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OBJECTIVE: To discuss the effects of single dose of cisplatin on renal interstitial fibrosis indicators in rats dynamically. METHODS: 72 SD rats were randomly divided into normal group and cisplatin group, with 36 rats in each group. Normal group and cisplatin group were given equal volume of normal saline and cisplatin 5 mg/kg intraperitoneally on the first day, respectively. Each 6 rats were sacrificed on 8th, 14th, 30th, 50th, 60th, 90th day. The serum levels of blood urea nitrogen (BUN) and creatinine (Cr) were determined, and the degree of renal tubulointerstitial injury and relative area of renal tubulointerstitial fibrosis were evaluated. The expression of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Col Ⅰ) and transforming growth factor β1 (TGF-β1) were determined in renal tissue. RESULTS: Compared with normal group, the serum levels of BUN and Cr, renal tubulointerstitial injury indexes, relative area of renal tubulointerstitial fibrosis, and the expression of α-SMA, Col Ⅰ and TGF-β1 in renal tissue were increased significantly (P<0. 05 or P<0. 01). In cisplatin group, within the 8th-90th days, serum level of BUN in rats had no significant change; serum level of Cr, renal tubulointerstitial injury indexes, renal tubulointerstitial fibrosis, the expression of a-SMA, Col Ⅰ and TGF-β1 in renal tissue increased first and then decreased. CONCLUSIONS: A single dose of clinical dose of cisplatin can induce renal interstitial fibrosis in rats, and its mechanism may be related to the expression of TGF-β1 in renal tissue.
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Objective The mechanisms of methylation acting on myocardial fibrosis are not yet clear at present. The aim of this study was to investigate the role of DNA methyltransferase 3A (DNMT3A) in regulating the expressions of collagens during the activation of cardiac fibroblasts.Methods Cardiac fibroblasts were obtained from 50 neonatal mice and divided into three groups: blank control, DNMT3A overexpression plasmid (mDNMT3A-pEGFP-C3) and small interference DNMT3A siRNA. The contents of collagens in the cell supernatant were detected by ELISA. The mRNA and protein expressions of type I collagen (Col Ⅰ), type Ⅲ collagen (Col Ⅲ) and DNMT3A in the cardiac fibroblasts were determined by real-time quantitative PCR and Western blot respectively and the proliferative activity of the cardiac fibroblasts measured by CCK8 assay.Results The contents of Col I and Col Ⅲ in the cell supernatant were significantly increased in the DNMT3A overexpression plasmid group but decreased in the DNMT3A siRNA group as compared with those in the blank control (P<0.05). The expressions of Col Ⅰ, Col Ⅲ and DNMT3A were remarkably higher in the DNMT3A overexpression plasmid group but lower in the DNMT3A siRNA group than in the blank control (P<0.05). The cell activity was markedly higher in the DNMT3A overexpression plasmid group than in the empty vector plasmid and control groups (2.087±0.317 vs 1.063±0.223 and 1.082±0.207, P<0.05) but lower in the DNMT3A siRNA group (0.463±0.087) than in the latter two (P<0.05).Conclusion DNMT3A can increase the proliferation and activation of cardiac fibroblasts, upregulate the expressions of collagens and thus promote myocardial fibrosis.
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Aim To explore the protective effects of in-terleukin-17 ( IL-17 ) monoclonal antibody ( mAb ) on bleomycin-induced pulmonary fibrosis rats and the re-lated mechanisms. Methods Seventy-five male SD rats were randomly divided into normal control group, sham operation group, model group, non-specific IgG group and IL-17 mAb group. Each group included fif-teen rats. Rats in the latter three groups were intratra-cheally administered with bleomycin A5 to establish pulmonary fibrosis model, whereas the ones in sham operation group were treated with the same volume of physiological saline. On day 7, 14 and 21, rats in non-specific IgG group and IL-17 mAb group were in-jected with non-specific IgG and IL-17 mAb, respec-tively,through the caudal vein. However,the ones in the other groups were administered with the same volume of physiological saline. All rats were sacrificed on day 28. Pulmonary tissues were then removed, and HE and Masson staining was performed. The contents of IL-17, IL-6 and tumor necrosis factor-α ( TNF-α) in pulmo-nary tissues were measured by enzyme linked immu-nosorbent assay ( ELISA ) . Western blot was used to analyze the pulmonary tissues protein expression of nu-clear factor-κB ( NF-κB) p65 in the nucleus as well as collagen type I ( Col Ⅰ) and collagen type III ( ColⅢ) in the whole cells. The levels of Col Ⅰ and ColⅢ in the pulmonary tissues were detected by fluores-cence real-time quantitative PCR. Serum was separa-ted, and the concentrations of procollagen type 1 carboxyterminal propeptide ( PICP ) and procollagen type III aminoterminal propeptide ( PIIINP ) in serum were then measured by ELISA. Results The severity of alveolitis and pulmonary fibrosis was lower in IL-17 mAb group than that in model group and non-specific IgG group ( P 0. 05 ) . Similar results were also seen between sham operation group and normal control group ( P >0. 05 ) . Conclusion IL-17 mAb protects rats from pulmonary fibrosis by inhibiting inflammatory response via downregulating NF-κB expression and decreasing collagen synthesis in the pulmonary tissues.
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Objective To investigate the effects of Hui-hui Gan-song Yin(HGY) on the accumulation of extracellular matrix of rat glomerular mesangial cells(MCs) induced by high glucose.Methods The 40 SD rats were randomly divided into normal control group(distilled water), glurenorm group(10 mg/kg), HGY high-dose group(10 g/kg) and HGY low-dose group(5 g/kg), 10 rats in each group.The rats in each group were treated with corresponding drugs, twice a day.After 3 days, the serum containing each drug were prepared to culture rat MCs in vitro.The MCs were divided into the normal control group( 10% serum of rats in normal control group ) , high glucose group ( 30 mmol/L glucose +10% serum of rats in normal control group), glurenorm group(30 mmol/L glucose+10% serum of rats in glurenorm group), HGY high-dose group(30 mmol/L glucose+10% serum of rats in HGY high-dose group) and HGY low-dose group(30 mmol/L glucose+10% serum of rats in HGY low-dose group).The fibronectin(FN), ColⅠand ColⅣ levels were detected by Western blot.Results Compared with normal control group, the expression of FN, ColⅠand ColⅣ in high glucose group increased(P<0.01).The HCY suppressed the protein expression of FN, ColⅠand ColⅣ significantly(P<0.05).Conclusion The serum containing HGY could suppressed protein expression of FN , ColⅠand ColⅣ and inhibit the accumulation of extracellular matrix of MCs induced by high glucose, which could protect glomerulus and delay the development of diabetic nephropathy.
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Aim To isolate and characterize the human circulating fibrocytes from human peripheral blood and explore the effects of curcumin on human circulating fi-brocytes.Methods The cells were isolated and puri-fied by density gradient centrifugation,and identified by flow cytometry and immunocytochemistry .Then , CCK-8 and flow cytometry were used to study the effect of curcumin on the proliferation as well as COL I ex-pression of human circulating fibrocytes,respectively. Results After being isolated the cells expressed CD34,CD45 and COLⅠ,among which 79.7% were both CD45 and collagen I positive,typical of human circulating fibrocytes.Curcumin could exert regulatory effects on proliferation of human circulating fibrocytes. Exposure of the cells to curcumin for as short as 24 hours promoted their growth,while prolonged treatment (72 h ) significantly inhibited cell propagation and downregulated the COLⅠ levels,best manifested at a concentration as high as 20 μmol · L-1 .Conclusion The proliferation of cells and COLⅠexpressions can be effectively inhibited by curcumin with the prolonged action period and high concentrations.
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Objective To explore the effect of Tangnaikang (TNK) on extracellular matrix expression of human tubular epithelial cell HK-2 induced by TGF-β1 and explore its mechanism on the renal fibrosis. Methods The HK-2 cells were cultured by DMEM/F12 (1∶1) with 10%fetal bovine serum and divided into control group, TGF-β1 group (TGF-β110 ng/mL), rat serum control group (TGF-β110 ng/mL +10% rat serum), TNK-containing rat serum therapy groups (TGF-β110 ng/mL+5% Tangnaikang, or +10%Tangnaikang, or +20% TNK). After 24 h of administration, the expression of ColⅠ, Col Ⅲ and FN mRNA were tested by fluorescence quantitative PCR assay. Results The expression of ColⅠ, Col Ⅲ and FN mRNA of HK-2 cultured with TGF-β1 were much higher than the control, and significantly decreased in HK-2 cultured with TGF-β1 plus Tangnaikang compared with only TGF-β1 (P<0.05), but rat serum control had no effect. Conclusion TNK could inhibit the expression of ColⅠ, Col Ⅲ and FN mRNA of HK-2 cell induced by TGF-β1, and prevent the development of renal fibrosis to some extent.
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Objective To investigate the effect of massage on quadriceps femoris repair after injury by external force and the expression of transforming growth factor β1 (TGF-β1) and collagen-Ⅰ (COL-Ⅰ) mRNA.To explore the molecular mechanisms inhibiting scar tissue formation and promoting muscle repair.Methods Forty New Zealand white rabbits weighing (2.0 ±0.5) kg were randomly divided into a normal control group (A) (n =4),a selfrepair group (B) (n =20,further divided into the 3rd,7th,11th,15th and 19th day time points),and a massage group (C) (n =16,further divided as in group B).In group A the rabbits were not treated,as normal controls.In groups B and C rabbit models of quadriceps femoris injury were prepared using a self-made beater.In group B no massage therapy was given as a natural recovery control; in group C,massage therapy was given after 5 days.Realtime quantitative PCRs were used to detect TGF-β1 and COL-Ⅰ mRNA expression.Resnlts There was no significant difference between groups B and C in the expression of TGF-β1 or COL-Ⅰ mRNA on day 7.At the later time points,expression of both mRNAs in group C was significantly less than in group B.Conclusion Massage can effectively reduce the expression of TGF-β1 and COL-Ⅰ mRNA,inhibit excessive scar formation and promote repair of injured tissue.
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Summary: The aim of this study was to investigate the effect and possible mechanism of all-trans retinoic acid (ATRA) on liver fibrosis induced by common bile duct ligation (CBDL) in rats. Fifty-three female Wistar rats were randomly divided into 5 groups: sham operation group (group J, 5 animals) and groups A, B, C and D (12 animals in each group). The rats in groups A, B, C and D were subjected to CBDL to induce liver fibrosis, while those in group J to sham operation. From the 3rd week the rats in groups B, C and D respectively received daily administration of ATRA via gastric tube at three different doses [0.1, 1.5 and 7.5 mg/kg body weight (BW)]. Animals were sacrificed at 6th week. Rats' liver tissues were observed for pathologic changes under a light microscope. The protein levels of type Ⅰ collagen (COL Ⅰ), matrix metalloproteinase-2 (MMP2), MMP13 and tissue inhibitors of metalloproteinase-1 (TIMP-1) in liver tissues were determined by immunohistochemical techniques. The expression levels of TGF-β1 and CTGF mRNA in liver tissues were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that loss of normal hepatic architecture and formation of obvious fibrosis were observed in group A, while ATRA treatment for 4 weeks notably alleviated the pathological changes of hepatocytes. The expression of COL Ⅰ and TIMP-1 proteins in group A was increased, while decreased in ATRA-treated CBDL groups (P<0.05). ATRA (1.5 and 7.5 mg/kg BW) reduced the expression levels of COL Ⅰprotein more greatly than that of 0.1 mg/kg BW (P<0.05). ATRA treatment increased the protein levels of MMP2 and MMP13. The expression levels of TGF-β1 and CTGF mRNA in group A were increased. In comparison with group A, the mRNA levels of TGF-β1 and CTGF in ATRA-treated CBDL groups were significantly decreased (P<0.05). It was concluded that ATRA could inhibit CBDL-induced liver fibrosis in rats by suppressing the expression of TGF-β1 and CTGF so as to diminish the inhibition of TIMP-1 on MMP2 and MMP13 and increase the activity of MMP2 and MMP13.