Expression and functional role of p38 MAPK in the kidney after unilateral ureteral obstruction in rats

AIM: To investigate the expression and functional role of p38MAPK in the kidney after unilateral ureteral obstruction in rats. METHODS: Unilateral ureteral obstruction (UUO) models were induced by ligating the left ureter. Rats were sacrificed at 1 h, 3 h, 6 h, 12 h, 1, 3, 5, 7, 14, 21, and 28 days after UUO was initiated. p38MAPK activity was assayed by immunohistochemical staining and specific substrate phosphorylation with immunoprecipitation and Western blotting. TGFβ mRNA and protein expression were analyzed with in situ hybridization and immunohistochemical stainning. RESULTS: A basic p38MAPK activity was detectable in the normal kidney(0.22±0.06).p38MAPK pathway was rapidly act ivated at 1 hour(0.45±0.14 vs control,P<0.05)and this was steadily in creased by 12 hours(0.91±0.07 vs control,P<0.01)after UUO.Afterwards,the activity of p38MAPK reduced grad ually,then increased again from 3 days and this was steadily increased by 7 days(0.93±0.06 vs control,P<0.01). Upregulation of TGFβ₁ was markedly tested at 3 days(13.55±6.33 vs control,P<0.05)and this was steadily in creased by 7 days(26.78 8.77 vs control,P<0.01).The activation of p38MAPK preceded markedly the expression of TGF 1.The early activity of p38 MAPK was positively related to the amount of TGFβ₁ expression.The amount of TGFβ₁ expressed in obstructed kidney also related significant ly to the late activity of p38MAPK(r=0.84,P<0.01). CONCLUSION: The activity of p38MAPK is increased significantly in the obstructed kidney, which may cause renal fibrosis via inducing the expression of TGFβ₁.     

Relationship between peritubular capillary injury and renal interstitial fibrosis in mice with unilateral ureteral obstruction

AIM: To observe the injury of peritubular capillary (PTC), hypoxia and interstitial fibrosis after unilateral ureteral obstruction (UUO), and to explore the effects of PTC injury and hypoxia on interstitial fibrosis in mouse model of UUO. METHODS: Forty-eight male KM mice were randomly divided into control group and UUO group. On the 1st, 3rd, 7th and 14th days, 6 mice in each group were sacrificed. The changes of pathomorphism in the kidney were observed by HE and Masson staining. The expression levels of thrombospondin-1 (TSP-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α), and PTC density were detected by immunohistochemistry. The protein expression of VEGF was also determined by Western blotting. RESULTS: No histological abnormalities of the kidneys were observed in sham-operated mice. The expression of TSP-1 was increased 1 day after UUO, and significantly increased on the 3rd, 7th and 14th days(P<0.05). The expression of VEGF was obviously decreased(P<0.05). PTC density was gradually decreased. The expression of HIF-1α was gradually increased, and renal interstitial area was gradually expanded. PTC density was negatively correlated with the expression of TSP-1 and HIF-1α (r=-0.874 and r=-0.930, respectively). VEGF expression was positively correlated with PTC density (r=0.745). PTC density was negatively correlated with the area of renal fibrosis (r=-0.787). HIF-1α expression was positively correlated with the area of renal fibrosis (r=0.835, P<0.05). CONCLUSION: In mouse UUO model, the expression of TSP-1 is increased. The expression of VEGF is reduced. The peritubular capillary injury and tissue hypoxia are aggravated, and renal interstitial fibrosis area is expanded. Ischemia and hypoxia may play an important role in the progression of UUO.     

Pancreatic kininogenase protects against renal fibrosis in rat model of unilateral ureteral obstruction

AIM To assess the beneficial effects of pancreatic kininogenase （PK） on renal fibrosis in rat model of unilateral ureteral obstruction （UUO）. METHODS Male Sprague-Dawley rats were randomly assigned to 5 groups and treated daily with PK for 7 d and 14 d. Masson trichrome and HE staining were used to assess the degree of tubulointerstitial fibrosis, and immunohistochemistry and Western blot were employed to evaluate the expression of profibrotic and proinflammatory cytokines, and apoptosis- and autophagy-related proteins. RESULTS PK treatment significantly decreased expression of profibrotic and proinflammatory cytokines, and these were paralleled with attenuation of tubulointerstitial inflammation ［monocyte chemoattractant protein-1 （MCP-1） and Toll-like receptor 2 （TLR-2）］ and fibrosis ［transforming growth factor β1 （TGF-β1） and connective tissue growth factor （CTGF）］ in a time-dependent manner. Oxidative stress induced by UUO manifested by augment of oxidant enzymes ［NADPH oxidase-2 （NOX-2） and NOX-4］ and decrease in antioxidant enzymes ［superoxide dismutase 1 （SOD1） and SOD2］, which was closely associated with dysregulation of apoptosis- and autophagy-related proteins subsequent excessive apoptotic （Bcl-2/Bax and cleaved caspase-3） and autophagy （LC3B, beclin-1 and P62）, and all of these were eliminated by administration of PK. CONCLUSION PK treatment protects against the progression of renal fibrosis in obstructed kidneys probably by interfering oxidative stress and programmed cell death.     

Increased fucosyltransferase 8 expression in rat kidney with unilateral ureteral obstruction

AIM: To investigate the effect of fucosyltransferase 8 (FUT8) on rat renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). METHODS: Ninety male Wistar rats were randomly divided into normal control group (control), sham operation group (sham) and UUO group, and sacrificed on day 1, 3, 7, 14 and 21 after operation. Serum creatinine and urea nitrogen were detected to assess renal function. PAS and Masson staining were used to observe histological changes of the rat kidneys. The time-correlated expression of FUT8 in the kidney was monitored by RT-PCR and Western blotting. The protein expression levels of FUT8 and activin receptor-like kinase 5(ALK5) were determined by immunofluorescence double-staining. Immunohistochemical staining was also used to assess the protein expression of fibronectin (FN), type I collagen (Col I) and α-smooth muscle actin (α-SMA). RESULTS: Compared with control group, serum creatinine and urea nitrogen in UUO group elevated on day 3 after operation (P<0.05) and reached the peak value on day 21 after operation (P<0.01). Renal tubule atrophy and renal interstitial fibrosis were observed in UUO group 7 and 14 days after operation. The mRNA and protein expression levels of FUT8 increased markedly in UUO group on day 3 (P<0.05) and reached its peak value on day 14 and 21 after operation (P<0.01). The results of immunofluorescence and immunohistochemistry showed that FUT8 and ALK5 were coexpressed in renal tubulointestitium. FN, Col I and α-SMA were significantly elevated in UUO group (P<0.05), and were positively correlated with the expression of FUT8 (P<0.05).CONCLUSION: The expression of FUT8 influences the progress of renal interstitial fibrosis, tubule atrophy and inflammatory cell infiltration in the kidney.     

Ginsenoside Rh1 attenuates unilateral ureteral obstruction-induced renal interstitial fibrosis in rats

AIM:To observe the effects of ginsenoside Rh1 (G-Rh1) on unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis and to investigate the underlying mechanisms. METHODS:Male Sprague-Dawley (SD) rats (n=40) were divided into the following 4 groups:UUO-operated group (UUO group), sham-operated group (sham group), UUO-operated plus a low dose (50 mg·kg^-1·d^-1) of G-Rh1 treatment (low G-Rh1 group) and UUO-operated plus a high dose (100 mg·kg^-1·d^-1) of G-Rh1 treatment group (high G-Rh1 group). The G-Rh1 treatment was carried out by gastric gavage from the next day after the UUO operation once a day for 2 weeks (14 d). Immediately after the final dose of G-Rh1, 24 h urine was collected for the urine protein test, and then the rats were euthanized. The blood was collected for the blood urea nitrogen (BUN) and serum creatinine (SCr) assays, and the kidney was removed for pathological and biochemical evaluations. RESULTS:The levels of 24 h urine protein did not show any significant diffe-rence among the groups, while significantly increased levels of BUN and SCr in UUO group were observed (P<0.05), which was prevented by the treatment with G-Rh1 at both doses in a dose-dependent manner. Pathological evaluation showed the renal tissue damage was obvious in UUO group, which was improved by the treatment with G-Rh1 at both doses. Immunohistochemcial analysis exhibited that UUO increased renal interstitial transforming growth factor-β₁ (TGF-β₁) expression, which was also inhibited by the treatment with G-Rh1 at both doses(P<0.05). Significantly increased protein expression of renal interstitial collagen type I, α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) in UUO group was detected, which was suppressed by the treatment with G-Rh1 at both doses. CONCLUSION:G-Rh1 improves UUO-induced renal dysfunction and attenuates interstitial fibrosis, which is mediated via modulation of TGF-β₁-related pro-fibrogenic signaling pathway.     

Relationship of microRNA-133a and TGF-β1 protein in myocardial tissues of spontaneously hypertensive rats

AIM：To observe the changes of microRNA-133a and transforming growth factor β1 (TGF-β1) protein in the myocardium of spontaneously hypertensive rats (SHR). METHODS：Male SHR (18 weeks old, n=12) and male Wistar-Kyoto rats (WKY, 18 weeks old, n=12) served as SHR group and control group, respectively. Caudal arterial blood pressure was detected by a noninvasive blood pressure measurement and analysis system. Myocardial collagen volume fraction (CVF) and perivascular collagen area ratio (PVCA) were determined by Masson staining. The level of miR-133a in the heart was detected by real-time quantitative PCR. The protein level of TGF-β1 in the heart was also analyzed by the methods of immunohistochemisty and Western blotting. RESULTS：Compared with control group, systolic and diastolic blood pressure, CVF and PVCA significantly increased, the expression of TGF-β1 protein was significantly up-regulated, and the level of miR-133a was significantly reduced in SHR group. In SHR group, the expression of miR-133a was decreased to (23.9±4.6)% in control group. A negative correlation between the levels of miR-133a and TGF-β1 protein in SHR group was observed (r=-0.791, P<0.01). CONCLUSION：The level of miR-133a is down-regulated along with the up-regulation of TGF-β1 protein expression and collagen synthesis in the myocardial tissues of SHR. miR-133a and TGF-β1 may be involved in myocardial fibrosis in SHR.     

Effect of erlotinib on renal injury in rats with STZ-induced diabetic nephropathy

AIM: To investigate the effect of epidermal growth factor receptor (EGFR) inhibitor erlotinib on kidney injury in diabetic nephropathy (DN) rat and the underlying mechanism. METHODS: The rat model of DN was induced by intraperitoneal injection of streptozotocin (STZ) at dose of 55 mg/kg. One week after STZ injection, the rats with blood glucose level exceeding 16.7 mmol/L were identified as diabetic. Diabetic rats were randomly divided into 2 groups:STZ group and STZ+erlotinib group. In addition, the normal rats were used as control group. The rats in STZ+erlotinib group were treated with erlotinib at 100 mg·kg^-1·d^-1 for 4 weeks(5th~8th week). The fasting blood glucose (FBG), serum creatinine (SCr) and 24 h urine protein were measured. The pathological changes of the kidney were observed by HE staining and Masson staining. The protein levels of EGFR, p-EGFR, transforming growth factor β1 (TGFβ1), Smad2/3, p-Smad2/3, collagen Ⅳ (ColⅣ) and fibronectin in the kidney tissues were determined by Western blot. The reactive oxygen species (ROS) level and malondialdehyde (MDA) content in the renal tissues were futher analyzed. RESULTS: Compared with control group, the levels of FBG, 24 h urine protein and Scr were significantly increased in STZ group (P<0.01). Compared with STZ group, the levels of FBG, 24 h urine protein and SCr in STZ+erlotinib group were markedly decreased (P<0.05). In additon, the glomerular structure was restored to normal, the proliferative degree of mesangial cells markedly attenuated, and the epithelial cells were in alignment in STZ+erlotinib group. Moreover, erlotinib significantly inhibited the protein levels of p-EGFR, TGFβ1, p-Smad2/3, ColⅣ and fibronectin in the kidney tissues of STZ rats. In addition, erlotinib also significantly inhibited the levels of ROS and MDA in the kidney tissues of STZ rats. CONCLUSION: Erlotinib ameliorates STZ-induced diabetic nephropathy possibly through inhibiting the activation of EGFR/TGFβ1-Smad2/3 signaling pathway in association with suppression of fibrosis and oxidative stress.     

Expression of BMP-7 and its receptors in rats with unilateral ureteral obstruction

AIM:To explore the expression changes of b one morphogenetic protein-7 (BMP-7) and its receptors (BMPR-Ⅱ,ALK-2,ALK-3,A LK-6) in the renal tubulo-interstitial lesions induced by unilateral ureteral ob struction (UUO).METHODS:Rats were divided into normal control,sham operation a nd UUO groups,and sacrificed at postoperative day 1,3,7 and 14.The mRNA leve ls of BMP-7,BMPR-Ⅱ,ALK-2,ALK-3 and ALK-6 were examined by RT-PCR.The protei n expression site and level of BMP-7 was detected by immunohistochemistry staini ng.RESULTS:Compared to sham operation group,the mRNA levels of BM P-7,BMPR-Ⅱ,ALK-2 and ALK-3 were significantly decreased,but the change of AL K-6 mRNA was not marked in UUO rats.The reduction of mRNA expression levels of BMP-7,BMPR-Ⅱ,ALK-2 and ALK-3 in the kidney tissue aggravated along with the d elayed post-operated days.The results of immunohistochemistry staining indicate d that BMP-7 mainly expressed in renal tubular and interstitial,rarely in glome ruli.In UUO rats,the protein expression of BMP-7 decreased in varying degrees according to the post-operated days.CONCLUSION:The loss of BMP-7 and its receptors (BMPR-Ⅱ,ALK-2,ALK-3) were observed in the early phase of fibrotic process and this may play a very important role in mediating the renal tubulointerstital fibrosis.     

Intervention effects of apyrase on silica-induced pulmonary fibrosis in mice

AIM: To investigate the effect of apyrase on the experimental silicosis. METHODS: C57BL/6 male mice were randomly divided into control group, silica treatment group, silica+apyrase group and silica+NS group. A mouse model of lung fibrosis was induced by crystalline silica particles (50 mg/kg, via oropharyngeal instillation), and were sacrificed at 3 h, 7 d, 14 d and 28 d. Apyrase was delivered by oropharyngeal aspiration at the same time and 4 h after silica challenge. The lung indexes were calculated and the concentration of ATP was detected by bioluminescent assay. The mRNA expression levels of collagen type Ⅰ(Col Ⅰ), collagen type Ⅲ (Col Ⅲ) and transforming growth factor β1 (TGF-β1) were examined by real-time PCR. The protein levels of TGF-β1 in bronchoalveolar lavage fluid were measured by ELISA. RESULTS: The elevated lung index and collagen levels showed that silicosis model was established successfully. Compared with silica group, apyrase treatment significantly alleviated silica-induced inflammation, reduced inflammation score on day 7, and decreased the lung index, collagen volume fraction and the mRNA expression of Col Ⅰand Col Ⅲ on day 28. Treatment with apyrase effectively down-regulated the mRNA levels of TGF-β1 in the lung tissues and TGF-β1 protein levels in bronchoalveolar lavage fluid on day 7.CONCLUSION: Apyrase attenuates the pulmonary inflammation and fibrosis of silicosis, which may be related with down-regulation of ATP and TGF-β1 in the lung tissues.     

Phosphorothioate-modified antisense TGF-β1 oligodeoxynucleotide inhibits neointimal hyperplasia after vascular balloon injury in rats

AIM: To evaluate the effects of antisense TGF-β1 oligodeoxynucleotide (AS TGF-β1) on the expression of TGF-β1, deposition of extracellular matrix (ECM) and the neointima formation in the arteries after balloon injury. METHODS: The unmodified and phosphorothioate-modified AS TGF-β1 which containing 15 bases and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed. At the same time, sense TGF-β1 oligodeoxynucleotide (S TGF-β1) with the base sequence complement to AS TGF-β1 was synthesized as a control. The oligodeoxynucleotides were introduced into in vivo and in vitro experiments, respectively. RESULTS: The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner, and S TGF-β1 did not have the same effect. Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was observed. Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis. Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs. Fibronectin (FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (001~1 μmol/L)-dependent manner. AS TGF-β1 significantly increased the mRNA expression of contractile marker SM22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla, especially at the concentration of 001 μmol/L and 01 μmol/L. After treatment with AS TGF-β1 (90 μg·kg-1·d-1) for 28 d, the neointima formation was significantly inhibited, and the area ratio of intima/media was markedly decreased by 68% compared with untreated group, but S TGF-β1 had no effect on neointimal formation. CONCLUSION：The AS TGF-β1 specifically inhibits the protein expression of TGF-β1 in the VSMCs derived from injured arteries. Moreover, it significantly inhibits DNA synthesis and cell proliferation, and decreases the expression of FN. Therefore, AS TGF-β1 dramatically attenuates neointima formation after balloon njury. The effects of AS TGF-β1 on the injured VSMCs may be associated with its reverse effects on the alteration of VSMC phenotype after balloon injury.     

Expression of connective tissue growth factor in the renal tissue of rats with unilateral ureteral obstruction

AIM: To detect the expression of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in rat unilateral ureteral obstructive (UUO) nephropathy animal model, and to observe the kinetic changes at different stages of firosis. METHODS: Male SD rats were subjected to either left ureteral ligation or sham operation, then killed at 3, 7, 14, 21 or 28 days after UUO or sham operation (n=6 at each time point). HE, Masson or PAS staining were applied to the renal tissue sections. The extent of tubulointerstitial injury was determined by Banff classification. RESULTS: The extent of tubulinterstitial fibrosis became serious with the time of obstruction. Tubules were mostly atropic and replaced by proliferative fibrous tissue at day 28. The expression of CTGF and α-SMA were consistent with the damage of tubulointerstitial. The positive correlation among CTGF or α-SMA and the tubulointerstitial injury scores were significant. The expression of TGF-β1 came to peak at day 7 to 14, and gradually decreased at day 21 and 28. CONCLUSION: These results indicate that the expression of CTGF may be upregulated by TGF-β in UUO rats, and CTGF may be involved in tubulointerstitial fibrosis through the development of myofibroblasts.     

Effect of lysophosphatidic acid on expression of integrin β6 in epithelial cells

AIM: To investigate the effect of lysophosphatidic acid (LPA) on the expression of integrin β₆ (ITGB6) for determining the role of transforming growth factor β (TGF-β) activation induced by LPA in this process. METHODS: Normal human bronchial epithelial (NHBE) cells were primarily cultured in 6 well plate and stimulated with LPA. The mRNA expression of ITGB6 and the level of cell surface ITGB6 protein were detected by RT-PCR and flow cytometry,respectively. The activity of active TGF-β induced by LPA was measured by the method of transformed mink lung epithelial cells (TMLC) transfected with TGF-β responsive plasminogen activator inhibitor 1(PAI-1) promoter fused with firefly luciferase reporter gene. RESULTS: After stimulated with LPA at concentration of 10 μmol/L for 2 h, the mRNA expression of ITGB6 in epithelial cells was significantly increased in a time-dependent manner. The results of flow cytometry showed that the protein level of ITGB6 on cell surface was obviously increased after treated with LPA at concentration of 10 μmol/L for 4 h. The active TGF-β induced by LPA in epithelial cells was blocked by an α_Vβ₆ blocking antibody. However, α_Vβ₆ blocking antibody failed to inhibit the mRNA expression of ITGB6 induced by LPA. CONCLUSION: LPA induces the mRNA and cell surface protein of ITGB6 in epithelial cells. The up-regulated ITGB6 expression by LPA is independent on LPA-induced TGF-β activation.     

Expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and its clinical significance

AIM: To evaluate the expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and the significance. METHODS: The patients with breast cancer (n=150) in our hospital from January 2015 to January 2017 were selected as study object. The tumor tissue samples of these patients were obtained from paraffin section of breast cancer by surgical resection with complete clinicopathological data. The corresponding paracancerous tissue sam-ples were taken from the non-tumor tissue samples from the above breast cancer patients, which were 0.5~1 cm away from the tumor tissue. The methods of real-time PCR and Western blot were performed to examine the expression of Wnt-1 and β-catenin at mRNA and protein levels. Human breat cancer MCF-7 cells were divided into 3 groups:control group (MCF-7 cells without treatment), agonist group[MCF-7 cells+Wnt3a (1 mg/L)] and antagonit group[MCF-7 cells+DKK1 (16 μmol/L)]. The expression of Wnt-1 and β-catenin at mRNA and protein levels was detected by real-time PCR and Western blot. RESULTS: Compared with the paracancerous tissues, the expression levels of Wnt-1 and β-catenin were higher in tumor tissues at mRNA and proteins levels (P<0.05). Notably, the positive expression rates of Wnt-1 and β-catenin were significantly higher in tumor tissues than that in the paracancerous tissues. Furthermore, Wnt-1 expression was associated with tumor metastasis (χ²=5.352, P=0.021), tumor stage (χ²=9.412, P=0.002) and tumor size (χ²=9.412, P=0.002). In addition, β-catenin expression was also associated with tumor metastasis (χ²=9.851, P=0.002) and tumor stage (χ²=5.661, P=0.017). Compared with control group, the expression of Wnt-1 and β-catenin at mRNA and protein levels in agonist group was increased (P<0.05),while that in antagonist group was decreased (P<0.05). CONCLUSION: The expression levels of Wnt-1 and β-catenin related with Wnt/β-catenin signaling pathway are increased in the breast cancer, which are closely related to the malignant state of the tumor.     

Effect of Yiqi Huayu Huatan decoction on expression of KLF15, HMGB1, NF-κB and its downstream inflammatory factors in rats with UUO-induced renal interstitial fibrosis

AIM: To observe the effect of Yiqi Huayu Huatan decoction (YHHD) on unilaterral ureteral obstruction (UUO)-induced renal interstitial fibrosis in rats, and to investigate the possible mechanism. METHODS: Female SD rats (n=48) were randomly divided into sham group, model group, telmisartan group, and low-, middle-and high-dose YHHD groups, with 8 rats in each group. The UUO model rats was established by ligating left ureter. The rats in sham group and model group were treated with equal volume of normal saline, others were treated with the corresponding drugs daily. After 12 weeks, the rats were sacrificed. The serum samples were collected for determining the concentrations of cystatin C (Cys-C) and uric acid (UA). The morphological changes of the renal tissue were observed by PAS staining. The collagen fiber was observed by Masson staining. The mRNA expression of Krüppel-like factor 15 (KLF15), high-mo-bility group box protein 1 (HMGB1), nuclear factor-κB (NF-κB), IκB, monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), fibronectin (FN), collagen type I (Col I) and Col-Ⅳ was detected by real-time PCR. The protein expression of KLF15, HMGB1 and NF-κB was detected by Western blot. The protein expression of MCP-1 was determined by the method of immunohistochemistry. RESULTS: Compared with sham group, the deposition rate of collagen fibers and the concentration of Cys-C in model group were significantly increased (P<0.05), the mRNA and protein expression of KLF15 was significantly down-regulated (P<0.05), while the mRNA expression of HMGB1, NF-κB, IκB, MCP-1, IL-1β, TNF-α, FN, Col I and Col Ⅳ and the protein expression of HMGB1, NF-κB and MCP-1 were significantly up-regulated (P<0.05). Compared with model group, the deposition rates of collagen fibers in middle-and high-dose YHHD groups and telmisartan group were significantly decreased (P<0.05), with down-regulated protein expression of HMGB1 and NF-κB and mRNA expression of IL-1β and TNF-α (P<0.05). The protein expression of KLF15 was significantly up-regulated in high-dose YHHD group and telmisartan group (P<0.05), while the protein expression of MCP-1 and the mRNA expression of FN were significantly down-regulated (P<0.05). The mRNA expression of KLF15 was significantly up-regulated in high-dose YHHD group (P<0.05), while the mRNA expression of MCP-1, Col I and Col IV was significantly down-regulated (P<0.05). The mRNA expression of NF-κB and IκB was significantly down-regulated and the concentration of Cys-C was significantly decreased in each dose of YHHD groups and telmisartan group (P<0.05). No significant difference of UA level among the groups was observed. CONCLUSION: YHHD alleviates renal interstitial fibrosis in a dose-dependent manner, and YHHD at high dose shows the most obvious effect. The mechanism may be associated with the up-regulation of KLF15 and the down-regulation of HMGB1, NF-κB and its downstream inflammation-related factors in the renal tissue.     

Role of hepatitis B virus in intrahepatic TGF-β1/Smads signaling pathway

AIM:To explore the effects of hepatitis B virus (HBV) on intrahepatic expression of transforming growth factor β1（TGF-β1） and Smads. METHODS:The expression of intrahepatic TGF-β1, HBsAg and HBcAg in control group and chronic hepatitis B (CHB) group was detected by immunohistochemical method.The serum HBV DNA content was determined by real-time PCR. The role of HBV in the expression of TGF-β1, Smad3 and Smad7 in human hepatic stellate cell line LX-2 in vitro was observed by cell culture and Western blotting. RESULTS:The average score of intrahepatic TGF-β1 expression in CHB group was higher than that in control group. With the increase in serum HBV DNA content, intrahepatic TGF-β1 expression was also enhanced. In the HBcAg positive hepatic tissue, there was higher TGF-β1 expression than that in the liver tissue of HBcAg negative. Compared with control group and HBV+anti-TGF-β1 group, HBV caused increased expression of TGF-β1 and Smad3 in HBV group in vitro. No difference of Smad7 protein among control group, HBV group and HBV+anti-TGF-β1 group was observed. CONCLUSION: The expression of intrahepatic TGF-β1 is related to serum HBV DNA and hepatocellular HBcAg in the patients with CHB. HBV-induced liver fibrosis mainly relies on positive regulatory mechanisms of Smad3,and the negative regulation by Smad7 almost does not function.     

Expression and modulation of connective tissue growth factor in renal interstitial fibrosis

CTGF, a member of the CCN family of immediate early genes, is a recently discovered profibrotic growth factor, which is involved in many pathophysiologic procedures. CTGF acts as a downstream effector of TGF-β acting on interstitial cells to enhance the progression of fibrotic renal diseases. It has been shown that CTGF gene expression can be induced or blocked by some kinds of cytokine and drugs. It is an interesting candidate target for future intervention strategies of renal interstitial fibrosis.     

Effects of curcumin analogue L6H4 on myocardial tissue of type 2 diabetic rats

AIM: To explore the effects of curcumin analogue L6H4 on the myocardial tissue of type 2 diabetic rats and its mechanism. METHODS: Male Sprague-Dawley rats were randomly divided into normal control (NC) group, high-fat (HF) group, high-fat treatment (FT) group, diabetes mellitus (DM) group and diabetes treatment (DT) group.The rats in the latter 4 groups were fed high-fat diet for 4 weeks, then the rats in DM groups and DT groups were intraperitoneally injected with streptozotocin (STZ) to induce type 2 diabetes, while the rats in FT group and DT group were given L6H4. The blood glucose and lipid levels were detected by biochemical method, and serum adiponectin (APN) levels were detected by ELISA. The serum insulin levels were measured by radioimmunoassay and homeostasis model assessment of insulin resistance (HOMA-IR) were calculated. The morphological changes of myocardium were observed by Masson staining and electron microscopy. The protein expression of adiponectin receptor 1 (AdipoR1) and transforming growth factor β1(TGF-β1) in myocardial tissue were determined by immunohistochemistry. The protein expression of adipoR1 was also detected by Western blot for verification. RESULTS: Compared with NC group, the blood glucose, lipids, insulin, HOMA-IR and TGF-β1 were increased in HF and DM group, but they were decreased after treated with L6H4. Compared with NC group, the concentration of serum APN were decreased and the expression of AdipoR1 in the myocardium were weakened in HF group and DM group, and they increased after treated with L6H4. The myocardial fibrosis was obvious in HF group and DM group, the mitochondria in cardiomyocytes expanded, and the cristae disordered, partial disappeared. These lesions were significantly reduced after L6H4 treatment. CONCLUSION: L6H4 exerts a protective effect on the heart in type 2 diabetic rats. The increased concentration of serum APN, the enhanced expression of AdipoR1, and the expression of TGF-β1 inhibited by APN may be involved in the mechanism of protection.     

Sedum sarmentosum Bunge extract inhibits epithelial-mesenchymal transition and collagen accumulation in aristolochic acid-treated rat renal tubular epithelial cells

AIM:To investigate the effect of Sedum sarmentosum Bunge (SSB) extract on epithelial-mesenchymal transition (EMT) and collagen accumulation induced by aristolochic acid (AA) in renal tubular epithelial cells. METHODS:Rat renal tubular epithelial NRK-52E cells were randomly divided into 3 groups, including control group (only treated with solvent), AA group (treated with AA at concentrations ranging from 1 to 100 mg/L) and SSB group (treated with AA at a concentration of 10 mg/L plus SSB extract at concentrations ranging from 10 to 2 000 mg/L). After cultured for 24 h, the morphology of the NRK-52E cells was observed under inverted phase-contrast microscope. The level of transforming growth factor β1 (TGF-β1) in the culture supernatant was measured by ELISA. Immunofluorescent analysis was performed to detect the expression of epithelial marker α-smooth muscle actin (α-SMA), mesenchymal marker E-cadherin, and extracellular cell matrix component type III collagen. The mRNA expression of E-cadherin, α-SMA, bone morphogenetic protein 7 (BMP-7) and type I collagen was also quantified by real-time PCR. RESULTS: Fibrosis-like reaction observed under microscope was obviously increased in AA-treated NRK-52E cells, and aggravated as the increase in the concentration of AA. AA at concentrations of 1 and 10 mg/L increased the expression of α-SMA, type I and type III collagens, and decreased the expression of E-cadherin. With SSB extract treatment, fibrosis in NRK-52E cells was alleviated, accompanied with the decreasing expression of α-SMA, type I and type III collagen, and the enhancing expression of E-cadherin and BMP-7.Moreover, SSB extract down-regulated TGF-β1 level in a concentration-dependent manner. CONCLUSION: AA-induced fibrosis-like reaction in renal tubular epithelial cells is reduced by the treatment with SSB extract. The possible mechanism is that SSB extract decreases TGF-β1 level, and inhibits renal EMT and collagen accumulation induced by AA.［KEY WORDS］Sedum sarmentosum Bunge|Aristolochic acid|Transforming growth factor β1|Epithelial-mesenchymal transition|Collagen     

PAI-1,TIMP-1 gene expression in renal tubulointerstitial fibrosis of ureteral obstruction and the interfering effects of HGF treatment

AIM: The aim of this study was to determine the relationship between PAI-1,TIMP-1 gene expression and renal tubulointerstitial fibrosis(TIF) of ureteral obstruction ,and the interfering effects of hepatocyte growth factor (HGF) treatment. METHODS: Sixty rats were divided into normal control, sham operation,unilateral ureteral obstruction(UUO),and rhHGF treated groups (received 0.5 mg·kg^-1·d^-1 HGF for twenty-one days), and were sacrificed at postoperative day 3, 7, 14, 21. The levels of PAI-1, TIMP-1 mRNA were measured by RT-PCR. MMP2 and MMP9 activities were detected by substrate zymography, and renal fibrosis was assessed by measuring tissue hydroxyproline. RESULTS: Compared to controls, expression levels of PAI-1,TIMP-1 mRNA were significantly increased in UUO rats, and this was accompanied by decreased activities of MMP2 and MMP9 and increase in tissue hydroxyproline content. HGF treatment significantly decreased expressions of PAI-1, TIMP-1 mRNA, increased MMP2 and MMP9 activities,and decreased tissue hydroxyproline content in the obstructive kidney. CONCLUSIONS: These results indicate that the increases in PAI-1, TIMP-1 mRNA expression may be the major cause of sustained decreased matrix degradation during the development of tubulointerstitial fibrosis after unilateral ureteral obstruction. rhHGF efficiently ameliorates renal tubulointerstitial injury by the reduction of PAI-1,TIMP-1 mRNA expression, and increasing MMP2, MMP9 activities.