Effects of obacunone on renal interstitial fibrosis and ferroptosis in unilateral ureteral obstruction model mice / 中国药房

OBJECTVE To study the improvement effects of obacunone on renal interstitial fibrosis （RIF） in unilateral ureteral obstruction （UUO） model mice， and to investigate its mechanism based on ferroptosis mediated by nuclear factor erythroid 2- related factor 2（Nrf2）/glutathione peroxidase 4 （GPx4） signaling pathway. METHODS Thirty mice were randomly divided into sham operation group， model group， irbesartan group （positive control， 20 mg/kg）， obacunone low-dose and high-dose groups （10， 40 mg/kg）， with 6 mice in each group. Except for sham operation group， UUO model was established by ligation of unilateral ureter in other groups. After operation， administration groups were given intraperitoneal injection of relevant medicine， and sham operation group and model group were given intraperitoneal injection of constant volume of normal saline， once a day， for 7 consecutive days. The levels of creatinine （Cr） and blood urea nitrogen （BUN）， the content of malondialdehyde （MDA） and the activities of total superoxide dismutase （T-SOD） in serum and the concentration of Fe2+ in renal tissue were all detected. HE staining and Masson staining were performed to observe the morphology and the fibrosis of renal tissues. Immunohisto- chemical staining was used to determine expressions of the fibronectin （Fn）， α-smooth muscle actin （α-SMA）， GPx4 964083717@qq.com and Nrf2 in renal tissue. Western blot and real-time quantitative polymerase chain reaction （RT-qPCR） were used E-mail：834300205@qq.com to detect the protein and mRNA levels of Fn， α-SMA， Nrf2， GPx4 and SLC7A11 in the renal tissues. RESULTS Compared with sham operation group， serum levels of Cr and BUN， the concentration of Fe2+ in renal tissue， the protein and mRNA levels of Fn and α-SMA in model group were increased significantly （P＜0.05）， while the activity of T-SOD in serum， protein and mRNA expressions of Nrf2， GPx4， SLC7A11 in kidney tissue were significantly decreased （P＜0.05）； in the kidney tissue， the renal tubules were dilated， the collagen deposition was obvious， the fibrous bands were thicker and darker， and the renal interstitial inflammatory cells infiltrated significantly. After intervened with obacunone， the levels of above indexes （except for mRNA expression of SLC7A11 in obacunone low-dose group） in serum and renal tissue were reversed significantly （P＜0.05）， and pathological damage and collagen deposition of kidney tissue were alleviated. CONCLUSIONS Obacunone can improve renal interstitial fibrosis of UUO model mice， the mechanism of which may be associated with activating the Nrf2/GPx4 pathway and then inhibiting ferroptosis to relieve RIF in UUO model mice.

Effect and Mechanism of Baoshen Prescription in Alleviating Renal Fibrosis in UUO Mice by Regulating ERK/p38 MAPK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the protective effect of Baoshen prescription against renal fibrosis and explore its underlying mechanism through network pharmacology， molecular docking， and in vivo experiments. MethodAll mice were randomly divided into sham surgery group， model group， low-， medium-， and high-dose Baoshen prescription groups， and a benazepril hydrochloride group. Unilateral ureteral obstruction （UUO） was performed to establish a renal fibrosis model， and the administration of Baoshen prescription at low， medium， and high doses （0.455， 0.91， and 1.82 g·kg-1）， and benazepril hydrochloride （1.68 mg·kg-1） or distilled water began on the same day as model preparation. Mice in the model group and the sham surgery group were given an equal volume of distilled water. The intervention was carried out once daily for 14 days. Mouse serum levels of blood urea nitrogen （BUN） and creatinine （Cr） were measured. Hematoxylin-eosin （HE） staining and Masson staining were used to observe renal pathological changes. Western blot and immunohistochemistry were used to assess the expression of fibronectin （FN）， α-smooth muscle actin （α-SMA）， and E-cadherin， which are related to renal fibrosis. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression of transforming growth factor-β1 （TGF-β1）， tumor necrosis factor-α （TNF-α）， NOD-like receptor protein 3 （NLRP3）， and monocyte chemoattractant protein-1 （MCP-1） in renal tissues. The mechanism of Baoshen prescription in improving renal fibrosis was explored through network pharmacology， molecular docking， and Western blot experiments. ResultCompared with the sham surgery group， the model group showed significantly increased levels of BUN and Cr （P<0.01）. The model group exhibited abnormal renal glomerular morphology， loss of tubular brush borders， tubular dilation， and an enlarged area of blue collagen fibers. Mice in the model group showed significantly elevated levels of FN and α-SMA （P<0.01）， significantly decreased expression of E-cadherin （P<0.01）， and significantly increased expression of TGF-β1， TNF-α， NLRP3， and MCP-1 mRNA （P<0.05， P<0.01）. Compared with the model group， the Baoshen prescription groups showed significantly reduced BUN and Cr levels （P<0.01）， alleviated renal pathological damage， improved fibrosis， reduced expression of FN and α-SMA （P<0.01）， increased E-cadherin expression （P<0.01）， and downregulated mRNA expression of TGF-β1， TNF-α， NLRP3， and MCP-1 （P<0.05， P<0.01）. Network pharmacology and molecular docking predicted that Baoshen prescription could potentially improve renal fibrosis through the extracellular signal-regulated kinase （ERK）/p38 mitogen-activated protein kinase （MAPK） signaling pathway. Pharmacological research showed that compared with the sham surgery group， the model group exhibited significantly increased expression of phosphorylated （p）-ERK and p-p38 （P<0.05， P<0.01）. Compared with the model group， medium- and high-dose Baoshen prescription groups showed significantly downregulated expression of p-ERK and p-p38 proteins （P<0.05， P<0.01）. ConclusionBaoshen prescription can effectively improve renal fibrosis induced by UUO in mice， and its mechanism of action may be related to the ERK/p38 MAPK signaling pathway.

Effect of Yishen Huoxue Prescription on Endothelial Structure and Function in Mice with Renal Interstitial Fibrosis / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Yishen Huoxue prescription in renal interstitial fibrosis （RIF） from the perspective of endothelial cell and cell energy metabolism. MethodThe model was successfully established by unilateral ureteral obstruction （UUO）. Seventy-five SPF C57BL/6 mice were randomly divided into a model group， a resveratrol group （50 mg·kg-1·d-1）， three Yishen Huoxue prescription low， medium， and high-dose groups （7.1， 14.2， 28.4 g·kg-1·d-1）， with 15 mice in each group. In addition， another 15 mice were used to prepare sham operation model. Mice in the sham operation group and the model group were gavaged with equal volume of normal saline. All mice were sacrificed on 7， 14， and 21 d after modeling. The protein expression of platelet endothelial cell adhesion molecule 31 （CD31） was detected by immunohistochemical S-P method. The expression of α-smooth muscle actin （α-SMA）， collagen Ⅳ （Col-Ⅳ）， angiopoietin 1（Ang-1） and tyrosine kinase receptors 2 （Tie-2）， vascular endothelial growth factor （VEGF）， vascular endothelial cadherin （VE-cadherin）， and occludin in renal tissues was detected by Western blotting. The mRNA expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin in renal tissues were detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the levels of reactive oxygen species （ROS） in mice were detected by enzyme linked immunosorbent assay （ELISA）. ResultAs compared with the sham operation group， the expression of CD31 in renal tissues of the model group was significantly decreased and worsened with the extension of modeling time （P<0.05）， α-SAM and Col-Ⅳ protein expression levels were significantly increased （P<0.01）， but the expression of CD31 was stable in 14-21 d. ROS levels were significantly increased （P<0.01）， and the protein and mRNA expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin were significantly down-regulated （P<0.01）. As compared with the model group， the expression of CD31 was increased （P<0.05）， and α-SAM and Col-Ⅳ in the resveratrol group and the medium and high-dose Yishen Huoxue prescription groups were significantly decreased （P<0.01）. The ROS content was significantly decreased （P<0.01）， and the protein and mRNA expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin were up-regulated （P<0.01）， As compared with the resveratrol group， the protein expressions of Ang-1/Tie-2， VEGF， VE-cadherin， and occludin in the medium and low-dose Yishen Huoxue prescription groups were significantly different （P<0.01）. There was no significant difference in the mRNA expressions of CD31 and Ang-1/Tie-2 in the high-dose Yishen Huoxue prescription group， and no significant difference in the ROS level in the medium-dose Yishen Huoxue prescription group. ConclusionThe anti-RIF effect of Yishen Huoxue prescription may be related to promoting vascular endothelial repair， regulating mitochondrial ROS to reduce oxidative stress， protecting the integrity of renal endothelial structure， delaying cell apoptosis， and maintaining cell energy metabolism.

MicroRNA-302b mitigates renal fibrosis via inhibiting TGF-ß/Smad pathway activation

Renal fibrosis is one of the most significant pathological changes after ureteral obstruction. Transforming growth factor-β (TGF-β) signaling pathway plays essential roles in kidney fibrosis regulation. The aims of the present study were to investigate effects of microRNA-302b (miR-302b) on renal fibrosis, and interaction between miR-302b and TGF-β signaling pathway in murine unilateral ureteral obstruction (UUO) model. Microarray dataset GSE42716 was downloaded by retrieving Gene Expression Omnibus database. In accordance with bioinformatics analysis results, miR-302b was significantly down-regulated in UUO mouse kidney tissue and TGF-β1-treated HK-2 cells. Masson's trichrome staining showed that miR-302b mimics decreased renal fibrosis induced by UUO. The increased mRNA expression of collagen I and α-smooth muscle actin (α-SMA) and decreased expression of E-cadherin were reversed by miR-302b mimics. In addition, miR-302b up-regulation also inhibited TGF-β1-induced epithelial mesenchymal transition (EMT) of HK-2 cells by restoring E-cadherin expression and decreasing α-SMA expression. miR-302b mimics suppressed both luciferase activity and protein expression of TGF-βR2. However, miR-302b inhibitor increased TGF-βR2 luciferase activity and protein expression. Meanwhile, miR-302b mimics inhibited TGF-βR2 mRNA expression and decreased Smad2 and Smad3 phosphorylation in vivo and in vitro. Furthermore, over-expression of TGF-βR2 restored the miR-302b-induced decrease of collagen I and α-SMA expression. In conclusion, this study demonstrated that miR-302b attenuated renal fibrosis by targeting TGF-βR2 to suppress TGF-β/Smad signaling activation. Our findings showed that elevating renal miR-302b levels may be a novel therapeutic strategy for preventing renal fibrosis.

Improvement for the construction of renal interstitial fibrosis model with unilateral ureteral obstruction in rats / 中国组织工程研究

BACKGROUND: Unilateral ureteral obstruction (UUO) is a traditional surgical method that can cause renal interstitial fibrosis in rats in a short period of time (1-2 weeks), but it can develop many postoperative complications and result in a high mortality. OBJECTIVE: To optimize and improve the UUO operation in rats for reducing the incidence of postoperative complications and improving the survival rate of animals, and to detect the pathophysiological indicators of rats modeled by UUO, providing background data for the basic research on functional pharmacology. METHODS: Twenty male Sprague-Dawley rats were randomly divided into four groups, followed by traditional UUO ligation, improved UUO ligation, undissociated ureteral ligation, and opening the abdominal cavity with no ureter ligation (sham group), respectively. At 14 days postoperatively, the successful rate of renal interstitial fibrosis model, mortality rate, and the incidence of postoperative complications were compared between groups. Another 28 Sprague-Dawley rats, half male and half female, were randomly divided into improved UUO group and control group. Physiological indexes, including urine analysis, blood cells analysis, blood biochemical analysis, were tested as background information. Besides, the pathological changes of kidney tissues were compared between groups using hematoxylin-eosin staining and Masson staining at 14 and 21 days postoperatively. RESULTS AND CONCLUSION: Compared with the undissociated ureteral ligation group, the successful rate of modeling was higher in the traditional and improved UUO groups. The improved UUO surgery with double ligation of the middle ureter and no cutting of the middle ureter was characterized by easier operation, smaller surgical wound, faster modeling and higher successful rate of renal interstitial fibrosis model, lower postoperative mortality and lower incidence of complications in animals as compared with the traditional UUO surgery. The improved UUO group showed statistically significant differences from the sham group in blood biochemical indexes such as urea nitrogen, glutamate aminotransferase, and albumin (P < 0.05). Results from hematoxylin-eosin staining and Masson staining of the obstructed kidney revealed typical pathological features of renal interstitial fibrosis.

Effect of Shenshuai Xiezhuo Decoction and Its Deficiency Tonifying and Pathogen Eliminating Components on Renal Interstitial Fibrosis in UUO rats / 中国实验方剂学杂志

Objective:To study the effect of Shenshuai Xiezhuo decoction and its deficiency tonifying and pathogen eliminating components on renal interstitial fibrosis in UUO rats. Method:A rat model of unilateral ureteral obstruction (UUO) was established through ligation of a unilateral ureter. The rats were divided into six groups: sham operation group, model group, benazepril group, Shenshuai Xiezhuo decoction group, Buxufang group, and phlegm group, with 24 rats in each group. On the third day after operation, the rats in the Shenshuai Xiezhuo decoction group, Buxufang group, and phlegm group were given Shenshuai Xiezhuo decoction concentrating agent at a dose of 8.0 g·kg-1·d-1, the rats in the benazepril group were given benazepril 1.5 mg·kg-1·d-1, and the rats in the sham operation group and the model group were given the same volume of saline. On the 7th, 14th and 21st days after operation, the expressions of peripheral cells and relevant signal pathway markers in renal tissue were detected by Western blot and Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) respectively. Result:The stromal damage score and the interstitial collagen accumulation on the 14th and 21st days after UUO were significantly lower in the Shenshuai Xiezhuo decoction group, Buxu prescription group and Qixie prescription group than those in the model group (P<0.05). Except for the sham operation group, the protein expressions of α smooth muscle actin (α-SMA), platelet derived growth factor A (PDGFA), nerve/glial type 2 chondroitin sulfate glycoprotein (NG-2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 1 (VEGFR1), platelet-derived growth factor -β (PDGF-β) and platelet-derived growth factor receptor-β (PDGFR-β) in the kidney tissues of the 14th and 21st days were significantly increased compared with those on the 7th day (P<0.05). On the 21st day after surgery, the expressions of NG-2, VEGFA, VEGFR1 and PDGFR-β in renal tissue of Shenshuai Xiezhuo decoction group were significantly lower than those in the model group (P<0.05). Compared with the model group, relevant expressions of α-SMA, PDGFA, NG-2, VEGFA, VEGFR1, PDGF-β, and PDGFR-β in kidney tissue of Shenshuai Xiezhuo decoction group decreased significantly on the 21st day after operation (P<0.05), and relative expressions of α-SMA, NG-2, VEGFA, VEGFR1 and PDGFR-β in the Shenshuai Xiezhuo decoction group were significantly lower than those in the benazepril group on the 21st day after surgery (P<0.05). Conclusion:Shenshuai Xiezhuo decoction and its deficiency tonifying and pathogen eliminating components have an antagonistic effect on renal interstitial fibrosis in UUO rats. Its mechanism is related to the inhibition of activation of peripheral cells and relevant cell signaling pathways.

Effect of Qizhi Zhenwutang in Improving Renal Interstitial Fibrosis and Its Mechanism on Unilateral Ureteral Obstruction in Rats / 中国实验方剂学杂志

Objective:To explore the effect of Qizhu Zhenwutang on renal interstitial fibrosis in rats ligated with unilateral ureter， transforming growth factor-β1 （TGF-β1）/Smads and oxidative stress. Method:A total of 30 male SD rats were randomly divided into sham operation group， model group， high-dose group， low-dose group and irbesartan group （n=6）. The left ureter ligation was performed in the model group and the treatment group. In the sham operation group， the ureter was not ligated， only the ureter was separated， and the abdominal cavity was closed. Rats in each group were given drugs by gavage on the next day after operation. Sham operation group and model group were given aseptic distilled water 10 mL·kg-1 by gavage， high-dose Qizhizhenwu Tang group was given 22.2 g·kg-1 by gavage， low-dose group was given 11.1 g·kg-1 by gavage， and irbesartan group was given 0.02 g·kg-1 by gavage. Rats in each group were sacrificed on the 14th day after operation， 24-hour urine was collected before sampling， and the total amount of 24 hour urine protein （24 h-Upr） was detected. Blood samples were collected from the abdominal aorta to detect serum creatinine（SCr） and blood urea nitrogen （BUN）. The tissues were stained with htoxylin eosin （HE） and Masson， and the pathological changes were observed under light microscope， immunohistochemical method was used to detect α-SMA， FN and Col-Ⅰ expressions. Western blot method was used to detect the expressions of TGF-β1， Smad3， p-Smad3 and NOX4. Result:Compared with sham group， SCr， BUN and collagen volume fraction （CVF），24 h-Upr in model group were all increased （P<0.05， P<0.01）. The expressions of α-SMA， Col-Ⅰ， FN， TGF-β1， p-Smad3， NOX4 were higher （P<0.05）. Compared with the model group， SCr， BUN and CVF were lower in high-dose group and irbesartan group （P<0.05）. 24 h-Upr was lower in high-dose group （P<0.05）， the expressions of α-SMA， Col-Ⅰ， FN， TGF-β1， Smad3， p-Smad3， NOX4 in traditional Chinese medicine treatment group were less （P<0.05）. Conclusion:Qizhi Zhenwutang can reduce the urinary protein of UUO rats， protect the renal function， and inhibit the occurrence and development of renal interstitial fibrosis， the mechanism may be related to the inhibition of TGF-β1/Smads signaling pathway and oxidative stress response.

Influence of hepatocyte growth factor-transfected bone marrow-derived mesenchymal stem cells towards renal fibrosis in rats

Background & objectives: Hepatocyte growth factor (HGF) produced by endothelial cells, fibroblasts, fat cells and other interstitial cells, can promote angiogenesis, repair damaged tissues and resist fibrosis. Mesenchymal stem cells (MSCs) are located in bone marrow and secrete a variety of cytokines and are often used in the repair and regeneration of damaged tissues. This study was aimed to investigate the influence of HGF-transfected bone marrow-derived MSCs towards renal fibrosis in rats. Methods: The HGF gene-carrying adenoviral vector (Ad-HGF) was transfected into MSCs, and the Ad-HGF-modified MSCs were transplanted into rats with unilateral ureteral obstruction (UUO). The localization of renal transplanted cells in the frozen section was observed with fluorescence microscope. The Masson's trichrome staining was performed to observe the renal collagen deposition, and the immunohistochemistry was performed to detect the expressions of ?-smooth muscle actin (?-SMA) and HGF in renal tissues. Reverse transcription (RT)-PCR was used to detect the mRNA expressions of ?-SMA, HGF and fibronectin (FN). Results: Ad-HGF-modified MSCs could highly express HGF in vitro. On the post-transplantation 3rd, 7th and 14th day, the 4',6-diamidino-2-phenylindole (DAP)-labelled transplanted cells were seen inside renal tissues. Compared with UUO group, the renal collagen deposition in transplantation group was significantly reduced, and the expressions of ?-SMA mRNA and protein were significantly decreased, while the expressions of HGF mRNA and protein were significantly increased, and the expression of FN mRNA was significantly decreased (P<0.001). Interpretation & conclusions: Trans-renal artery injection of HGF-modified MSCs can effectively reduce the renal interstitial fibrosis in UUO rat model.

Fluorofenidone inhibits apoptosis of renal tubular epithelial cells in rats with renal interstitial fibrosis

This study aimed to investigate the mechanism of fluorofenidone (AKF-PD) in treating renal interstitial fibrosis in rats with unilateral urinary obstruction (UUO). Thirty-two male Sprague-Dawley rats were randomly divided into sham, UUO, UUO + enalapril, and UUO + AKF-PD groups. All rats, except sham, underwent left urethral obstruction surgery to establish the animal model. Rats were sacrificed 14 days after surgery, and serum was collected for renal function examination. Kidneys were collected to observe pathological changes. Immunohistochemistry was performed to assess collagen I (Col I) protein expression, and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining to observe the apoptosis of renal tubular epithelial cells. The expression of Fas-associated death domain (FADD), apoptotic protease activating factor-1 (Apaf-1), and C/EBP homologous protein (CHOP) proteins was evaluated by immunohistochemistry and western blot analysis. AKF-PD showed no significant effect on renal function in UUO rats. The pathological changes were alleviated significantly after enalapril or AKF-PD treatment, but with no significant differences between the two groups. Col I protein was overexpressed in the UUO group, which was inhibited by both enalapril and AKF-PD. The number of apoptotic renal tubular epithelial cells was much higher in the UUO group, and AKF-PD significantly inhibited epithelial cells apoptosis. The expression of FADD, Apaf-1, and CHOP proteins was significantly upregulated in the UUO group and downregulated by enalapril and AKF-PD. In conclusion, AKF-PD improved renal interstitial fibrosis by inhibiting apoptosis of renal tubular epithelial cells in rats with UUO.

Rhein alleviates renal interstitial fibrosis by inhibiting tubular cell apoptosis in rats

BACKGROUND: Ureteral obstruction causes injury of the renal tissues and can irreversibly progress to renal fibrosis, with atrophy and apoptosis of tubular cells. The goal of the current study was to examine the effects of rhein on the apoptosis o renal tubular cells as well as renal fibrosis using a rodent model of unilateral ureteral obstruction (UUO). METHODS: UUO was induced through ureteral ligation, then animals received treatments with rhein or vehicle. The control rats only received sham operation. The renal tissue was harvested 1 week after surgery for assessment of kidney fibrosis. RESULTS: The expressions of collagen I and α-smooth muscle actin (α-SMA), as well as the severity of renal tubular apoptosis and fibrosis were time-dependently increased following UUO. Treatments with rhein partially inhibited such responses. Renal interstitial fibrosis was associated with STAT3 (signal transducer and activator of transcription 3) phosphorylation as well as altered expressions of Bax and Bcl2, both apoptosis-related proteins. Treatment with rhein also partly blocked these responses. CONCLUSION: These findings demonstrated that rhein mitigated apoptosis of renal tubular cell as well as renal fibrosis in a UUO rodent model. This curative effect is likely mediated via suppression of STAT3 phosphorylation.

Progress in Research of Kangxianling Decoction in Delaying Renal Fibrosis / 中国实验方剂学杂志

Renal fibrosis is a common pathological change in the later stages of all kidney diseases. It is a multi-cytokine, multi-signal pathway, multi-factor driven chronic kidney disease. It includes renal interstitial fibrosis, tubular sclerosis and glomerular sclerosis, which eventually leads to chronic renal failure. From health through injury to loss of function, the disease process is closely related to the degree of deterioration of renal function and the prognosis of chronic kidney disease. There is no effective western medicine for the treatment of renal fibrosis. Professor HE Liqun from Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine has summarized the Kangxianling decoction through decades of long-term practical experience. It has the effects in strengthening the spleen and replenishing Qi, clearing away dampness and heat, promoting blood circulation and removing phlegm, and strengthening turbidity. It is composed of Salviae Miltiorrhizae Radix et Rhizoma 15 g, Persicae Semen 12 g, Angelicae Sinensis Radix 12 g, Achyranthis Bidentatae Radix 9 g, Rhei Radix et Rhizoma 15 g. Kangxianling can affect the synthesis and secretion of cytokines and inflammatory factors by expanding blood vessels, and can improve renal tubular fibrosis. It has a good multi-channel, multi-target and multi-directional protective effect on renal function. It also can delay the progress of chronic renal fibrosis by significantly alleviating such symptoms as fatigue and edema in patients with chronic renal fibrosis, and reducing serum creatinine, urea nitrogen and urine protein. In this paper, 5/6 nephrectomy, ischemia reperfusion injury, adriamycin-induced nephropathy, unilateral ureteral obstruction and other different modeling methods are listed. The mechanism of Kangxianling decoction in antagonizing renal fibrosis is discussed and summarized, which further provided new ideas and directions for future clinical and scientific research.

Effect of curcumol on the apoptosis inhibition of traf2 and xbp1 controled by ire1 in rats with obstructive nephropathy / 中国药学杂志

OBJECTIVE: To investigate the effect of curcumol on the apoptosis inhibition of TRAF2 and XBP1 controled by IRE1 in rats with obstructive nephropathy. METHODS: Rats were randomly divided into sham control group, model control group and enalapril group as well as high, medium and low formononetin groups. Animal model of RIF was established by unilateral ureteral obstruction (UUO). The rats were sacrificed at 14 d after UUO, and blood samples were collected. Levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were examined. Renal tubular damage index was determined by H&E staining. The area of RIF was determined by Masson staining. Expressions of GRP78,IRE1,p-IRE1,TRAF2 and XBP1 in kidney were determined by Western blotting analysis. Apoptosis of tubular epithelial cells was detected by TUNEL assay. RESULTS: Levels of Scr and BUN, tubulointerstitial injury index, RIF and apoptotic index as well as expressions of GRP78,IRE1,p-IRE1,TRAF2 and XBP1 were different between the model control and treatment groups (P<0.05, P<0.01). Compared with the enalapril group, tubulointerstitial injury index and RIF as well as the levels of Scr and BUN were decreased (P < 0.05) in the high curcumol group. CONCLUSION: The curcumol can block the apoptosis of renal tubular epithelial cells through interfering IRE1-XBP1 signaling pathway by inhibiting TRAF2 protein expression mediated by IRE1 phosphorylation. Ultimately, development of RIF was postponed.

Effects of resveratrol on expression of renal fibrosis proteins ORP150, GRP78, and GRP94 in rat kidney tissue / 中草药

Objective To assess the effect of resveratrol on the expression of endoplasmic reticulum stress molecular partners150-KD oxygen-regulated protein (ORP150) in renal tissues of rats with unilateral ureteral obstruction (UUO). Methods The UUO rat model of renal interstitial fibrosis was established. The rats were randomly divided into sham operation group, model group, enalapril group, the high-dose, medium-dose and low-dose groups of resveratrol, each group of 10. After 14 d of surgery, rats were sacrificed to collect serum and kidney tissue to detect the level of serum Scratinine (Scr) and blood urea nitrogen (BUN); Pathological changes were stained by HE to evaluate of renal tubular damage index, renal interstitial collagen deposition area was detected by Masson staining, apoptosis of in situ cell in renal tissue was determined by TUNEL assay, and using the western blot for the detection of protein expression of ORP150, GRP78, and GRP94 in renal tissue. Results After comparison of the treatment groups with model group, BUN and Scr levels in serum were significantly reduced in high-dose resveratrol group, the damage degree of renal tubules was significantly reduced, the relative area of the renal interstitial collagen decreased, In addition, the expression of ORP150 was increased (P < 0.05, 0.01), GRP78 and GRP94 proteins expression were significantly reduced, (P < 0.05, 0.01). Moreover, the resveratrol high-dose group seems more effective than enalapril groups in reversing the phenotype (P < 0.05). Conclusion Resveratrol was able to protect renal function, alleviate hydronephrosis, reduce interstitial injury of renal tubular, induce the expression of the key endoplasmic reticulum stress signal molecule ORP150 and the down-regulated molecular partner GRP78 and GRP94, delay the apoptosis of renal tubular epithelial cell of rats after UUO and inhibit renal interstitial fibrosis caused by apoptosis deposition.

Effect of formononetin on protein expression of ASK1 and JNK in rats after unilateral ureteral obstruction / 中草药

Objective To study the effect of formononetin on the expression of ASK1 and JNK on the protein level in rat after unilateral ureteral obstruction (UUO). Methods Rats were then randomly divided into control group, model group, the enalapril group, and the high, medium, and low dose groups of formononetin (100, 50, and 25 mg/kg). Renal interstitial fibrosis (RIF) rats model was established by unilateral ureteral obstruction except the control group. The rats were sacrificed 14 d after surgery, and blood samples were collected to detect serum creatinine (Scr) and blood urea nitrogen (BUN) levels. HE staining was used to observe renal pathologic change and determine renal tubular damage index. The area percentage of RIF was detected by Masson staining. Expressions of ASK1 and JNK protein in kidney were determined by Western blotting. Tubular epithelial cell apoptosis was detected by TUNEL assay. Results Serum Scr, BUN level, tubulointerstitial injury index, RIF, the expressions of ASK1 and JNK protein, and apoptotic index were significantly decreased in the treatment groups when compared with the model group (P < 0.05, 0.01). The high dose group of formononetin was more effective than enalapril group. Conclusion Formononetin inhibited the expressions of ASK1 and JNK protein in UUO rats model. Ultimately renal tubular epithelial cell apoptosis was suppressed and the progression of obstructive nephropathy pathologic process was retarded.

Mechanism of Shenluotong regulating NR3C2/SGK-1/Smad pathway in inhibiting renal interstitial fibrosis / 中草药

Objective To investigate the mechanism of Shenluotong inhibiting renal interstitial fibrosis by regulating NR3C2/SGK-1/Smad pathway. Methods A total of 48 Wistar rats were randomly divided into sham operation group, model group, Eplerenone group, and Shenluotong group (n = 12). Model group, Eplerenone group, and Shenluotong group used unilateral ureteral obstruction (UUO) method to establish rat renal interstitial fibrosis model. After the operation, the rats in the eplerenone group were treated with eplerenone at a dose of 100 mg/(kg∙d). Rats in the Shenluotong group were oral given Shenluotong decoction at a dose of 26 g/(kg∙d) and Sham operation group and model group were administrated equal volume of saline once daily for continuous 10 d. Laser confocal microscopy was used to detect mineralocorticoid receptor NR3C2 expression. The expressions of SGK-1, TGF-β1, Smad4, and Smad7 in renal tissues were detected by immunohistochemistry, Western Blot and Real time-PCR. Results In the sham operation group, NR3C2 was expressed in the cytoplasm of renal tubular epithelial cells and was not expressed in the nucleus. The expression of NR3C2 in the UUO rat was significantly up-regulated in cytoplasm and positive expression was observed in the nucleus. The expression of NR3C2 in the nucleus of cells in the Eplerenone group and Shenluotong group was significantly decreased when compared with the model group. Compared with the sham-operated group, the expression of SGK-1, TGF-β1, and Smad4 was significantly up-regulated and the expression of Smad7 was significantly decreased (P < 0.05, 0.01) in the other groups. Compared with model group, the expression range and intensity of TGF-β1 and Smad4 were significantly decreased in Eplerenone group and Shenluotong group (P < 0.05, 0.01), and the expression range and intensity of Smad7 were significantly increased (P < 0.01). Conclusion Shenluotong can inhibit renal interstitial fibrosis through blocking the activation of mineralocorticoid receptor, reducing the level of SGK-1, and regulating the Smads signal pathway to inhibit the overexpression of TGF-β1.

Effect of GP130 small molecular inhibitor SC144 on renal interstitial fibrosis in mice with unilateral ureteral obstruction / 上海交通大学学报（医学版）

Objective: To investigate the effect of glycoprotein 130 (GP130) inhibitor SC144 on extracellular matrix accumulation and JAK2/STAT3 signaling pathway in unilateral ureteral obstruction (UUO) mouse model, and explore its mechanism. Methods: Eighteen female BALB/c mice were randomly divided into 3 groups i.e. sham group, UUO group and SC144 group. All mice were sacrificed at day 14 and kidneys were harvested for further analysis. The changes of renal tissue morphology and pathology were observed by H-E and Masson staining. The expression of α-smooth muscle actin (α-SMA) and infiltration of macrophage cells were assayed by immunohistochemical staining. The levels of collagen-I, collagen-IV, monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β (TGF-β) mRNA were analyzed by real-time PCR. The activation of JAK2 and STAT3 was measured by Western blotting. Results: There was a trend toward decreased renal tubular lesion and renal interstitial fibrosis in SC144 group (H-E, P=0.052; Masson, P=0.063). SC144 significantly inhibited the levels of α-SMA, type I/type IV collagen and TGF-β mRNA (all P<0.05). Compared with UUO group, the phosphorylation levels of JAK2 and STAT3 were significantly decreased in SC144 group (both P<0.05). Conclusion: The treatment of UUO mouse model with SC144 can inhibit the activation of α-SMA, attenuate the phosphorylation of STAT3, reduce extracellular matrix protein deposition following injury and renal tubular epithelial-mesenchymal transition (EMT) via JAK2/STAT3 signaling pathway, indicating its potential in attenuating interstitial fibrosis in obstructive nephropathy.

Effects of Polydatin on Protein Expression of MMP-9 and TIMP-1 in Renal Tissue of Renal Fibrosis Model Rats / 中国药房

OBJECTIVE:To investigate the effects of polydatin on protein expression of MMP-9 and TIMP-1 in renal tissue of renal fibrosis model rats,and to investigate the mechanism of delaying renal fibrosis in rats. METHODS:A total of 40 rats were randomly divided into sham operation group(normal saline),model group(normal saline),polydatin group(100 mg/kg)and benazepril group(positive control,5 mg/kg). Except for sham operation group,renal fibrosis rat model was induced by unilateral ureteral obstruction in other groups. 1 h after surgery,each group was given relevant medicine intragastrically,once a day.After 4 weeks of consecutive treatment,the contents of β 2-microglobulin(β 2-MG)and N-acetyl-β-D-glucosaminidase(NAG)in urine, serum contents of BUN and Cr were detected in each group. HE staining was used to observe and score pathological changes of renal tissue in rats. Protein expressions of MMP-9 and TIMP-1 in renal tissue of rats were determined by immunohistochemistry. RESULTS:Compared with sham operation group,the contents of β2-MG and NAG in urine,serum contents of BUN and Cr were increased significantly in model group(P<0.05),and pathological score and protein expressions of MMP-9 and TIMP-1 were also increased significantly(P<0.01). Compared with model group,protein expression of MMP-9 in renal tissue were increased continuously in polydatin group and benazepril group(P<0.01);other indexes were decreased significantly(P<0.05 or P<0.01),and there was no statistical significance in each index between polydatin group and benazepril group(P>0.05). CONCLUSIONS:Polydatin can delay renal fibrosis,the mechanism of which may be associated with up-regulating protein expression of MMP-9,down-regulating protein expression of TIMP-1 and increasing the ratio of MMP-9/TIMP-1.

Effect of GP130 small molecular inhibitor SC144 on renal interstitial fibrosis in mice with unilateral ureteral obstruction / 上海交通大学学报（医学版）

Objective·To investigate the effect of glycoprotein 130 (GP130) inhibitor SC144 on extracellular matrix accumulation and JAK2/STAT3 signaling pathway in unilateral ureteral obstruction (UUO) mouse model, and explore its mechanism. Methods·Eighteen female BALB/c mice were randomly divided into 3 groups i.e. sham group, UUO group and SC144 group. All mice were sacrificed at day 14 and kidneys were harvested for further analysis. The changes of renal tissue morphology and pathology were observed by H-E and Masson staining. The expression of α-smooth muscle actin (α-SMA) and infiltration of macrophage cells were assayed by immunohistochemical staining. The levels of collagen-I, collagen-IV, monocyte chemoattractant protein-1(MCP-1),transforming growth factor-β(TGF-β)mRNA were analyzed by real-time PCR.The activation of JAK2 and STAT3 was measured by Western blotting. Results·There was a trend toward decreased renal tubular lesion and renal interstitial fibrosis in SC144 group (H-E, P=0.052;Masson,P=0.063).SC144 significantly inhibited the levels of α-SMA,type I/type IV collagen and TGF-β mRNA(all P<0.05).Compared with UUO group, the phosphorylation levels of JAK2 and STAT3 were significantly decreased in SC144 group (both P<0.05). Conclusion·The treatment of UUO mouse model with SC144 can inhibit the activation of α-SMA, attenuate the phosphorylation of STAT3, reduce extracellular matrix protein deposition following injury and renal tubular epithelial-mesenchymal transition(EMT)via JAK2/STAT3 signaling pathway,indicating its potential in attenuating interstitial fibrosis in obstructive nephropathy.

Selenium and benazepril inhibit renal interstitial fibrosis in rat with unilateral ureteral obstruction / 基础医学与临床

Objective To study the protective effect of the sodium selenite and benazepril on renal interstitial fibro-sis(RIF) in rat model of unilateral ureteral obstruction(UUO) and its mechanism. Methods The male SD of clean grade rats were randomly divided into sham-operation group,UUO group(UUO model was established by li-gating unilateral ureter), UUO+ sodium selenite group group(sodium selenite 0.2 mg/kg·d gavage), UUO+benazepril group(benazepril 10 mg/kg·d gavage),with 18 in each group.At day 7,14 and 21 after thetreatment, 6 rats selected randomly from each group were killed.The extent of RIF was evaluated by HE and Masson staining of the renal tissue. The expression of connective tissue growth factor(CTGF),transforming growth factor-β1(TGF-β1), alpha smooth muscle actin(α-SMA) andⅢ collagen(ColⅢ) were detected by immunohistochemical method.The protein expression of CTGF and TGF-β1 were detected by Western blot. Chemical colorimetric method was used to detecte the contents of supper oxide dismutase (SOD),malondialdehyde (MDA) and glutathione peroxidase (GSH-px) in renal cortex. Results The extent of RIF and the expression of CTGF,TGF-β1,α-SMA and ColⅢin renal cortex were significantly lower in sodium selenite group and benazepril group at day 7,14 and 21 after the op-eration compared with that in UUO group(P<0.05 or P<0.01). In sodium selenite group and benazepril group,the contents of SOD and GSH-px in renal cortex were higher significantly than those in UUO group at day 7,14 and 21 after the operation respectively(P<0.05),but the MDA in renal cortex was significantly decreased(P<0.05).There were no significant differences in the indexes between the two groups of sodium selenite and benazepril. The expres-sion of CTGF,TGF-β1,α-SMA,ColⅢand the extent of RIF were positively correlated to the level of MDA in UUO group(P<0.05,respectively),and negatively correlated to the level of SOD and GSH-Px(P<0.05,respectively). The expression of CTGF was positively correlated to the expression of α-SMA and ColⅢin UUO group(P<0.05).The expres-sion of CTGF,α-SMA and ColⅢwere positively correlated to RIF in UUO group(P<0.05).Conclusions Sodium sele-nite and benazepril can reduce the extent of RIF in rat model with unilateral ureteral obstruction.

Research of mesenchymal stem cells transfected with hepatocyte growth factor transplantation in the rat's kidney with partial unilateral ureteral obstruction / 国际儿科学杂志

Objective To observe the phenotypic transformation of bone marrow mesenchymal stem cells(MSC) transfected with hepatocyte growth factor(HGF) after their transplantation in the rat's kidney with partial unilateral ureteral obstruction(PUUO).Methods We isolated,cultured bone MSCs of the male rats,and transfected them with Ad-HGF in vitro.Thirty-six female rats with PUUO were randomly divided into control group(A) and the experimental groups(B,C).Saline,Ad-GFP transfected MSCs or Ad-HGF transfected MSCs were respectively injected into the parenchyma of kidney on the 7th day with PUUO.We released the ureteral obstruction of rats after transplantation.Kidney tissue of the rats were collected on the 7th day after transplantation.The distribution and phenotypic transformation of MSCs in the kidney were determined by immunofluorescence method.Results The green fluorescent-labeled MSCs mainly distributed in the tubular cells,and a part of bone MSCs underwent phenotypic transformation after transplantation.Compared with group B,the number of bone MSCs with phenotypic transformation significantly increased in group C.Conclusion After transplantation,MSCs can survive and differentiate into renal tubular epithelial cells,and HGF may promote survival and differentiation of MSCs.