VPA alleviates bleomycin-induced pulmonary fibrosis in rats

AIM: To investigate the effect of sodium valproate (VPA) on bleomycin-induced pulmonary fibrosis (PF) and its mechanism. METHODS: SD rats (n=42) were randomly divided into model group and treatment group. Bleomycin at dose of 5 mg/kg was intratracheally injected to establish a rat PF model. The rats in treatment group were given normal saline (NS, 0.5 mL/d), VPA (300 mg·kg^-1·d^-1) or dexamethasone (DEX, 0.6 mg·kg^-1·d^-1) via intraperitoneal injection from the 14th day to the 28th day after modeling. The rats in model group were sacrificed on 0 d, 14 day and 28 d after modeling . The rats in treatment group were killed at 14th day after treatment. The effects of VPA on PF were evaluated by HE and Masson staining. The hydroxyproline (HYP) content in the rat lung tissues was detected, and the expression of α-smooth muscle actin (α-SMA) and E-cadherin was determined by Western blotting. RESULTS: HE staining showed that the alveolar structure and interstitial morphology in VPA group were better than those in NS group and DEX group. The level of collagen in VPA group was significantly lower than that in DEX group and NS group by determining the HYP content and Masson staining. VPA reduced the expression of α-SMA, a mesenchymal marker protein of PF, while increased the expression of epithelial marker protein E-cadherin. CONCLUSION: VPA reduces collagen content and distribution, and up-regulates the expression of the epithelial marker protein E-cadherin, while down-regulates mesenchymal marker protein α-SMA, thereby preventing the rat lung tissues from bleomycin-induced PF.     

Matrine attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in mice

AIM: To investigate the influence of matrine (MA) on the phenotype switching of mouse monocytes and alveolar macrophages induced by bleomycin (BLM).METHODS: All mice were randomly divided into normal saline (NS) group, BLM group, BLM+NS group and BLM+MA group. The mice were administered with BLM at 2.5 mg/kg via oropharyngeal instillation. The mice in BLM+MA group were treated with MA (15 mg·kg^-1·d^-1) by oral gavage following BLM administration. The mice were sacrificed on days 3, 7, 14, and 21. The lungs were removed for pathological analysis. The circulating monocyte subsets and polarization state of bronchoalveolar lavage fluid (BALF)-derived alveolar macrophages were analyzed by flow cytometry.RESULTS: The results of HE and Masson trichrome staining in BLM and BLM+NS groups exhibited classical pathological stages of lung fibrosis, including acute inflammation phase and later fibrosis phase. Compared with BLM+NS group, MA treatment alleviated the inflammatory response and the degree of fibrosis induced by BLM (P<0.05). There was a rapid change of circulating Ly6C^hi monocytes and its magnitude was positively associated with the pulmonary inflammatory response. An expansion of M2-like alveolar macrophages was positively correlated with the magnitude of lung fibrosis. Moreover, MA treatment partially normalized the phenotype switching of monocytes and alveolar macrophages.CONCLUSION: Matrine treatment attenuates BLM-induced pulmonary injury partially via modulating the phenotype switching of monocytes and alveolar mocrophages.     

Role of mineralocorticoid receptor in bleomycin-induced pulmonary fibrosis

AIM:To investigate the role of mineralocorticoid receptor (MR) in the lungs of experimental fibrotic mice. METHODS:C57BL/6 male mice (6～8 weeks old) were randomly divided into control group, bleomycin treatment group (Bleo) and bleomycin+spironolactone treatment group (Bleo+Spiro). For induction of pulmonary fibrosis, the mice were administered bleomycin at dose of 2.5 mg/kg dissolved in 50 μL saline by the intratracheal route or given 50 μL sterile saline as control. The mice in Bleo+Spiro group were treated with spironolactone (20 mg/kg) daily by oral gavage throughout the experiment. The mice were sacrificed at 12 h, 1 d, 2 d, 3 d, 7 d, 14 d and 28 d after administration of bleomycin. HE staining and Masson’s trichrome staining were used to conduct histopathologic examination. The mRNA expression levels of collagen 1 (Col1), collagen 3 (Col3), transforming growth factor beta (TGF-β), monocyte chemoattractant protein 1 (MCP-1), and MR were examined by real-time PCR. RESULTS:The results of histological analysis revealed the classical pathological stages of bleomycin-induced lung fibrosis, including acute inflammation phase (from 12 h to 3 d), progressive fibrosis phase (14 d) and late fibrosis phase (28 d). Compared with Bleo group, the inflammatory responses of the lungs in Bleo+Spiro group were attenuated in the acute inflammation phase and the degree of fibrosis was significantly reduced at 14 d after administration of bleomycin. Treatment with spironolactone effectively down-regulated the mRNA expression of MR. The levels of MCP-1 (in the acute inflammation phase), TGF-β (at 14 d), Col1 and Col3 (at 14 d) were also significantly reduced. CONCLUSION: Blockage of MR significantly attenuates the degree of bleomycin-induced pulmonary fibrosis by regulating the production and secretion of MCP-1 and TGF-β, thus reducing the degree of inflammation and inhibiting the expression of TGF-β in the progressive fibrotic phase.     

Gefitinib inhibits epithelial-mesenchymal transition by up-regulating Foxo3a expression in mice with bleomycin-induced lung fibrosis

AIM：To identify the effect of gefitinib on the expression of forkhead box protein O3a (Foxo3a), α-smooth muscle actin (α-SMA) and related signal pathway molecules in the mice with bleomycin-induced lung fibrosis and to investigate the inhibition mechanism of gefitinib on lung epithelial-mesenchymal transition. METHODS：Thirty Kunming female mice were randomly divided into 3 groups:control group (received normal saline intratracheally), bleomycin group (received bleomycin intratracheally, 3 mg/kg), and bleomycin plus gefitinib group (received bleomycin intratracheally and gefitinib orally, 20 mg/kg). All the mice were sacrificed 14 d after the treatments. Pulmonary histological changes were evaluated by hematoxylin-eosin staining and Masson trichrome staining. The mRNA levels of Foxo3a and α-SMA in the lung tissues were detected by RT-PCR. Nuclear Foxo3a, α-SMA, and phosphorylation of EGFR, Akt and Foxo3a in the lung tissues were determined by Western blotting. RESULTS：Gefitinib inhibited bleomycin-induced lung fibrosis and significantly decreased the scores of lung inflammation and fibrosis. Foxo3a mRNA expression and total Foxo3a protein expression were increased, while the phosphorylated Foxo3a was decreased. Nuclear Foxo3a was increased significantly. Meanwhile, phosphorylated EGFR and Akt were decreased. The level of α-SMA was observably increased. CONCLUSION:Gefitinib restores Foxo3a activity and reduces α-SMA expression by modulating EGFR/Akt activity, thus inhibiting bleomycin-induced lung fibrosis.     

Expression and probable role of extracellular signal-regulated protein kinases in renal fibrosis associated with diabetic in mice

AIM: To investigate the expression and probable role of extracellular signal-regulated protein kinase (ERK1/2) in renal fibrosis associated with diabetic in mice.METHODS: Male homozygous C57BL/6 mice were divided at random into control group (intraperitoneally injected with citrate buffer) and diabetes group (received 5 consecutive daily intraperitoneal injections of streptozotocin at dose of 50 mg·kg^-1·d^-1).All mice were followed up for 16 weeks.Diabetes was confirmed by serum glucose levels exceeding 16.7 mmol/L.Mice were killed at 0,4,8,12 and 16 weeks respectively after streptozotocin injection.The kidney tissues were obtained from diabetic and control mice.Serum glucose,kidney weight/body weight (KW/BW),24 h albumin excretion rate (UAE) and the serum creatinine (Scr) were measured.The kidney tissue was used for histological and morphometric studies of glomerular size,glomerular matrix expansion (PAS),and the expression of TGF-β₁,phosphorylated ERK1/2 and collagen Ⅲ by immunohistochemical staining.RESULTS: The serum level of glucose in streptozotocin -induced diabetic mice increased significantly.The kidney weight/body weight ratio,glomerular volume and glomerular matrix expansion in diabetic mice were obviously higher than those in control mice.Serum creatinine and 24 h albumin excretion rate in diabetic mice increased significantly compared with control mice.TGF-β₁,phosphorylated ERK1/2 and collagen Ⅲ levels were obviously increased in the kidney of diabetic mice compared with those in control mice (P<0.01).TGF-β₁ expression was positively related to the expression of phosphorylated ERK1/2.CONCLUSION: The overexpression of phosphorylated ERK1/2 in diabetic kidney may play an important role in the development of renal fibrosis associated with diabetic nephropathy in mice.     

Astragalus membranaceus inhibits bleomycin-induced pulmonary fibrosis in mice by antioxidation

AIM:To investigate the effect of Astragalus membranaceus on the balance of oxidation and antioxidation in bleomycin-induced pulmonary fibrosis in mice and the possible anti-fibrosis mechanism of Astragalus membranaceus.METHODS:Female KM mice (n=36) were randomly divided into 3 groups.The mice in control group were administered with saline aerosol intratracheally.The mice in fibrosis group were administered with bleomycin at dose of 3 mg/kg aerosol intratracheally.The mice in Astragalus membranaceus group were administered with bleomycin at dose of 3 mg/kg aerosol intratracheally and then intraperitoneal injected with Astragalus membranaceus parenteral solution at daily dose of 1.7 g/kg.All mice were sacrificed 14 d after the treatment,and the lungs and serum were collected for detection.Hematoxylin-eosin staining was performed in the lung tissue.The mRNA expression of superoxide dismutase 1/2/3(SOD1/2/3),catalase (CAT),NADPH oxidase 2/4(NOX2/4) and α-smooth muscle actin (α-SMA) was detected by RT-PCR,and the protein expression of α-SMA and NOX2/4 was determined by Western blot.The concentration of malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in the serum were measured by a colorimetric method.RESULTS:The pathological injury was obviously observed in bleomycin group compared with control group,but was attenuated in Astragalus membranaceus group.The α-SMA mRNA and protein expression,MDA/T-AOC,NOX2,NOX4 and SOD3 mRNA expression,and NOX2 protein expression in bleomycin group were significantly higher than those in control group,while those in Astragalus membranaceus group were significantly lower than those in bleomycin group.The protein expression of NOX4 in bleomycin group was significantly lower than that in control group,while that in Astragalus membranaceus group was higher than that in bleomycin group.The mRNA expression of SOD1 and CAT in Astragalus membranaceus group and bleomycin group were decreased compared with control group.No significant difference of SOD2 mRNA expression among the 3 groups was observed.CONCLUSION:Astragalus membranaceus inhibits bleomycin-induced pulmonary fibrosis in mice by maintaining the balance of oxidation and antioxidation.     

Effect of epigallocatechin-3-gallate on LPS-induced p38 MAPK pathway in mouse macrophages

AIM: To investigate the effect of epigallocatechin-3-gallate (EGCG) on lipopolysaccharide (LPS)-induced p38 MAPK activation and tumor necrosis factor-α (TNF-α) secretion in macrophages. METHODS: Western blotting was used to detect the phosphorylation of p38 MAPK in mouse macrophages cultured in vitro. Enzyme linked immunosorbent assay was used to determine the secretion of TNF-α in macrophages. Electron microscopy was used to study the effect of EGCG on the structure of LPS. RESULTS: LPS caused activation of p38 MAPK and more production of TNF-α, EGCG inhibited LPS-induced phosphorylation of p38 MAPK and TNF-α production and had no effect on the structure of LPS. CONCLUSIONS: EGCG has no direct effect on LPS, but blocks cellular signal pathway. The inhibition of EGCG on LPS-induced TNF-α production is mediated, at least in part, through blocking of p38 MAPK pathway.     

Rhein attenuates bleomycin-induced rats pulmonary fibrosis through TGF-β1/Smad pathway by inhibiting miR-21 expression

AIM: To investigate the effect of rhein on bleomycin-induced pulmonary fibrosis and the expression of microRNA-21 (miR-21) and transforming growth factor-β1 (TGF-β1)/Smad signaling molecules in rats. METHODS: A single dose of bleomycin was intratracheal injected into the SD rats to induce pulmonary fibrosis. After injection of bleomycin, the rats were randomly divided into low-, medium-and high-dose rhein treatment groups and model group. The rats that were instilled with normal saline intratracheally served as control group. After the treatment for 28 d, the pulmonary pathologic changes were observed under microscope with hematoxylin-eosin staining. The lung coefficient and hydroxyproline content were also measured. The expression of miR-21 and the mRNA levels of TGF-β1 and Smad7 in the lung tissues were detected by real-time PCR. The protein levels of TGF-β1 and Smad7 were determined by Western blot. RESULTS: Rhein significantly attenuated the experimental alveolitis, pulmonary fibrosis, lung coefficient and hydroxyproline contents in the rats. Rhein obviously decreased the expression of miR-21,and the mRNA and protein levels of TGF-β1, but significantly increased the mRNA and protein levels of Smad7 in the lung tissues. CONCLUSION: Rhein effectively prevents bleomycin-induced pulmonary fibrosis by inhibiting the expression of miR-21 and promoting the expression of Smad7, thus regulating the TGF/Smad signaling pathway to decrease extracellular matrix deposition.     

Effects of Danshensu on TGF-β1/Smads signaling pathways in rats with pulmonary fibrosis

AIM: To study the preventive and curative roles of Danshensu (DA) in bleomycin (BLM)-induced pulmonary fibrosis in rats. METHODS: Pulmonary fibrosis was induced in SD rats by intratracheal instillation of BLM. The rats were intraperitoneally injected with dexamethasone (1 mg·kg-1·d-1, DXM group), DA (15 mg·kg-1·d-1, DA group), or physiological saline (2 mL·d-1, BLM group). Normal controls (NC group) received physiological saline both intratracheally and intraperitoneally. At the 28th day after modeling, the histological changes of the lungs were evaluated by hematoxylin-eosin (HE) and Masson’s trichrome staining. The protein levels of α-smooth muscle actin (α-SMA) in the lung tissues were detected by the method of immunohistochemistry. The mRNA expression of transforming growth factor beta 1 (TGF-β1), Smad3 and Smad7 was assessed by real-time fluorescence quantitative PCR. RESULTS: Compared with BLM group, the degree of inflammation and fibrosis of the lung in DA group was obviously reduced, and so was the expression of α-SMA in the lung tissues. The mRNA expression of TGF-β1 and Smad3 in the lung tissues of the rats decreased and the mRNA expression of Smad7 increased. CONCLUSION: DA alleviates BLM-induced pulmonary fibrosis in rats in the early stage by inhibiting the expression of TGF-β1/Smad3 and stimulating the expression of Smad7 in the lung tissues.     

Inhibition of plasminogen activator inhibitor-1 by small interfering RNA attenuates pulmonary fibrosis in rats

AIM: To examine the effects of silencing of plasminogen activator inhibitor-1 (PAI-1) expression by small interfering RNA (siRNA) on bleomycin (BLM)-induced rat pulmonary fibrosis. METHODS: Total 72 Wistar rats were divided into 4 groups: control, BLM, BLM+non-specific siRNA (BLM+N), and BLM+ PAI-1 siRNA (BLM+P). Pulmonary fibrosis was induced by intratracheal injection of BLM (5 mg/kg), whereas equal volume of normal saline was used in control group. After the administration of BLM or normal saline, the rats were treated with tracheal injection of PAI-1-siRNA (7.5 nmol/0.2 mL per rat) in BLM+P group, non-specific siRNA (7.5 nmol/0.2 mL per rat) in BLM+N group, and 0.2 mL normal saline in BLM group and control group, twice a week, 8 times in 28 d. On day 7, 14, and 28, the rats (n=6 at each time point) were sacrificed. The bronchoalveolar lavage fluid (BALF) from the left lung was harvested to examine the activity of PAI-1. The mRNA expression of collagen type Ⅲ, α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the middle lobe of the right lung was detected by RT-PCR. RESULTS: PAI-1 activity and the expression of collagen type Ⅲ, α-SMA and TIMP-1 were increased in BLM group on day 7, 14 and 28. Intratracheal injection of PAI-1 siRNA twice a week continuously reduced PAI-1 activity in the BALF (P<0.05),and decreased the expression of collagen type Ⅲ, α-SMA and TIMP-1 in the fibrotic lung tissues on day 7, 14 and 28. Statistical differences in the expression of collagen type Ⅲ, α-SMA and TIMP-1 between BLM+P group and BLM group at the same time point were observed. CONCLUSION: Intratracheal injection of PAI-1 siRNA twice a week continuously inhibits the expression of PAI-1. PAI-1 siRNA ameliorates BLM-induced pulmonary fibrosis by down-regulation of TIMP-1 expression.     

Activation of α7nAChR promotes wound healing in diabetic mice by suppressing TNF-α expression

AIM: To explore the role of α7 nicotinic acetylcholine receptor (α7nAChR)-specific agonist PNU-282987 in promoting wound healing in diabetic mice by suppressing the expression of tumor necrosis factor α （TNF-α）.METHODS: A model of incised wound was established in diabetic mice or normoglycaemic mice (control). Skin samples were taken on 1 d, 3 d, 5 d, 10 d, 14 d and 21 d post-injury (5 mice in each posttraumatic interval). The numbers of macrophages and fibroblasts, the expression of TNF-α and the deposition of collagen were detected by the methods of immunohistochemistry, Western blotting and Masson staining, respectively. After incised wound was performed in the diabetic mice, PNU-282987 was applied by intraperitoneal injection at suitable posttraumatic interval. The above indexes were investigated again.RESULTS: Compared with control group, the diabetic mice presented delayed wound healing. In diabe-tic mice, the infiltration of macrophages and the expression of TNF-α were significantly reduced in the early phase during wound healing, while they were significantly increased from 5 d post-injury. Besides, from 5 d to 21 d post-injury, the wounds in diabetic mice showed decreased number of fibroblasts and deposition of collagen. From 5 d post-injury, PNU-282987 was applied to diabetic mice, which significantly down-regulated the expression of TNF-α, and increased the number of fibroblasts and the content of collagen in the wounds, eventually promoted wound healing.CONCLUSION: Inflammatory reactions delay wound healing in diabetic mice. Activation of α7nAChR promotes wound healing in diabetic mice by suppressing the expression of TNF-α.     

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.     

Effect of Kupffer cell blockade on activation of mitogen-activated protein kinases in response to lipopolysaccharide

AIM: Using the mouse model of lipopolysaccharide(LPS) attack,we study the effect of Kupffer cell (KC) blockade on the activation of mitogen-activated protein kinases(MAPKs) signal transduction pathway induced by LPS.METHODS: GdCl₃ (10 mg/kg) or the same volume of NS was continually injected intravenously at 48 h and 24 h before LPS (5 mg/kg) was injected into the male mice of Kunming species.The liver was then took out and KCs were isolated 30 minute after LPS was injected.The KCs isolated from the mice were cultured,and pretreated with GdCl₃ (100 μmol/L) for 1 h.The culture medium containing LPS (100 μg/L) was added and continuously incubated for 30 minute.The protein expression and phosphorylation level of ERK1/2 and p38MAPK in liver or KCs were assayed in vivo and in vitro,and effect of GdCl₃ on the phagocytosis function was observed,respectively.RESULTS: LPS induced the protein phosphorylation of ERK1/2 and p38MAPK in KCs or liver,no effect on the protein expression was observed.GdCl₃ treatment inhibited LPS-induced KCs activation and secretion of TNF-α,however,it had no effect on ERK1/2 and p38MAPK in KCs or liver,neither at the protein expression nor the phosphorylation.KCs secreted a few TNF-α with short time treatment with GdCl₃ alone in vitro.CONCLUSION: KC blockade with GdCl₃ alleviates LPS-induced KCs activation and the release of TNF-α not through modulating intracellular ERK1/2 or p38MAPK signal transduction pathways.We presume that GdCl₃ might reduce liver injury through cross talk of other intracellular signal transduction pathways (JNK,NF-кB,GPCR,etc).     

Aortic expression of HSP22, TNF-α and eNOS in rats with hyperlipidemia and effects of atorvastatin

AIM:To establish a rat hyperlipidemia model for studying the aortic expression of heat shock protein 22 (HSP22), tumor necrosis factor alpha (TNF-α) and endothelial nitric oxide synthase (eNOS) and the effect of atorvastatin intervention. METHODS:Hyperlipidemia model was established in SD rats. Afterwards, the rats were divided into normal control group, high fat group and high fat+atorvastatin intervention group. The expression of HSP22 and TNF-α in the rat aortas was detected by immunohistochemical assay and the expression of eNOS was assessed by Western blotting. RESULTS:No detectable expression of HSP22 and TNF-α in the normal control group was observed. However, the expression of HSP22 and TNF-α was positive in the high fat group and the atorvastatin intervention group. The mean densities of HSP22 and TNF-α positive particles were significant lower in the atorvastatin intervention group as compared with high fat group (both P＜0.05). The expression of eNOS protein in the high fat group and atorvastatin intervention group was significantly lower than that in normal control group (P<0.01). However, no marked difference of eNOS protein expression between high fat group and atorvastatins intervention group was observed. CONCLUSION: The expression of HSP22 and TNF-α in the rat aortas is increased in the hyperlipidemia rat model. This effect can be restored by atorvastatin treatment. The expression of eNOS in the rat aortas is decreased in the hyperlipidemia rat model, but this tendency could not be attenuated by atorvastatin.     

Chronic alcohol intake-induced epithelial-mesenchymal transition contri-butes to hepatic fibrogenesis in mice

AIM：To evaluate the effect of chronic alcohol intake on the histopathological changes of the liver and to determine the contribution of epithelial-mesenchymal transition (EMT) to hepatic fibrogenesis. METHODS：Thirty male C57BL/6 mice were randomly divided into 3 groups as following: the mice in control group was given (ig) water; the mice in low-dose alcohol group (2.0 g·kg -1·d -1) and high-dose alcohol group (4.0 g·kg -1·d -1) were given (ig) alcohol for 5 months. Alcohol-induced histopathological changes of the liver or development of hepatic fibrosis were evaluated using the histological methods with HE and Masson trichrome staining. The apoptosis of the liver was detected by TUNEL fluorometric staining (counterstained with DAPI). The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was measured by an automated biochemical analyzer. The expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA) and E-cadherin in the hepatic tissues was detected by immunofluorescence examination. The protein levels of E-cadherin, α-SMA, FSP-1, transforming growth factor β 1 (TGF-β 1) and hypoxia-inducible factor 1α (HIF-1α) were analyzed by Western blotting. RESULTS：Compared with control, the activity of serum ALT and AST, and apoptotic index of liver tissues were increased in the mice treated with alcohol for 5 months. The histopathological changes of the livers in the mice of low-dose alcohol group included steatosis and mild liver fibrosis, while severe liver fibrosis was observed in the high-dose alcohol-treated mice. Chronic alcohol consumption induced the increase in malondialdehyde (MDA) level, and the decreases in the activity of superoxide dismutase (SOD) and catalase (CAT) in the livers. It also reduced E-cadherin expression and increased α-SMA expression. FSP-1 immunostaining and albumn immunostaining positive cells were co-localized in the hepatocytes of low-dose alcohol group, but only FSP-1 positive hepatocytes were observed in high-dose alcohol group. Chronic alcohol consumption decreased E-cadherin expression and increased α-SMA, FSP-1, TGF-β 1 and HIF-1α expression in a dose-dependent manner, but the HIF-1α expression was not altered between the 2 alcohol-treated groups. CONCLUSION：Chronic alcohol intake induces the progression of hepatic fibrosis. Some fibroblasts derive from hepatocytes in liver fibrosis via EMT. The underlying mechanism is associated with the changes of the redox state, and increased TGF-β 1 generation and HIF-1α expression.     

Influence of 8-week swimming on peripheral neuropathy in type 2 diabetic rats

AIM: To explore the influence of long-term swimming on peripheral neuropathy in type 2 diabetic rats. METHODS: Male Wistar rats were fed with a high-fat and high-fructose diet, and injected with streptozocin to establish a model of type 2 diabetes mellitus. The rats were randomly divided into 4 groups: blank control group (C group), exercise control group (CE group), diabetes mellitus group (DM group) and diabetes mellitus+exercise group (DME group). The rats in CE group and DME group received 8-week swimming training (6 d/week). The training time was 20, 30 and 45 min in the first 3 d,respectively, and then it increased to 60 min a day. Eight weeks later, the motor nerve conduction velocity (MNCV) and the levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and C-reactive protein (CRP) in sciatic nerve tissues of the rats were measured. The morphological changes of the sciatic nerve were also observed under light microscope. RESULTS: Compared with DM group, 8-week swimming obviously accelerated the MNCV (P<0.05), decreased the levels of TNF-α, IL-6 and CRP in DME group (but no significant difference, P>0.05). The obvious nerve injury in DM group was observed. However, the pathological change of the sciatic nerve in DME group was relieved. CONCLUSION: Eight-week swimming training significantly accelerates the MNCV, attenuates the nerve injury in diabetic rats and has protective effect on peripheral nerve, which may be correlated with relieving the inflammatory reaction.     

Macrophage migration-inhibitory factor induces myofibroblastic phenotype transformation of human embryonic lung fibroblasts via Rho-kinase in vitro

AIM: To investigate the influence and the mechanism of recombinant macrophage migration-inhibitory factor (rMIF) on fibroblasts. METHODS: Cultured human embryonic lung fibroblasts (MRC-5 cells) were divided into 2 groups: the cells in treatment group were treated with rMIF (25~100 μg/L) for 24 h or 48 h, and the control cells were without rMIF treatment. The mRNA expression of α-SMA was examined by RT-PCR. The protein level of α-SMA induced by rMIF was quantified by Western blotting. Half an hour before 100 μg/L rMIF challenge, Y27632 was added to the cells of 2 groups. After challenged for 48 h, the total RNA and protein were extracted,and the expression of α-SMA at mRNA and protein levels was determined by means of RT-PCR and Western blotting. After challenged by 100 μg/L rMIF for 6 h, 12 h, 24 h and 48 h, the cell total protein was extracted, and the protein level of α-SMA induced by rMIF was quantified by Western blotting. RESULTS: After stimulation for 24 h, rMIF did not increase the α-SMA mRNA synthesis compared with control. After stimulation for 48 h, rMIF significantly increased the expression of α-SMA mRNA and protein in a dose-dependent manner compared with control (r=0.697 and r=0.957, both P<0.01). Y27632 pretreatment prevented the increase (P<0.01). The amount of phosphorylated MYPT1 increased at 6 h (P<0.01), reached the maximum at 12 h (P<0.01), and decreased but still higher than the control at 24 h and 48 h (P<0.01). CONCLUSION: rMIF increases the α-SMA synthesis in MRC-5 cells and Rho-kinase regulates this process, suggesting that MIF may play important roles in the pathogenesis of airway remodeling.     

Pretreatment with external trigeminal nerve electrostimulation inhibits seizures and decreases expression of IL-1β and TNF-α in hippocampus with status epilepticus induced by pentylenetetrazol in rats

AIM:To observe the effect of pretreatment with external trigeminal nerve electrostimulation (eTNS) on behavioral changes and the expression of interleukin-1β (IL-1β) and 　tumor necrosis factor α (TNF-α) in hippocampus of pentylenetetrazol (PTZ)-treated rats. METHODS:The rats were randomly divided into control group, PTZ group and eTNS group, and kindled by PTZ after administered 7 d, 14 d and 28 d of consecutive fake electrostimulation or eTNS. Subsequently, the severity and duration of seizure were quantitatively evaluated. The concentrations of IL-1β and TNF-α in hippocampus were detected by the methods of ELISA and immunohistochemisty. RESULTS:Compared with PTZ group, treatment with eTNS significantly inhibited the severity and duration of seizure (P<0.05), and significantly reduced the content of IL-1β and TNF-α in the hippocampus after status epilepticus (P<0.05 or P<0.01). CONCLUSION:Pretreatment with eTNS may provide a new approach for prevention and treatment of epileptogenesis.     

Relationship between TNF-α gene polymorphism-308 and type 2 diabetes with periodontitis

AIM: To investigate the gene polymorphism-308 of tumor necrosis factor alpha (TNF-α) in the relation with the susceptibility to periodontitis combined with type 2 diabetes mellitus (DM) and its severity.METHODS: Human DNA samples were obtained from 240 DM patients with periodontitis (periodontitis group, n=120) and without periodontitis (control group, n=120). All patients were genotyped by PCR-RFLP analysis. The frequencies of genotypes and alleles were analyzed. Sulcus bleeding index (SBI) and probing depth (PD) in all patients were measured. The polymorphism-308 of TNF-α gene in the relation with the susceptibility to periodontitis combined with DM and its severity was analyzed.RESULTS: No significant difference in the frequency of genotype and allele was found between DM patients with mild periodontitis and DM patients without periodontitis (P>0.05). However, the frequencies of these genotypes and alleles in DM patients with moderate and severe periodontitis were significantly higher than those in DM patients without periodontitis (P<0.01). The findings showed that the level of TNF-α was associated with SBI and PD (P<0.01).CONCLUSION: TNF-α -308 G/A polymorphism is not associated with DM patients with mild periodontitis, whereas it may have a role in pathogenesis and prognosis of moderate and severe periodontitis combined with DM through TNF-α level.