| Objective Urolithiasis is a common clinical disease.It can cause great pain and high recurrence rate.The pathogenesis of the disease has not been fully clarified.Paishi Granule is an empirical formula of a famous TCM scholar in Hubei,the compatibility is rigorous,the clinical effect is definite.In this study,calcium oxalate calculus rats were used as models.Based on the P38 MAPK pathway,the effects of Paishi Granule on oxidative stress and inflammation,changes of RAS system and tight junction in kidneys of rats induced by oxalic acid and calcium oxalate crystallization were observed.The molecular mechanism and target of Paishi Granule on calcium oxalate calculi formation were explored to provide theory for its further development and clinical application.Method1 Animal Grouping,Modeling and Sampling1.1 Animal grouping and modeling: 48 male SPF Wistar rats(weighing180g-220g)were randomly divided into four groups: group A,B,C and D.Group A,blank group,12 rats(normal rat feed and saline infusion,1ml/100g/time,once a day);group B,model group,12 rats(normal rat feed,1% ethylene glycol free drinking water and 2% ammonium chloride solution infusion,lml/100g/time).Group C,Paishi Granule Group,12 rats(0.58g/ml of suspension was prepared before using Paishi Granule(close to clinical dosage according to body surface area).The model was made by oral administration of Chinese medicine suspension at the same time,1ml/100g/time,once a day).Group D,potassium citrate group,12 rats(potassium citrate,25% potassiumcitrate solution,lml/100g/time,once a day).1.2 Extraction of animal specimens: On the 28 th day of sampling experiment,rats were placed in metabolic cages to collect 24-hour urine,which was divided into four markers.Blood samples were collected from abdominal vessels(abdominal aorta and inferior vena cava)of rats and placed in non-anticoagulant specimens tube for clinical use.Rat kidneys were removed and fixed in 10% formaldehyde solution on the right kidney;liquid nitrogen was placed on the left kidney after labeling,and then moved into the refrigerator at-80℃ for storage.2 Specimens Detection2.1 Urinary oxalic acid and urinary biochemistry(urinary calcium,phosphorus and magnesium)were detected by biochemical examination,and serum samples were separated to detect serum BUN and Cr levels,in order to understand the physical and chemical risk factors of stone formation and kidney function of Paishi Granule.2.2 Paraffin sections were made from the fixed spare renal tissue specimens to observe the renal crystalline precipitation and renal pathological changes(Von Kossa’s staining).2.3 Immunohistochemical method was used to detect the expression of TGF-beta1,MCP-1,Ang II,AT1 R,ACE,ACE 2 and p38MAPK/p-p38 MAPK,occludin and ZO-1 protein in kidney tissue of rats in each group.2.4 Real Time PCR was used to detect the expression of TGF-beta 1,MCP-1,Ang II,AT1 R,ACE,ACE 2 and p38 MAPK,occludin and ZO-1m RNA in kidney tissue of rats in each group.2.5 Western blotting was used to detect the expression of p38 MAPK,occludin and ZO-1 in kidney tissue of rats in each group.Results1 General condition,body weight,urine,serum biochemistry andmorphological changes of kidney in rats1.1 Generally speaking,rats in group B suffered from loss of appetite,reduced drinking water,unglorious hair,mental fatigue,curled up body and reduced urine volume at the 4th week.In group D and C,urine volume were increased,mental state was good and dietary activities were basically normal.The effect of group C was more obvious than that of group D.1.2 There was no significant difference in body weight of rats before treatment(P > 0.05).The body mass of rats after treatment was compared.Body weight after treatment: Compared with group A,group B and group C had lower body weight(P<0.01).Compared with group B,group C and group D had higher body weight(P<0.01).1.3 The levels of oxalic acid,calcium and phosphorus in urine in group B were significantly higher than those in group A,while the levels of magnesium in urine were significantly lower than those in group A.The levels of BUN and Cr in serum of rats in group B were higher than those in blank group.The levels of oxalic acid,calcium and phosphorus in urine in group C were decreased,and the levels of BUN and Cr were also decreased compared with group B.1.4 The Ca Ox crystallization score of kidney tissue in group B,C and D was significantly higher than that in group A(P<0.01);compared with group B,the Ca Ox crystallization score in group C and D was significantly lower(P<0.01).2 Effect of Paishi Granule on oxidative stress and inflammatory damage in rats by intervention of P38 MAPK signaling pathway: The level of SOD in group B decreased,the level of MDA increased;and the expression levels of p/p-p38 MAPK,MCP-1,TGF-beta 1 increased,while the expression levels of MDA,p/p-p38 MAPK,MCP-1,TGF-beta 1 in group C decreased and SOD level increased.It is suggested that Paishi Granule may alleviate oxidativestress and inflammation in rat kidney induced by oxalic acid and calcium oxalate crystals by interfering with P38 MAPK pathway.3 Effect of Paishi Granule on the changes of local RAS system in rats by interfering with P38 MAPK signaling pathway: Compared with group A,p/p-p38 MAPK,Ang II and AT1 R,ACE m RNA and protein levels were increased,and ACE2 m RNA and protein expression levels were decreased.Compared with group B,p38 MAPK,Ang II and AT1 R,ACE m RNA and protein levels were decreased,and ACE2 m RNA and protein expression levels were increased.It is suggested that Paishi Granule may affect the changes of RAS system induced by oxalic acid and calcium oxalate crystals,alleviate renal injury and inhibit the formation of stones in rats by interfering with P38 MAPK pathway.4 The effect of Pai Shi Granule on the changes of rats tight junction protein by interfering with P38 MAPK signaling pathway: The expression of p/p-p38 MAPK protein and m RNA was increased in group B compared with group A,and the expression of ZO-1 and occludin protein and m RNA was higher than group A.The expression of p/p-p38 MAPK protein and m RNA was lower in group C than in group B.The expression of ZO-1 and occludin was higher than that in group B,suggesting that Paishi granules may affect oxalic acid and calcium oxalate crystal induced tight junction changes in model rats by interfering with P38 MAPK pathway.conclusion Paishi Granule may affect the oxidative stress and inflammation response of kidney in rats induced by oxalic acid and calcium oxalate crystals,the change of RAS system in kidney and the change of renal tight junction by interfering with P38 MAPK pathway,thus inhibiting the formation of calcium oxalate stones and protecting kidney. |
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