,Bcl-2 and caspase3 in renal tissue of each group was detected by Western blot.The expressions of GRP78,CHOP and caspase-3 were detected by immunohistochemistry(IHC)in renal tissues.Experiment 4: Western blot was used to detect the expression of NF-κB in renal tissue of each group.The expression of TNF-α and MCP-1mRNA in renal tissue of each group was detected by real-time PCR.The expression of TNF-α and MCP-1 mRNA was detected by enzyme-linked immunosorbent assay(ELISA).Results:1.Effects of Shenshuaiyin Decoction on Renal Function and Kidney Morphology of Rats with Chronic Renal Failure1.1 Effects of Shenshuaiyin Decoction on the General State of Chronic Renal Failure RatsIn the normal group,the color,mental state,reactive sensitivity,activity,behavior,food intake,water consumption and stool discharge were normal and the weight gain was faster.Compared with the normal group,the rats in the model group were lighter and fluffy and easy to hair removal,apathetic,poor ability to stress,reduced activity,arch curled up,and like to curl up together,intaking less amount of food,drinking more water,watery stool,pale red eyes,pale and whitish color around the mouth,wet tail,almost normal weight gain(P<0.01).Compared with the model group,the rats in the Shenshuaiyin group and the Niaoduqing group were slightly dry and mildly dispirited,the reaction and the activity were almost normal,eat and drink more water than rats in the model group do,the quality of faeces was slightly thin,weight increased significantly(P<0.01).The mental status,sensitivity and food intake in the Shenshuaiyin group are better than those in the Niaoduqing group,especially on weights(P<0.01).1.2 Effects of Shenshuaiyin Decoction on Rats with Chronic Renal FailureCompared with the normal group,the serum creatinine and urea nitrogen in the model group were significantly higher(P<0.01).Compared with the model group,the serum creatinine and urea nitrogen were significantly decreased(P<0.01).Compared with the Niaoduqing group,the levels of serum creatinine and urea nitrogen in Shenshuaiyin group were significantly decreased(P<0.01).1.3 Effects of Shenshaiyin Decoction on Rats with Chronic Renal Failure in KidneyMorphology1.3.1 HE DyeingNormal group: Glomerular structure,size,shape,glomerular cysts and the proportion of blood vessels were normal;normal proximal and distal small tube lumen size,normal thickness of wall,no inflammatory cell infiltration.Model group: Glomerular structure,shape and size changed.Vascular ball atrophy and glomerular cysts become larger.Imbalance of proportion,proximal tubules and distal tubule wall thickness was uneven,epithelial degeneration with necrosis and swelling,a large amounts of brown and yellow adenine metabolites crystal deposition were visible in renal interstices and a large number of infiltration of inflammatory cells were visible.Shenshuaiyin group: Glomerular morphology,structure and size were close to normal.A slight atrophy of blood vessels.Glomerular cysts have changed.The proportion was basically normal.Thickness of proximal tubule and distal tube were more even.Swellingwere relieved.There are occasionally a small amount of adenine metabolites crystal deposition and infiltration of inflammatory cells in interstices.Niaoduqing group: Glomerular morphology,structure and size were close to normal.Filling of blood ball,swelling and necrosis of glomerular cyst.the proportion has changed,thickness of proximal tubule and distal tube wall and lumen size were basically normal.A mild amount of adenine metabolites crystal deposition and infiltration of inflammatory cells were visible in interstices.1.3.2 Masson Dyeing ResultsNormal group: There were no blue collagen fibers in the renal tissue,most of them were red or pale red,and the blue area was limited to the part of the blood vessels.Model group: The kidney tissue was blue in large area,and the color was uneven.Coloration in different structures was not clear.It is difficult to distinguish between the collagen fibers and muscle fiber.Shenshuaiyin group: The results showed that the color of the collagen fibers in the renal tissue was similar to that in the normal group,while most of the muscle fibers were red or pale red,and the collagen fibers were clear.Niaoduqing group: Collagen fiber of kidney tissue under the microscope presented blue in small area.Cytoplasm of muscle fiber is mostly red or pale red.collagen fiber boundary was less clear.Perivascular areas were occasionally blue in some parts,and the coloration is pale.2.Effects of Shenshuaiyin Decoction on Rats with Chronic Renal Failure in Expression of MAPK Pathway Associated Protein in Renal Tissue2.1 Effects of Shenshuaiyin Decoction on Phosphorylation of p38 Protein in Renal Tissue of Chronic Renal Failure RatsWestern blot was used to detect the expression of p38 and p-p38 protein in renal tissue of rats.Compared with the normal group,the expression of p-p38 protein in the model group was significantly higher than that in the normal group(P<0.01).Compared with the model group,the expression of p-p38 protein was significantly lower in Niaoduqing and Shenshuaiyin group(P<0.01).Compared with the Niaoduqingl group,the expression of p-p38 protein was lower in Shenshuaiyin group(P<0.05).2.2 Effects of Shenshuaiyin Decoction on JNK Protein Phosphorylation in Renal Tissue of Chronic Renal Failure RatsWestern blot was used to detect the expression of JNK and p-JNK protein in renal tissue of rats.Compared with the normal group,the expression of p-JNK protein in the model group was significantly higher.(P<0.01).Compared with the model group,the expression of p-JNK protein in the Shenshuaiyin and Niaoduqing group was significantly lower(P<0.01).Compared with the Niaoduqing group,the expression of p-JNK protein in Shenshuaiyin group presented no significant difference(P>0.05).2.3 Effects of Shenshuaiyin Decoction on ERK Protein Phosphorylation in Renal Tissue of Chronic Renal Failure RatsWestern blot was used to detect the expression of ERK and p-ERK protein in renal tissue of rats.Compared with the normal group,the expression of p-ERK protein in was significantly higher in model group(P<0.01).Compared with the model group,the expression of p-ERK protein in the Shenshuaiyin and Niaoduqing group was significantly lower(P<0.01).Compared with the Niaoduqing group,the expression of p-ERK protein in Shenshuaiyin group presented no significant difference(P>0.05).3.Effects of Shenshuaiyin Decoction on Endoplasmic Reticulum Stress and Apoptosis-related Factors in Renal Tissue of Chronic Renal Failure Rats3.1 Effects of Shenshuaiyin Decoction on Rats with Chronic Renal Failure in GRP78 Protein Expression in Renal Tissue3.1.1 The Expression of GRP78 Protein in Rats Kidney was Detected by Western BlotCompared with the normal group,the expression of GRP78 protein in the renal tissue of the model group was significantly increased(P<0.01).Compared with the model group,the expression of GRP78 protein was significantly lower in Shenshuaiyin and Niaoduqing group(P<0.01).Compared with the Niaoduqing group,the expression of GRP78 protein was decreased in Shenshuaiyin group(P<0.01).3.1.2 Expression and Distribution of GRP78 in Rats Renal Tissue by Immunohistochemistry(IHC)Microscopic display: The GRP78 protein of normal group in the distal renal tubules was only expressed in a small amount.Compared with the normal group,the model group showed a large number of brown and yellow positive granules in the distal and proximal renal tubules.The GRP78 of Shenshuaiyin group and Niaoduqing group was mainly expressed in the distal renal tubules,and the content was decreased compared with the model group.Compared with the Niaoduqing group,the expression of GRP78 in the distal renal tubular in Shenshuaiyin group was slightly decreased.Integral optical density:Compared with the nomal group,the integral optical density of the model group was significantly higher(P<0.01).Compared with the model group,the optical density of the Shenshuaiyin and Niaoduqing group was significantly lower(P<0.01,P<0.05).Compared with the Niaoduqing group,the integral optical density of Shenshuaiyin group decreased significantly(P<0.05).3.2 Effects of Shenshuaiyin Decoction on Rats with Chronic Renal Failure in ATF6 Protein Expression in Renal TissueWestern blot was used to detect the expression of ATF6 protein in rat kidney.Compared with the normal group,the expression of ATF6 protein in the model group was significantly higher(P<0.01).Compared with the model group,the expression of ATF6 protein in the Shenshuaiyin and Niaoduqing group wassignificantly lower(P<0.01).There was no significant difference in the expression of ATF6 protein in Shenshuaiyin compared with that in Niaoduqing group(P>0.05).3.3 Effects of Shenshuaiyin Decoction on Rats with Chronic Renal Failure in CHOP Protein Expression in Renal Tissue3.3.1 The Expression of CHOP Protein in Rats Renal Tissue was Detected by Western BlotCompared with the normal group,the expression of CHOP protein in the model group was significantly increased(P<0.01).Compared with the model group,the expression of CHOP protein was significantly lower in the Shenshuaiyin and Niaoduqing group(P<0.01).Compared with the Niaoduqing group,the expression of CHOP protein in Shenshuaiyin group was decreased(P<0.01).3.3.2 Expression and Distribution of CHOP in Rats Renal Tissue by IHCMicroscopic display: The expression of CHOP in the renal tubular epithelial cell cytoplasm was slight in the normal group.Compared with normal group,the expression of CHOP in the renal tubular epithelial cell cytoplasm was significantly higher than that in the model group.Compared with the model group,the expression of CHOP in the cytoplasm of renal tubular epithelial cells was decreased in the renal Shenshuaiyin group and the Niaoduqing group.Compared with the Niaoduqing group,the expression level of CHOP in the cytoplasm of renal tubular epithelial cells in Shenshuaiyin group was slightly decreased.Integral optical density:Compared with the nomal group,the integral optical density of the model group was significantly higher(P<0.01).Compared with the model group,the integral optical density of the Shenshuaiyin group and the Niaoduqing group was significantly lower(P<0.01).Compared with the Niaoduqing group,the integral optical density of Shenshuaiyin group was significantly decreased(P<0.05).3.4 Effects of Shenshuaiyin Decoction on Expression of Apoptosis-related Factors in Renal Tissue of Chronic Renal Failure Rats3.4.1 Effect of Shenshuaiyin Decoction on Bcl-2 / Bax Ratio in Renal Tissue of Chronic Renal Failure RatsWestern blot was used to detect the ratio of Bcl-2/Bax in renal tissue of rats.Compared with the normal group,the ratio of Bcl-2/Bax in the renal tissue of the model group was significantly lower(P<0.01).Compared with the model group,the ratio of Bcl-2/Bax significantly increased greatly in the Niaoduqing and Shenshuaiyin group(P<0.01).Compared with the Niaoduqing group,the ratio of Bcl-2/Bax in renal tissue of Shenshuaiyin group was significantly increased(P<0.01).3.4.2 The Effects of Shenshuaiyin Decoction on caspase-3 in Renal Tissue of Chronic Renal Failure Rats3.4.2.1 The expression of caspase-3 protein in rat kidney was detected by Western blotCompared with the normal group,the expression of caspase-3 protein in the renal tissue of the model group was significantly higher(P<0.01).Compared with the model group,the caspase-3 protein significantly decreased in the Shenshuaiyin and Niaodqing group(P<0.05,P<0.01).Compared with the Niaoduqing group,the caspase-3 protein of Shenshuaiyin group was significantly decreased(P< 0.05).3.4.2.2 IHC detected caspase-3 expression and distribution in rat kidneyMicroscopic display: The caspase-3 in the interstitial cells of the cytoplasm and renal tubular epithelial cells have a very small amount of expression in normal group.Compared with the normal group,the caspase-3 in the glomerular mesangial cells and endothelial cells are brown granules of nuclear staining in model group.Compared with the model group,caspase-3 in Shenshuanyin group and Niaoduqing group showed a small amount of expression in glomerular RTEC pulp.Compared with the Niaoduqing group,the expression of caspase-3 in renal decay group was slightly decreased in RTEC pulp.Integral optical density:Compared with the normal group,the integral optical density of the model group was significantly higher(P<0.01).Compared with the model group,the optical density of the Shenshuaiyin and Niaoduqing group was significantly lower(P<0.01).Compared with the Niaoduqing group,the integral optical density of Shenshuaiyin group was significantly decreased(P<0.05).4.Effects of Shenshuaiyin Decoction on Inflammatory Related Factors in Renal Tissue of Chronic Renal Failure Rats4.1 Effects of Shenshuaiyin Decoction on NF-κB Protein in Renal Tissue of ChronicRenal Failure RatsCompared with the normal group,the expression of NF-κB protein in the renal tissue of the rats in the model group was significantly higher(P<0.01).Compared with the model group,the expression of NF-κB protein in the renal tissue of the rats was significantly decreased in Shenshuaiyin group and Niaoduqing group(P<0.01).Compared with the Niaoduqing group,the expression of NF-κB protein in renal tissue of Shenshuaiyin group was decreased(P<0.05).4.2 Effects of Shenshuaiyin Decoction on TNF-α Gene and Protein in Renal Tissue of Chronic Renal Failure Rats4.2.1 Real-time PCR was used to detect the expression of TNF-αmRNA in rat kidney Compared with the normal group,the expression of TNF-αmRNA protein in the renal tissue of the model group was higher(P<0.01).Compared with the model group,the expression of TNF-αmRNA protein in the Shenshuaiyin group and Niaoduqing group was decreased(P<0.01).Compared with the Niaoduqing group,the expression of TNF-αmRNAprotein in renal tissue of Shenshuaiyin group was decreased(P<0.05).4.2.2 ELISA was used to detect the expression of TNF-α protein in rat kidney Compared with the normal group,the expression of TNF-α protein in the renal tissue of the model group was significantly higher(P<0.01).Compared with the model group,the expression of TNF-α protein in the renal tissue of the rats in the Shenshuaiyin group and Niaoduqing group was decreased(P<0.01).Compared with the Niaoduqing group,the expression of TNF-α protein in renal tissue of Shenshuaiyin group was decreased(P<0.05).4.3 Effects of Shenfu Decoction on MCP-1 Gene and Protein in Renal Tissue of Chronic Renal Failure Rats4.3.1 Real-time PCR was used to detect the expression of MCP-1 mRNA in rat kidneyCompared with the normal group,the protein expression of MCP-1 in the renal tissue of the model group was increased(P<0.01).Compared with the model group,the expression of MCP-1 protein in the renal tissue of the rats in the Shenshuaiyin group and Niaoduqing group was decreased(P<0.01).Compared with the Niaoduqing group,There is no significant difference in the expression of MCP-1 protein in renal tissue ofShenshuaiyin group(P>0.05).4.3.2 The expression of MCP-1 protein in rat kidney was detected by ELISACompared with the normal group,the expression of MCP-1 protein in the r enal tissue of the model group was increased(P<0.01).Compared with the mode l group,the expression of MCP-1 protein in the renal tissue of the rats in the S henshuaiyin group and Niaoduqing group was decreased(P<0.01).Compared wit h the Niaoduqing group,there is no significant difference in the expression of MCP-1 protein in Shenshuaiyin group(P>0.05).Conclusion:1.Innovative the theory of "kidney deficiency and toxic evil stasis"."Poison evil",includes dampness,phlegm poison,toxic evil stasis,and the toxic evil stasis is the focus of the pathogenesis,kidney deficiency is the basis of the disease,the syndrome is root deficiency and branch excess.2.Found methods of "Invigorating spleen and kidney,Removing toxic evil s tasis".Invigorating spleen and kidney,aiming to reinforcing healthy qi to anti-to xic;Eliminating stasis and dampness for detoxification,focusing on removing sta sis and activating collaterals;Smoothing fu-organs to remove toxin,anti-toxic and detoxification.Supplementing healthy qi and eliminating pathogenic factors are c ombined together with Shenshuaiyin to treat early and mid-term CRF.3.Shenshauiyin can be used to improve general state of chronic kidney failure induced by adenine gavage,reduce inflammatory reaction and inhibit renal interstitial injury.4.Shenshauiyin can inhibit the phosphorylation of p38,JNK and ERK in the MAPK signal pathway of rats with chronic renal failure induced by adenine gavage.5.Shenshuaiyin could decrease the expression of GRP78,ATF6 and protein in rats with chronic renal failure induced by adenine and inhibit the ER stress response.By reducing the expression of Caspase3 protein and increasing the ratio of Bcl-2/Bax to regulate the level of apoptosis.6.The expression of NF-κB,TNF-α and MCP-1 in chronic renal failure model ratswas induced by adenine,and the inflammatory response was inhibited.7.Shenshuaiyin can treat CRF rats via adjusting MAPK-related signal pathway,improve the endoplasmic reticulum stress response and apoptosis levels,inhibit inflammatory factor expression. |
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