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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.
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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.
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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.
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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.
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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.
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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.
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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.
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BACKGROUND: Unilateral ureteral obstruction(UUO) is an experimental model of tubulointerstitial injury, characterized by progression of interstitial fibrosis and tubular atrophy. In the pathogenic mechanism of renal injury, activation of renin-angiotensin system may play an important role. METHODS: We tested the hypothesis that tubulointerstitial injury begins with infiltration of macrophage, followed by enhanced expression of chemoattractants such as MCP-1 and osteopontin, which are affected by local angiotensin II activity. We examined the beneficial effects of ACEI and AT1RB and combination of these two drugs, with kidney after 5 days of ureter ligation. RESULTS: Monocyte/macrophage infiltration demonstrated by ED-1 staining was markedly increased in UUO group comparing to that of sham operared group, and it was reduced by administeration of ACEI, AT1RB and combination of both, but not statistically significant. MCP-1 mRNA expression increased significantly after 5 days of UUO. ACE inhibitor, enalapril treatment had suppressed significantly the MCP-1 mRNA expression level, but AT1RB, candesartan had not significance. Combination of both made no more reduction of MCP-1 mRNA level compared to ACEI alone. The other macrophage chemoattractant protein, osteopontin expression was examined by immunohistochemistry, and evaluated with image analysis. Marked up-regulated osteopontin expression was observed on the brushborder of proximal tubules after 5 days UUO. There was a great reduction of osteopontin expression in a enalapril treated group and more significant reduction was noted in the group, treated with combination of both. candesartan did not reduce osteopontin expression. CONCLUSION: This study suggest that activation of renin-angiotensin system has a major role in the pathogenesis of tubulointerstitial renal injury in UUO through expression of chemoattractants and infiltration of inflammatory cells. Blocking of this process may inhibit renal injury process and ACEI halts these process but AT1RB does not. Combination use of both drugs did not show more additive effect compare to ACEI use alone in the phase of early inflammatory process of renal injury.