Effect of salvianolic acid B on high glucose-induced phenotypic transition and extracellular matrix secretion in human glomerular mesangial cells

AIM:To investigate the effect of salvianolic acid B (Sal B) on high glucose-induced phenotypic transition and extracellular matrix (ECM) secretion in human glomerular mesangial cells (HGMCs) and the underlying mechanisms. METHODS:HGMCs were randomly divided into control group, high glucose group and high glucose plus high dose, medium dose and low dose of Sal B groups. The HGMCs except those in control group were exposed to high glucose (33.3 mmol/L) for 72 h, while those in Sal B groups were co-incubated with indicated concentrations of Sal B. The protein levels of α-smooth muscle actin (α-SMA), transforming growth factor-β₁ (TGF-β₁) and phosphorylated Smad2 and p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. The secretion levels of collagen type I (Col I), collagen type Ⅲ (Col Ⅲ), fibronectin (FN) and laminin (LN) were measured by ELISA. RESULTS:Exposure to high glucose markedly increased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs (P<0.01). The phosphorylation levels of Smad2 and p38 MAPK were also significantly increased (P<0.01). Co-incubation with Sal B evidently decreased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs induced by high glucose (P<0.05 or P<0.01). The phosphorylated levels of Smad2 and p38 MAPK were also reduced noticeably (P<0.05 or P<0.01). CONCLUSION:Sal B significantly suppresses high glucose-induced phenotypic transition and ECM secretion in the HGMCs, which might be attributed, at least partly, to inhibition of TGF-β₁/Smad signaling pathway and p38 MAPK activation.     

Effect of rosiglitazone on SREBP-1 and TGF-β1 expressions and accumulation of ECM in renal tubular cells of Wistar rats treated with high fat diet

AIM: To study the effect of high fat diet on the expression of sterol regulatory element biding protein-1 (SREBP-1) and transforming growth factor β1 (TGF-β1) in renal tubular cells and rosiglitazone intervention. METHODS: Wistar rats were treated with high fat diet and rosiglitazone for 3 months. The serum glucose, serum insulin and serum triglyceride were detected. Oil Red O staining was used to observe the renal lipid deposit and Masson staining was for the detection of ECM accumulation. SREBP-1, TGF-β1 and FN protein were determined by the methods of immunohistochemistry and Western blotting. SREBP-1 mRNA was detected by in situ hybridization. RESULTS: Rosiglitazone prevented effectively the increase in serum glucose, serum insulin and serum triglyceride resulted from high fat diet. High fat diet led to lipid droplet formation in renal tubular cells and interstitial ECM accumulation, which was decreased by rosiglitazone treatment. Compared to normal rats, SREBP-1 protein and SREBP-1 mRNA showed high expressions in high fat diet rats that were lowered by rosiglitazone. The precursor segment and mature segment of SREBP-1 protein were decreased by 27.39% and 27.32%. Similarly, the high expressions of TGF-β1 and FN protein in kidney of high fat diet rats were also prevented by rosiglitazone intervention. Compared to high fat diet rats, the expression of TGF-β1 in rosiglitazone treatment rats was lowered by 19.14%. CONCLUSION: Rosiglitazone prevents effectively the over-expression of SREBP-1 and TGF-β1 in renal tubular cells, and decreases lipid accumulation and ECM production in rats fed with high fat diet.     

Estradiol stimulated proliferation and differentiation of prostatic stromal cells through regulation of BPH-1 paracrine

AIM: To characterize the effect of estradiol on proliferation, differentiation and extracellular matrix (ECM) accumulation in stromal cells through regulation of BPH-1 paracrine. METHODS: BPH-1 cells were stimulated with different concentrations of estradiol. Conditioned media (CM) were harvested and their effects on stromal cell cultures were tested. Cell proliferation was determined by MTT assay. mRNA of smoothelin, fibronectin, collagen Ⅳ and transforming growth factor β₁(TGF-β₁) were analyzed by real-time RT-PCR. Western blotting was used to determine smooth muscle myosin heavy chain (SMMHC). ELISA and radioimmunoassay were respectively used to measure fibronectin, TGF-β₁ and collagen Ⅳ protein expressions.RESULTS: Estrodiol stimulated the expression and secretion of TGF-β₁ in BPH-1 cells. The proliferation of stromal cells increased when they were cultured with CM harvested from estrogen treated BPH-1 cells. The mRNA levels of collagen Ⅳ and smoothelin increased in stromal cells treated with CM from BPH-1 cells. The results of radioimmunoassay also showed that the collagen Ⅳ protein level up-regulated in the supernatants and cell extracts of CM-treated stromal cells. A neutralizing antibody to TGF-β₁ inhibited the stimulation of collagen Ⅳ and SMMHC by BPH-1 CM. The expression of fibronectin was only marginally changed in stromal cells cultured in the presence of BPH-1 CM. CONCLUSION: The BPH-1 cells increase ECM accumulation and differentiation of stromal cells through TGF-β₁. Estradiol stimulate differentiation of stromal cells by induction of TGF-β₁ expression. Estradiol stimulate proliferation by influencing the factors secreted from prostatic epithelial cells.     

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.     

Effect of astragalus polysaccharides on expression of MMP2 and MMP9 in annulus fibrosus of cervical intervertebral discs from cervical spondylosis model rats

AIM: To investigate the effect of astragalus polysaccharides (AP) on the expression of matrix metalloproteinase 2 (MMP2) and MMP9 in the annulus fibrosus of cervical intervertebral discs from cervical spondylosis model rats. METHODS: The model rats were randomly divided into model group (M group), and low-dose and high-dose AP treatment groups (L-AP and H-AP groups). The rats in sham operation group were used as negative control group (NC group). In addition, all the annulus fibrosus tissues were used for primary cell culture. Histological analysis was performed using HE staining and Safranin O staining. The expression of MMP2, MMP9, tissue inhibitor of metalloproteinase 2 (TIMP2) and collagen Ⅳ at mRNA and protein levels was analyzed by immunohistochemistry, Western blot and RT-qPCR. Cell-collagen adhesion assay was used to detect annulus fibrosus cell-collagen adhesion. RESULTS: The intervertebral discs of M group were degenerated, while astragalus polysaccharide improved the degenerative disc disease in the rats with cervical spondylosis. Compared with NC group, the expression of MMP2 and MMP9 in the annulus fibrosus tissues of M group increased significantly, while the expression of TIMP2 and collagen Ⅳ was decreased significantly (P<0.05). Compared with M group, the expression of MMP2 and MMP9 in L-AP group and H-AP group was significantly decreased, while the expression of TIMP2 and collagen Ⅳ was significantly increased (P<0.05). The cell-collagen adhesion in M group was significantly lower than that in NC group (P<0.05). Compared with M group, the cell-collagen adhesion in L-AP group and H-AP group was increased significantly (P<0.05). Compared with NC group, the expression of MMP2 and MMP9 in annulus fibrosus cells of M group was increased significantly, while the expression of TIMP2 and collagen Ⅳ was decreased significantly (P<0.05). Compared with M group, the expression levels of MMP2 and MMP9 in L-AP group and H-AP group of fibrocytes were significantly decreased, while the expression of TIMP2 and collagen Ⅳ was significantly increased (P<0.05). CONCLUSION: Astragalus polysaccharides inhibit the expression of MMP2 and MMP9 in the annulus fibrosus of cervical intervertebral discs from cervical spondylosis model rats and regulate the dynamic balance of MMPs and TIMPs in the extracellular matrix, thus inhibiting the degradation of collagen in the intervertebral disc matrix and having the potential research value for the treatment of intervertebral disc degeneration.     

Effects of angiotensionⅡon metalloproteinases and matrix in hypertrophied myocardium from infarcted rats

AIM: To investigate the roles of angiotensionⅡ (AngⅡ) receptors (AT₁, AT₂) antagonists on matrix metalloproteinases (MMPs) and extracellular matrix (ECM) system in septal myocardium from infarcted rats.METHODS: The model of rat myocardium infarction (MI) was established by permanent ligation of the left coronary artery. The treatments of the AT₁ receptor antagonist valsartan (10 mg·kg^-1·d^-1) or AT₂ receptor antagonist PD123319 (30 mg·kg^-1·d^-1) were started 7 days prior to surgery. On day 14 after MI, protein levels of MMP-2, 3, 9, fibronectin (FN), tenascin-C (TN-C) in interventricular septum (IS) were determined. The distributions of FN and TN-C were also determined by immunofluorescence.RESULTS: Pathological changes of IS on day 14 after MI showed typical myocardial hypertrophy. Protein expressions of MMP-2, 3, 9 and TN-C of IS in banding group were higher than those in sham-operation group (P<0.01). The expressions of TIMP-1 and FN were lower than those in sham-operation group (P<0.01). Protein expressions of MMP-2, 3, 9 and TN-C in valsartan group were obviously lower than those in banding and PD123319 groups (P<0.01). TIMP-1 and FN protein expressions in valsartan group were higher than those in banding and PD123319 groups (P<0.01). No difference between banding and PD123319 groups was observed (P>0.05).CONCLUSION: AngⅡis involved in myocardium remodeling in infarcted rats, which is mediated via AT₁ receptor to degrade matrix by MMPs. The heart protection of AT₁ receptor antagonists may relate to inhibition of MMPs.     

Preventive effects of anti-IGFBPrP1 antibody on hepatic fibrosis in mice

AIM: To investigate the preventive effect and mechanism of anti-insulin-like growth factor binding protein related protein 1(IGFBPrP1) antibody on hepatic fibrosis induced by thioacetamide (TAA) in mice.METHODS: Twenty-four male C57BL/6 wild-type mice were randomly divided into 3 groups (n= 8 in each group): normal control group, TAA group (4 weeks) and TAA+anti-IGFBPrP1 antibody group (4 weeks). The morphological changes of liver tissues were observed. The expression levels of α-smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β₁), Smad3, phosphorylated Smad2/3 (p-Smad2/3), fibronectin (FN), collagen I, collagen Ⅲ and IGFBPrP1 were detected by the methods of immunohistochemistry and Western blotting.RESULTS: In TAA group (4 weeks), obvious injury of liver was observed, and the expression levels of α-SMA, TGF-β₁, Smad3, p-Smad2/3, FN, collagen Ⅰ, collagen Ⅲ and IGFBPrP1 were significantly increased as compared with normal control group (P<0.01). Compared with TAA group (4 weeks), the injury of the liver was alleviated and the expression levels of the proteins above were decreased in TAA+anti-IGFBPrP1 antibody group (4 weeks, P<0.01). IGFBPrP1 was positively correlated with TGF-β₁, Smad3, p-Smad2/3, FN and collagen I (P<0.01). CONCLUSION: Anti-IGFBPrP1 antibody prevents TAA-induced hepatic fibrosis in mice by inhibiting the activation of hepatic stellate cells, reducing the expression of p-Smad2/3 and inhibiting the TGF-β₁/ Smad3 signal transduction, thereby depressing the deposition of extracellular matrix in liver tissues.     

Effect of IGFBP-rP1 on hepatic stellate cells and the activity of nuclear factor-κB

AIM:To identify the effect of insulin-like growth factor binding protein-related protein 1(IGFBP-rP1) on the activation of hepatic stellate cells (HSC) and the synthesis of extracellular matrix (ECM), and to investigate the possible pathway of signal transduction. METHODS:Rat HSC-T6 was cultured in vitro and established respectively the control group (incubated with PBS) and the groups treated with different concentrations of IGFBP-rP1. Cell-coated dishes were attained, and the single cell suspension was prepared after 24 h, then the expression of alpha-smooth muscle actin (α-SMA), collagen I and fibronectin (FN) were detected by immunocytochemical staining. The DNA binding activity of NF-κB p65 was detected by flow cytometry. RESULTS:The results of immunocytochemical staining showed that the positive stainings of α-SMA, collagen I and FN in the groups of IGFBP-rP1 were significantly higher than those in control group, and a dose-dependent relationship in a certain range was observed. Flow cytometry analysis showed that the percentage of NF-κB p65 positive cells in the groups of IGFBP-rP1 markedly increased compared with control group. CONCLUSION:HSC is activated by IGFBP-rP1 and the level of HSC activation gradually enhances in a certain dose range of IGFBP-rP1. The synthesis of both collagen I and FN is increased by IGFBP-rP1. IGFBP-rP1 enhances the DNA binding activity of NF-κB p65 in HSC.     

Inhibitory effect of fluvastatin on intimal hyperplasiaafter balloon injury

AIM: To investigate the effects of fluvastatin on intimal hyperplasia following balloon injury of rabbit iliac artery. METHODS: The iliac arteries of twelve white New Zealand rabbits were injured with an inflated balloon catheter, six rabbits were given fluvastatin 8 mg·kg-1·d-1 by gavage, the others served as controls. Serum concentrations of endothelin, thromboxane A2 and prostacyclin were measured by radioimmunoassay at time points before, 3 hours and 3 days after balloon injury. After 28 days, ratio of intima-to-media area (I/M) of the injured arteries were calculated using a computer image analysis system and the alteration of TGF-β1 and extracellular matrix (FN, LN) were quantified with immunohistochemistry. RESULTS: ① Serum endothelin-1 and thromboxane A2 significantly increased after injury, whereas serum prostacyclin markedly decreased. The alterations were completely reversed by the treatment with fluvastatin. ② The expression of TGF-β1 and deposition of ECM in intimal were much lower than that in the untreatment animals. ③ Intimal hyperplasia after balloon injury was significantly attenuated by fluvastatin treatment. CONCLUSION: The inhibitory effects of fluvastatin on intimal hyperplasia after balloon injury may attribute to its inhibiting thrombosis and deposition of ECM.     

Effects of IGFBPrP1 and thioacetamide on liver tissues in mice

AIM: To investigate the effects of insulin-like growth factor binding protein related protein 1(IGFBPrP1) and thioacetamide (TAA) on the liver tissues, and to identify the role of IGFBPrP1 in liver fibrosis. METHODS: Thirty-two male C57BL/6 wild-type mice were randomly divided into 4 groups (n=8 in each group): control group, recombinant murine IGFBPrP1(rmIGFBPrP1) 4 weeks group, TAA 2 weeks group and TAA 4 weeks group. The methods of hematoxylin-eosin (HE) staining, picric acid-Sirius red staining, immunohistochemistry and Western blotting were performed. RESULTS: The extensive fatty degeneration of liver cells in rmIGFBPrP1 4 weeks group was observed. The collagen deposition was found in TAA 2 weeks group. In TAA 4 weeks group, the degree of hepatic fibrosis was more serious than that in TAA 2 weeks group. The expression levels of IGFBPrP1, transforming growth factor beta 1(TGF-β₁), Smad3, p-Smad2/3, collagen Ⅲ, collagenⅠand fibronectin (FN) in liver tissues were higher in rmIGFBPrP1 4 weeks group, TAA 2 weeks group and TAA 4 weeks group than those in control group. No significant difference of the expression levels of IGFBPrP1, collagen I and FN between rmIGFBPrP1 4 weeks group and TAA 2 weeks group was observed. CONCLUSION: IGFBPrP1 plays an important role in the process of thioacetamide-induced liver fibrosis. Meanwhile, IGFBPrP1 induces excessive deposition of extracellular matrix through TGF-β₁/Smad3 pathway.     

Chronic metabolic acidosis markedly induces proliferation of mesangial cells in rats

AIM: To study the effects of chronic metabolic acidosis on glomerulus, mesangial cells and the production of extracellular matrix. METHODS: Chronic metabolic acidosis was induced by addition of 0.28 mol/L NH4Cl to drinking water for 3, 7, or 14 days in male Wistar rats (n=10). Light microscope combined with computer software (Motic Images Advanced 3.2) was used to determine the effect of chronic acid loading on renal morphologic changes. The expressions of proliferation cell nuclear antigen (PCNA) and p27 in glomeruli were detected by Western blotting or immunohistochemistry. Fibronectin (FN) mRNA was detected by real-time PCR. The proliferation of mesangial cells in vitro was determined by ［3H］-TdR incorporation. The concentration of FN in cultured supernatant was detected by ELISA. RESULTS: On day 1, 3, 7 and 14, the arterial pH and plasma ［HCO3-］ in experimental rats were significantly decreased. There was a significantly increased in the kidney weight and the ratio of kidney to body weigh in experimental rats on day 3, 7 and 14. The glomerular area and cell numbers also increased significantly. Immunoblotting demonstrated decreased p27 expression and increased PCNA expression in isolated glomeruli, and the expression of PCNA increased in a time-dependent manner following the time of chronic metabolic acidosis. Immunohistochemistry showed increased positive PCNA expression mainly localized to mesangial cells. The expression of FN mRNA was significantly elevated in experimental rats on day 7 and 14. In vitro, acid loading induced mesangial cell proliferation and synthesis of FN. CONCLUSION: These results suggest that chronic metabolic acidosis induces mesangial cell proliferation, and its mechanism may be associated with the downregulation of cell cycle kinase inhibitor p27.     

Expression of SGK isoforms in human proximal tubular epithelial cells under the condition of high glucose concentration

AIM: To investigate the effect of high glucose concentration on serum and glucocorticoid induced protein kinase (SGK) mRNA and protein expressions in human proximal tubular epithelial cells (HKC) and the possible role of SGK in the production of extracellular matrix (ECM) of HKC under the condition of high glucose. METHODS: HKC was divided into 3 groups: control glucose group (CG group, 5.5 mmol/L D-glucose); high glucose group (HG group, 25 mmol/L D-glucose) and osmotic control group (MG group, 19.5 mmol/L mannitol and 5.5 mmol/L D-glucose). The expressions of SGK mRNA and protein were assessed by semi-quantitative RT-PCR and Western blotting respectively. The level of secretary and cytoplasmic fibronectin (FN) were detected by enzyme-linked immunoabsordent assay (ELISA) and indirect-immunofluorescence. RESULTS: HKC expressed SGK1, SGK2 and SGK3 at mRNA and protein levels. Their mRNA level were up-regulated since 2 hours after cells exposed to D-glucose and this up-regulation persisted to the end of 8th hour, and SGK1 protein level elevated simultaneously. On the other hand, the increased FN secretion by high glucose was in a time-dependent manner and its improved secretion threshold was just followed by the high expression of SGK1. CONCLUSIONS: In response to high glucose, the expression of SGK1, SGK2 and SGK3 in human proximal tubular epithelial cells were up-regulated which was accompanied with FN accumulation. The high expression of SGK may mediate overproduction of ECM in proximal tubular epithelial cells and contribute to the diabetic nephropathy.     

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 p38MAPK activation in high glucose-induced collagen Ⅲ synthesis in normal rat kidney tubular epithelial cells NRK52E

AIM: To study the role of p38 mitogen-activated protein kinase (p38MAPK) activation in high glucose-induced collagen Ⅲ synthesis in NRK52E cells. METHODS: Normal rat tubular epithelial cell line NRK52E was cultured in D-glucose of different concentrations, pretreated with SB203580 and collected at different time points. The levels of phospho-p38MAPK and extracellular matrix collagen Ⅲ were examined by Western blotting. RESULTS: The activation of p38MAPK was shown to be dependent upon D-glucose concentration and the time-course. Pretreatment with SB203580 blocked p38MAPK activation induced by high concentration of D-glucose in NRK52E cells. CONCLUSIONS: The activation of p38MAPK induced by high concentration of glucose may play a role in diabetic interstital renal fibrosis. SB203580 has a potential value of clinical applications in the prevention and treatment of diabetic nephropathy.     

Role of caveolae in high glucose-induced extracellular matrix production in rat mesangial cells

AIM：To investigate the role of caveolae in high glucose (HG)-induced extracellular matrix (ECM) production in rat mesangial cells (MCs). METHODS：Synchronized rat MCs were divided into normal glucose group, HG group, HG+methyl-β-cyclodextrin (β-MCD) group and HG+β-MCD+cholesterol (Chol) group. Western blotting was used to detect the protein expression of caveolin-1 (Cav-1), phosphorylated caveolin-1 (p-Cav-1-Y14) and collagen type 1 (Col I). The mRNA expression of Cav-1 was determined by real-time PCR. ELISA was used to measure the level of fibronectin (FN) in the supernatant. RESULTS：High glucose significantly increased the expression of FN and Col I. In HG 12, 24 and 48 h groups, the mRNA and protein levels of Cav-1 were not significantly different from those in HG 0 h group, whereas the level of p-Cav-1-Y14 was significantly increased. β-MCD significantly attenuated HG-induced elevation of p-Cav-1-Y14 and FN production, but had no effect on HG-induced Col I expression. All these responses to β-MCD were abolished by Chol. CONCLUSION：High glucose significantly increases the production of Col I and FN in rat MCs. FN production induced by high glucose is mediated by p-Cav-1-Y14.     

Effect of alpha-lipoic acid on expression of miR-21/Smad7 and fibrosis in kidney of diabetic rats

AIM: To investigate the protective effect of alpha-lipoic acid (ALA) on the kidney of the rats with diabetes mellitus (DM), and to discuss the mechanism. METHODS: The DM rats were divided into normal control (NC) group, DM group and ALA group. After treated with ALA for 6 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and the pathological changes of the kidney tissues were observed by HE staining and Masson staining. The protein levels of transforming growth factor-β1 (TGF-1), p-Smad2/3, Smad7, collagen I and collagen Ⅲ were determined by Western blot. In addition, the expression of microRNA-21 (miR-21) was detected by RT-qPCR. RESULTS: Compared with NC group, the kidney weight/body weight, blood glucose (BG), total cholesterol, triglyceride and 24-h urine protein were remarkably increased in DM group (P<0.05). The pathological observation of the kidney tissues showed fibrosis changes in DM group. The level of Smad7 was reduced in DM group, while the levels of TGF-β1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were increased (P<0.05). After treatment with ALA for 6 weeks, all the relevant biochemical parameters were reduced except BG, and the renal fibrosis lesions were obviously alleviated. Compared with DM group, the levels of TGF-1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were reduced in ALA group, while the level of Smad7 was increased (P<0.05).CONCLUSION: ALA may prevent the development of renal fibrosis in rats through restraining the expression of TGF-β1 and miR-21, increasing the levels of Smad7 protein, and reducing the deposition of extra cellular matrix.     

Expression of cathepsin B and cystatin C and its potential role in diabetic rats

AIM: To observe the expressions of cathepsin B (CB) and cystatin C (CC) in different stage of diabetic rats and to investigates their potential roles.METHODS: Sixty rats were divided into diabetes mellitus group induced by intravenous injection of streptozotocin (55 mg/kg) and normal group injected with citrate buffer. Ten rats were sacrificed respectively at the end of fourth week, eighth week and sixteenth week in both groups. 24 h urine excretion was collected in rats before sacrifice. The blood and the kidney were also collected. The mRNA and protein expressions of CB and CC in kidney were detected by real time PCR and immunohistochemical staining, respectively.RESULTS: At the end of eighth week, the expression of Ccr, 24 h urinary protein excretion, CB, CC in diabetic rats increased significantly, compared to the results at the fourth week (P<0.01 or P<0.05). With the aggravation of diabetic nephropathy, the expressions of CC, colⅣ, FN and 24 h urinary protein excretion were up-regulated significantly (P<0.01 or P<0.05). The expression of CB in diabetic rats was up-regulated at eighth week significantly (P<0.01), whereas, at the end of sixteenth week it was down-regulated significantly (P<0.01). The 24 h urinary protein excretion, the expressions of colⅣ at protein and mRNA levels and FN were negatively correlated with CB (P<0.01).CONCLUSION: The unbalance of CB and CC exists in diabetic nephropathy renal tissue, which is likely to lead to the accumulation of extracellular matrix.