Effect Of Paishi Granule On Oxidative Stress Injury And Occludin、ZO-1 Expression Of Renal Tubular Epithelial Cell In The Rat Of Nephrolithiasis

ObjectiveNephrolithiasis is a common and frequently-occuring disease in the clinic.It is a focal issue to seek a safe and efficacy of new therapies actively to prevent and treat calculus in the medical field.It has been has a history of over 30 years in treating nephrolithiasis with Paishi granules,Paishi granules have marked anti-inflammatory effect for treatment nephrolithiasis and the total effective rate of treatment is 86%.Calcium oxalate(CaOx) is the main component of nephrolithiasis.It is the key to the mechanism of nephrolithiasis that CaOx crystal induced oxidative stress(OS) and inflammatory injury in renal tubular epithelial cells. To observe the effects of Paishi granules to OS and inflammatory injury and tight junction(TJ) proteins occludin、 ZO-1 expression in renal tubular epithelial cells,the study established the calcium oxalate nephrolithiasis models in rats and stimulate renal epithelial cells of the rats by using oxalate(Ox) and CaOx crystal in vitro,meanwhile to intervene them with Paishi granules,in order to discussed the molecular mechanisms and targets that Paishi granules prevent and treat calculus and prevent its recurrence from new perspectives,and provided a theoretical basis that the prescription can be more widely used in clinic. Methods1 Animal experimentThe rats were administered intragastrically with ammonium chloride(AC) and ethylene glycol(EG) as stone agents to establish the model of nephrolithiasis.The SPF level 48 male and healthy 8-9-week-old Wistar rats, were randomly divided into 4 groups,12 rats in each group,after feeding applicability one week.The control group was given intragastric administration with normal saline,2ml/d;The model control group was given stone agents;The Paishi granules treatment group was given intragastric administration with stone agents and Paishi granules of 1.2g/ml suspension,2ml/day;The potassium citrate treatment group was given intragastric administration with stone agents and 25% potassium citrate,2ml/d. The experiment lasted for 28 days.At the end of the experiment,collecting and recording 24 h urine of the rats,and collecting blood samples from abdominal aortic,determine BUN、Cr、Ca2+、Mg2+、P3+ content in sersum by biochemical analyzer;eviscerating left kidney and immobilizing it in 4% paraformaldehyde solution.After paraffin embedding,the kidney of rats will be made into pathology slice,and staining with HE and Von Kossa’s,examinating the expression of TGF-β1、NF-κB、KIM-1、MCP-1、IL-6、OPN、AngII、AT1R、AT2R、ACE、ACE2、occludin and ZO-1 protein in kidney tissue of the rats by immunohistochemistry.Right kidney placed in liquid nitrogen.After tissue homogenating, the homogenate was detected as follows:①detecting activity of SOD and MDA content in kidney tissue;②extracting total RNA,detecting the amount of TGF-β1、NF-κB、KIM-1、MCP-1、IL-6、OPN、AngII、AT1R、AT2R、ACE、ACE2、occludin and ZO-1 in kidney tissue of rats by RT-PCR method;③extracting total protein,measuring the expression of occludin and ZO-1 protein in rats by Western blotting technique.2 In-vitro cell culture experimentsAfter the recovery and passage,the renal tubular epithelial cells of rats(NRK52E cells) were divided into five groups.The control group were still given 5ml DMEM culture medium.The injury group of calcium oxalate monohydrate(COM) were put 1.0mmol/L Ox plus 500μg/mL COM in 5ml DMEM culture medium. The treatment control group were put 1.0mmol/L Ox plus 500μg/mL COM and Low、medium and high concentration drug serum(contain 10%) in 5ml DMEM culture medium.48 h later,removing the above culture medium,douching the NRK52 E cells in all groups twice with neutral pH7.4 PBS solution,extracting total protein and total RNA of the NRK52 E cells,detecting the amount of occludin,ZO-1 mRNA and protein levels by Western Blotting and real time PCR. Results1 The change of general condition in the rat:During the experiment, all the rats survived.In the fourth week,the rats of model control group were anorexia,the hair was not glory,water reduction,urine output decreased,mental fatigue,body curled up and weight loss;the group of rats intervented by Paishi granules,whose weight and urine volume increased,were in good spirit,diet and activities were normal.2 The change of blood and urine biochemical indexes in the rat:the model group were more than the control group in the content of Ox、Ca2+、P3+ in urine and BUN、Cr、Ca2+、P3+ in serum,but less than them in the content of Mg2+ in urine and serum;After the intervention of Paishi granules, the content of Ox、Ca2+、P3+ in urine and BUN、Cr、Ca2+、P3+ in serum decreased,but the content of Mg2+ in urine and serum increased. The results show that Ox and CaOx crystals can lead to increased levels of Ox、Ca2+、P3+ in rat urine, decreased levels of stone formation inhibitor Mg2+,in order to promote stone formation; Paishi granule could inhibit stone formation may be by increasing levels of Ox、Ca2+、P3+ in rat urine, and decreasing levels of Mg2+ content increased.3 The pathomorphological changes of kidney in rats:compared with the control group,the colour and lustre of kidney in the model group rats is dim,and kindey tissue showed swelling markedly,prominent and scattered particles existing,white setion center,plenty of CaOx crystal formation,renal tubular expanding obviously;But after the intervention of Paishi granules,the surface colour of kidney tissue is normal,setion center is paler in color,and a small amount of CaOx kidney crystals scattered,few renal tubular dilated in varying degrees. The results showed that Ox and CaOx crystals could stimulate the kidneys of rats and make the kidney pathological changes in rats. Paishi granule may protect the kidney and prevent the formation of stone by intervening the process of pathological changes.4 The change of OS and inflammatory injury in the rat: compared with the control group, SOD activity was decreased,MDA content and the expression of TGF-β1、NF-κB、KIM-1、MCP-1、IL-6、OPNmRNA and protein in renal tissue of rats of the model group were increased significantly;After the intervention of Paishi granules,SOD activity was increased, MDA content and the expression of TGF-β1、NF-κB、KIM-1、MCP-1、IL-6、OPNmRNA and protein were decreased. The results showed that Ox and CaOx crystals could increase ROS production and the expression of TGF-β1、NF-κB、MCP-1、IL-6,etc,and promot OS and inflammation injury of kidney epithelial cell,increase the expression of OPN,promote crystal adhesion,nucleation and aggregation.The intervention of Paishi granules could directly or indirectly reduce production of ROS and expression of TGF-β1、NF-κB、MCP-1、IL-6,etc,and reduce OS and inflammation injury of kidney epithelial cell,to protect kidney epithelial cells; In addition,Paishi granules can regulate the level of OPN protein expression of renal tissue,interfere in the adhesion, nucleation and aggregation of CaOx crystals,thereby inhibiting the Ca Ox crystals from precipitating.5 The change of RAS related indicators in the rat: compared with the control group,the expression of AngII、AT1R、ACE2mRNA and protein in renal tissue of rats of the model group increased obviously,the expression of AT2R、ACE2 mRNA and protein decreased;But after the intervention of Paishi granules, the expression of AngII、AT1R、ACEand protein in renal tissue of rats of the model group increased obviously,the expression of AT2 R and ACE decreased. The results showed that Ox、CaOx crystals can activate the local RAS in the kidney of rats,increase the expression of ACE to turn AngI into AngII.The plentiful AngII and AT1 R plays the important role in the promotion of OS and inflammatory lesions.Meanwhile,AngII also mediate the expression of OPN to promote the nucleation and aggregation of crystal.The intervention of Paishi granules on CaOx stones may be through upregulation of ACE2, which can turn AngI into Ang-(1-9) or degradate AngII into Ang-(1-7),in order to descend the level of AngII and increase the expression of AT2 R in kidney,consequently,antagonise the AngII effect by AT1 R mediated to relieve OS and inflammation injury of kidney epithelial cell,which aim to protect the kidneys.6 The change of TJ of the rat’s renal tubular epithelial cells in vitro and in vivo: ①Animal experiment:compared with the control group,the expression of occludin 、 ZO-1reduced significantly;After the intervention of Paishi granules,the expression of occludin、ZO-1 increased.②In-vitro cell culture experiments:compared with the control group,the expression of occludin、ZO-1 in NRK52 E cells reduced significantly;After the intervention of Paishi granules,the expression of occludin、ZO-1 in NRK52 E cells increased, and following the increasing concentrations of the drug serum,the expression was increased. The results showed that Ox and CaOx crystals may result in OS and inflammation injury of kidney epithelial cell by activating cytokines(TGF-β1、NF-κB、MCP-1、IL-6,etc.),which led to the damage of intercellular TJ. TJ damage can lead to the migration of CaOx crystal、Ox、plasma calcium and phosphate into epithelial cells in the renal tubules,and start the renal tubular interstitial inflammatory injury and Randall’s plaque formation,which may be a foundation of stone formation and growth.After the intervention of Paishi granules,the expression of occludin and ZO-1 increased, which indicated that Paishi granules acted directly or indirectly on TJ to promote the recovery of the structure and function of TJ in order to achieve the purpose of prevention and treatment of nephrolithiasis. Conclusion1 Establishing models of rats with calcium oxalate stones successfully by glycol and ammonium chloride solution gavage.2 Ox and CaOx crystals could induce the expression of related cytokines, result in the OS and inflammatory injury of renal tubular epithelial cells, promote the adhesion、nucleation and aggregation of crystals; Paishi granules may interfere with the process to achieve the purpose of prevention calculus formation and protecting the kidney.3 Ox and CaOx crystals could activate the local RAS of rat kidney, promote OS and inflammatory injury,accelerate nucleation and aggregation of crystals;Paishi granule may have antagonistic effect on the expression of the related effector molecules,to alleviate the OS and inflammatory injury,reaching the purpose of prevention calculus formation and protecting the kidney.4 Ox and CaOx crystals could destroy the structure of TJ in renal tubular epithelial cells in vivo and in vitro,promote cell injury and calculus formation;Paishi granule may alleviate the damage and inhibit the adhesion of crystals by restoring the expression of TJ related proteins, to achieve the purpose of prevention calculus formation and protecting the kidney.