Effects of mesenchymal stem cells,fusion protein of tumor necrosis factor receptor Ⅱ-IgG Fc and mesalazine on disease activity index and tissue damage index of TNBS-induced colitis in rats

AIM: To study the effects of mesenchymal stem cells (MSCs), the fusion protein of tumor necrosis factor receptorⅡ-IgG Fc (TNFRⅡ-IgG) and mesalazine on the disease activity index (DAI) and tissue damage index (TDI) in the rat model of colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). METHODS: MSCs were cultured in low-glucose DMEM containing 10% FBS. Eighty-one Sprague-Dawley rats were used in the study and the model of colitis induced by TNBS/ethanol was established. The rats were randomly divided into 6 groups: normal control group (A), colitis group (B), MSCs treatment 1 (3×106 MSCs) group (C), MSCs treatment 2 (5×106 MSCs) group (D), TNFRⅡ-IgG treatment group (E) and mesalazine treatment group (F). The scores of DAI were used to record the manifestations of the rats, colon macroscopic damage index (CMDI) was used to describe the macroscopic features of the colon, and the scores of TDI were estimated by determining the pathological changes of the colon under microscope. RESULTS: Pure MSCs were gained by 3 times of passages. Compared with group A, the scores of DAI, CMDI and TDI in group B were always significantly increased. On day 6, these scores in every group except group A were not different obviously. On day 9, the scores in group C,group D and group F were lower than those in group B, and no statistic difference between group C and group D was observed. On day 14, the scores in group C, group D, group E and group F were lower than those in group B, and the scores in the groups were group F > group E > group C > group D. CONCLUSION: MSCs, TNFRⅡ-IgG and mesalazine used for 14 d significantly improve the scores of DAI, CMDI and TDI in the rats with colitis induced by TNBS. The method using MSCs is better than those using TNFRⅡ-IgG and mesalazine.     

Escherichia coli promotes recovery from dextran sulfate sodium-induced colitis in mice

AIM: To investigate the important role of intestinal microflora in the pathogenesis of inflammatory bowel disease by studying the effects of E.coli on dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: BALB/c mice were given 3.5% DSS in the drinking water for 5 days to induce acute colitis. The control mice (n=6) did not drink DSS water. The mice subject to drinking DSS were randomly divided into 3 groups: (1) simple DSS treatment group; (2) bacteria-depleted mice treated with DSS alone; (3) bacteria-depleted mice treated with DSS+E.coli. Four aspects of the treatment response were evaluated in each group: (1) general conditions, including body weight, disease activity index (DAI) score, colon length and weight; (2) histological score; (3) chemical colorimetric detection of myeloperoxidase (MPO) activity in the diseased tissues; (4) the activation of NF-κB detected by immune histochemistry. RESULTS: The bacteria-depleted mice that did not treat with E.coli were difficult to recover from DSS-induced colitis. Compared with non-treatment group, the general score, histological score and MPO activity in E.coli treatment group improved significantly (P<0.05). The activity of NF-κB in E.coli treatment group was significantly higher than that in non-E.coli treatment group (P<0.05). CONCLUSION: Intestinal microbiota is necessary during the recovery from DSS-induced colitis. E.coli promotes the recovery from DSS-induced colitis in mice. The mechanism may be associated with NF-κB activation.     

Protective effect of Zhenrenyangzang decoction on intestinal mucosal barrier function in ulcerative colitis rats

AIM: To investigate the protective effect of Zhenrenyangzang decoction (ZRYZ) on the intestinal mucosal barrier function and the expression of tight junction proteins, zonula occludens-1 (ZO-1) and occludin, in the colon of rats with trinitrobenzenesulfonic acid (TNBS)-induced ulcerative colitis in order to explore the possible mechanism involved. METHODS: Male Wistar rats were randomly divided into normal group, model group, salazosulfapyridine (SASP) positive control group, high-dose ZRYZ (ZRYZ-H) group and low-dose ZRYZ (ZRYZ-L) group. Except the normal group, the rats in other groups were given the TNBS/50% ethanol mixed solution by enema to make the ulcerative colitis model. The rats in positive control group were given SASP suspension liquid. The rats in ZRYZ-H group and ZRYZ-L group were orally administered with ZRYZ at doses of 30.4 g/kg and 15.2 g/kg, respectively, while the rats in normal group and model group were orally administered with saline. All the drugs were administered to the rats for consecutive 21 d. Disease activity index (DAI) was investigated during the drug treatment, and colon samples were collected after drug administration for evaluating morphological damage score. Intestinal mucosal permeability was measured by detection of lactulose/mannitol (L/M) in urine. Colonic myeloperoxidase (MPO) activity and goblet cell number, serum diamine oxidase (DAO) and D-lactic acid (D-LA) levels, and the colonic expression of ZO-1 and occludin were also measured. RESULTS: Compared with normal group, DAI, morphological damage score, L/M value, colonic MPO activity as well as serum D-LA and DAO levels in model group significantly increased (P<0.05), while goblet cell number as well as the expression levels of ZO-1 and occludin in model group were significantly reduced (P<0.05). Compared with model group, DAI, morphological damage score, L/M value, colonic MPO activity as well as serum DAO and D-LA levels in ZRYZ-H group and ZRYZ-L group were reduced significantly (P<0.05), while goblet cell number as well as the expression of ZO-1 and occludin in ZRYZ-H group and ZRYZ-L group increased significantly (P<0.05). CONCLUSION: ZRYZ protects the intestinal mucosal barrier function in ulcerative colitis rats by reducing intestinal mucosal permeability, and its mechanisms may be related to increasing the expression of ZO-1 and occludin.     

Expression of hepatocyte nuclear factor 4α in experimental colitis in mice

AIM: To investigate the role of hepatocyte nuclear factor 4α (HNF4α) in the pathogenesis of ulcerative colitis (UC) by measuring the expression of HNF4α in the colon tissues in experimental colitis mice. METHODS: BALB/c mice were exposed to 2% or 2.5% (W/V) dextran sulfate sodium (DSS) to induce acute colitis, and the severity of colitis was assessed by observation of disease activity index (DAI), histological injuries and inflammatory cytokines. The correlation between the expression of HNF4α and the severity of disease as well as E-cadherin (E-CAD), junctional adhesion molecule 1 (JAM-1) and desmocollin 2 (DSC-2) was analyzed. RESULTS: Compared with the normal controls, DAI, histological injuries and the mRNA expression of inflammatory cytokines in DSS-treated mice were significantly elevated (P<0.05). The expression of HNF4α at protein and mRNA levels was significantly decreased (P<0.01). The result of Pearson analysis indicated an inverse correlation between the protein expression of HNF4α and the severity of disease (P<0.01). The positive correlation between the mRNA expression of HNF4α and E-CAD/JAM-1/DSC-2 (P<0.01) was also observed. CONCLUSION: There is a close relationship between the expression of HNF4α and the severity of colitis as well as the intercellular linking proteins. The low expression of HNF4α in intestine might aggravate the function of intestinal mucosal barrier, thus promoting the development of UC.     

TL1A promotes development of intestinal fibrosis associated with chron?ic experimental colitis by regulating IL-17 and IFN-γ

AIM To explore how tumor recrosis factor ligand-related molecule 1A (TL1A) promotes the development of intestinal fibrosis associated with chronic experimental colitis by regulating interleukin-17 (IL-17) and interferon-γ (IFN-γ). METHODS Aexperimental colitis-associated wild-type (WT) and TL1A (L-Tg) transgenic intestinal fibrosis model was established by dextran sulfate sodium (DSS) induction.The severity of colitis was evaluated by detecting the disease activity index (DAI). HE staining was used to observe the histopathological changes and pathological score of the colitis.Myeloperoxidase (MPO) was measured in each group. The collagen deposition was detected by Masson’s trichrome staining and Sirius red staining. The lamina propria, spleen and mesenteric lymph nodes(MLN) mononuclear cells were isolated and counted, and the levels of IL-17 and IFN-γ weremeasured by ELISA, andthe percentages of CD4⁺IFN-γ⁺T cells and CD4⁺IL-17⁺T cells were analyzed by flow cytometry. RESULTS After drinking DSS water,the body weight of the mice in DSS/Tg group was decreased significantly as compared with WT group(P<0.05). The DAI score, histology score and MPO activity were significantly increased(P<0.05). Thelevels of IL-17 and IFN-γ, LPMC, spleen and MLN were significantly increased. The percentages of CD4⁺IFN-γ⁺T cells and CD4⁺IL-17⁺T cells were significantlyincreased.The thickness and collagen deposition of the colon were increased inTg group. CONCLUSION TL1A promotes the development of intestinal fibrosis associated with chronic experimental colitis by regulating IL-17 and IFN-γ.     

Roles of novel cannabinoid chemicals O-1602 and cannabidiol in DSS-induced mouse colitis

AIM:To investigate the roles of O-1602 and cannabidiol(CBD) in dextran sulfate sodium(DSS)-induced mouse colitis. METHODS:The model of colitis was induced in C57BL/6 mice by drinking water containing 4% DSS for 7 days. The model mice were treated with O-1602(5 mg/kg), CBD(1 mg/kg) or SB203580. A colitis scoring system was used to evaluate the colon local lesion, and the systemic inflammatory responses were observed by detecting the plasma levels of tumor necrosis factor α(TNF-α), interleukin 6(IL-6) and cytokine-induced neutrophil chemoattractant 1(CINC-1), and the activity of myeloperoxidase(MPO) in the lung tissues. The expression of G protein-coupled receptor 55(GPR55) was detected by the method of immunohistochemistry. The expression of p38 and phosphorylated p38(p-p38) in colon tissues was determined by Western blotting. RESULTS:O-1602 and CBD improved the pathological changes in the mice with DSS-induced colitis and decreased the plasma levels of TNF-α, IL-6 and CINC-1, and the activity of MPO in the lung tissues(P<0.05). Lower expression of p-p38 was observed after treatment with O-1602, CBD and SB203580(P<0.05). The expression of GPR55 was mainly in the submucosa of mouse colon tissues. CONCLUSION:O-1602 and CBD show protective effect on the mice with experimental colitis, and the anti-inflammatory roles of O-1602 and CBD are related to the inhibition of p38 MAPK. The expression level of GPR55 in the submucosa of mouse colon tissue is low.     

Effects of cannabinoids WIN and O-1602 on expression of HSPs in mice with colitis or acute pancreatitis

AIM: To investigate the effects of cannabinoids WIN55, 212-2 (WIN) and O-1602 on the expression of heat-shock proteins (HSPs), such as HSP27, HSP60 and HSP70, in the inflammatory tissues of mice with experimental colitis or acute pancreatitis (AP). METHODS: Mouse colitis was induced by feeding the C57/BL with 4% dextran sulfate sodium (DSS) for 7 d, and AP was induced by intraperitoneal injection of ceruline in the mice (50 μg/kg hourly, with a total of 6 times). The mice were intraperitoneally administered with WIN or O-1602 for the therapeutic evaluation by observing the following parameters: pathological changes of the tissues, plasma activity of amylase, plasma levels of IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), and HSP expression in the colonic and pancreatic tissues. RESULTS: Compared with normal control mice, the colonic tissues from colitis mice and the pancreatic tissues from AP mice appeared obvious signs of inflammation and injury. The plasma levels of IL-6 and CINC-1 significantly increased in colitis mice and AP mice. In DSS group of colitis mice, HSP27 expression increased, but HSP60 and HSP70 were reduced in the colonic tissues. WIN showed anti-inflammatory effects on the pathological changes of colonic tissues and the plasma cytokine levels. WIN also improved the expression of HSP70 (P<0.05). In AP group, the expression of HSP27 and HSP70 in pancreatic tissues increased, and HSP60 decreased. O-1602 also showed some anti-inflammatory effects on the changes of the pathological tissues and the plasma parameters, but had no obvious effects on the HSP expression (P>0.05). CONCLUSION: The experimental colitis and acute pancreatitis in mice were induced by feeding DSS and injection of cerulein, respectively. The changes of HSP expression were different in the inflammatory tissues. WIN and O-1602 show anti-inflammatory effects and increase the expression of several HSPs to some extent.     

Role of CXCR4 in changes of protein C system in ulcerative colitis mice

AIM: To explore the role of chemokine receptor CXCR4 in the pathogenesis of protein C system (PCS) in ulcerative colitis (UC).METHODS: In vivo, the mice were divided into control group and UC group. The macroscopic score, microscopic score and ulcer index were assessed. The mRNA levels and activity of myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), stromal cell-derived factor-1α (SDF-1α) and monocyte chemotactic protein 1 (MCP-1) both in colonic tissue and plasma were determined. The expression and location of CXCR4, β-arrestin, p-JNK, endothelial cell protein C receptor (EPCR) and thrombomodulin (TM) were detected. The activity of protein C (PC) and protein S (PS) was measured in each group. In vitro, mouse colonic microvascular endothelial cells were isolated, cultured and identified. Both CXCR4-overexpressing and CXCR4-silencing colonic mucosa microvascular endothelial cells were constructed. The effects of SDF-1α on the protein levels of EPCR, TM, β-arrestin and p-JNK, and on the activity of PC, PS and activated protein C (APC) were observed.RESULTS: Compared with control group, UC mice showed increased gross score, histopathological score and ulcer index (P<0.05). The mRNA levels and activity of MPO, COX-2, SDF-1α and MCP-1 in colon and plasma were increased (P<0.01). The protein levels of CXCR4, β-arrestin and p-JNK were up-regulated, EPCR expression was down-regulated in colon, and the activity of PC and PS in plasma was decreased (P<0.05 or P<0.01). CXCR4 overexpression further aggravated SDF-1α-induced PCS inhibition in colonic mucosa microvascular endothelial cells, and further up-regulated the protein levels of β-arrestin and p-JNK (P<0.05).CONCLUSION: PCS is inhibited in UC. CXCR4 is involved in the regulation of PCS inhibition by mediating chemokines and acting on colonic mucosa microvascular endothelial cells through β-arrestin-JNK pathway.     

Inhibitory effect of cholecystokinin-octapeptide on production of hydrogen sulfide in lung of LPS-induced lung injury rats

AIM: To investigate the role of hydrogen sulfide (H2S) in the cholecystokinin octapeptide (CCK-8) attenuating lipopolysaccharide (LPS)-induced lung injury. METHODS: A rat model of lung injury induced by intravenous injection of LPS was developed. Male Wistar rats were divided into normal control group, LPS group, LPS+CCK-8 group and CCK-8 group. Six hours after LPS injection, partial pressure of oxygen in the arterial blood (PaO2), H2S content and cystathionine-γ-lyase (CSE) activity in lung tissue were detected. The mRNA expression of CSE in lung tissue was determined by RT-PCR; the structure of lung tissues was observed under optical microscope. RESULTS: Compared to normal control rats, the LPS-treated rats had significantly decreased PaO2 level, increased index of quantitative assessment (IQA) score, while H2S content, CSE activity and the mRNA expression of CSE in lung tissue were significantly increased (all P<0.05). Administration of CCK-8 into LPS-treated rats increased the PaO2 level and alleviated the degree of lung injury (measured by IQA score). In addition, CCK-8 decreased H2S content, CSE activity, and the mRNA expression of CSE (all P<0.05). No significant difference of the above-mentioned parameters between CCK-8 group and normal control group was observed. CONCLUSION: CCK-8 reduces LPS-induced lung injury through inhibiting the generation of endogenous H2S.     

Effects of oxidative stress on high-fat,high-salt diet and sucrose solution-induced metabolic syndrome in nephropathy rats

AIM: To establish a suitable animal model of nephropathy associated with metabolic syndrome (MS) induced by abnormal diet, and to investigate the effects of oxidative stress on renal damage in MS rats. METHODS: Normal 7-week-old male SD rats were randomly divided into 2 groups.The animals were fed with normal chow (control group, n=10) or high-fat and high-salt diet plus 20% sucrose solution (MS model group, n=10) for 20 weeks. Systolic blood pressure (SBP) was measured monthly. The levels of blood glucose, serum and urinary creatinine (Cr), total cholesterol (TC), triglycerides (TG), fasting insulin (FIns), urinary protein, urinary albumin and urinary sodium were determined. Insulin resistance (HOMA-IR), creatinine clearance (Ccr), urinary protein excretion (UPE), urinary albumin excretion (UAE) and urinary sodium excretion (USE) were calculated. Renal total-antioxidant capacity (T-AOC), inhibiting superoxide anion capacity (ISAC), malondialdehyde (MDA) content, and antioxidant enzyme activity were measured. Renal protein expression of Cu/Zn-SOD, NADPH oxidase subunit p47^phox and p22^phox was detected by Western blotting. In addition, pathological changes of the kidney were observed with PAS and Masson staining,and degree of glomerulosclerosis (GS) and tubulointerstitial injury was evaluated. RESULTS: Compared with control rats, SBP, TC, TG, FIns, USE and UAE were increased in MS rats. Furthermore, the MS rats showed a significant elevation of renal MDA content, p47^phox protein expression and GS score, and reduction of T-AOC, ISAC, SOD activity, and Cu/Zn-SOD protein expression in the kidney. CONCLUSION: SD rats fed with abnormal diet produce a suitable animal model of MS nephropathy that mimics the major features of human MS. Oxidative stress caused by up-regulation of NADPH oxidase expression and down-regulation of SOD expression may be one of the mechanisms leading to MS renal damage.     

Repair effect of purple sweet potato anthocyanin on intestinal barrier injury in mice with ulcerative colitis induced by DSS

AIM To explore the repair effect of purple sweet potato anthocyanin on intestinal barrier injury of ulcerative colitis mice induced by dextrin sulfate sodium （DSS）. METHODS The mice were randomly divided into normal drinking group， DSS model group， different doses of purple sweet potato anthocyanin （12.5， 25， 50 and 75 mg/kg） groups， and 5-aminosalicylic acid （5-ASA） positive drug control group. Except using normal drinking water for control group， the mice in the other groups were treated with 2.5% DSS in drinking water for 7 days to induce the ulcerative colitis model. The mice in purple sweet potato anthocyanin treatment group and the 5-ASA positive drug control group were given the drug by intragastric gavage on the first day of modeling. The body weight of the mice and the hematocheziawere recorded every day. After continuous administration for 8 days， the mice in each group were killed and colon tissue was retained. Immunohistochemical technique （IHC） was used to detect the expression of tight junction protein ZO-1 and occludin and inflammatory factors tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the colon of mice. The expression of mucin in goblet cells of colon tissue was observed by glycogen PAS staining. The protein expression of ZO-1， occludin， TNF-α were determined by Western blot， Sirius red staining was used to detect colonic fibrosis in mice. RESULTS Compared with control group， the disease activity index and histological injury of DSS model mice were significantly increased（P<0.01）. Compared with model group， the disease activity index scores of the mice in different dose groups of purple sweet potato anthocyanin were decreased. The expression and distribution of ZO-1 and occludin in colon tissues were increased， and the expression and distribution of TNF-α and IL-6 in colon tissues were decreased. Glycogen PAS staining showed a significant increase in the distribution and expression of mucin in goblet cells of colon tissues in the purple sweet potato anthocyanin treatment group. Sirius red staining also showed that the degree of fibrosis in the purple sweet potato anthocyanin treatment groups was lower than that in model group. CONCLUSION Purple sweet potato anthocyanins has therapeutic effect on ulcerative colitis in mice induced by DSS， mainly through up-regulating the expression of tight junction proteins ZO-1 and occludin， to protect the integrity of intestinal barrier， inhibiting the expression of inflammatory cytokines TNF-α and IL-6 and intestinal fibrosis， to suppress the development of colonic inflammation.     

Yellow wine and red wine inhibit matrix metalloproteinase-2 and improve pathological changes of atherosclerosis in LDL receptor knockout mice

AIM: To study the possibility that yellow wine improves the pathological changes of atherosclerosis in vivo. METHODS: Six weeks old LDL receptor knockout mice (n=48) on a high-fat and L-methionine diet developed plasma hyperhomocysteinemia and atherosclerosis. The animals were randomly divided into yellow wine group, red wine group, ethanol group and control group (n=12 in each group) and were sacrificed after 14 weeks. The levels of plasma lipids and homocysteine in serum were examined. The morphological changes of aorta artery and the atherosclerosis of aorta sinus were observed under microscope. The expression and activation of matrix metalloproteinase-2 (MMP-2) were determined by the method of immunohistochemistry. RESULTS: No significant difference of plasma total cholesterol, triglyceride or high density lipoprotein cholesterol among groups was observed. Plasma homocysteine was significantly decreased in yellow wine group as compared to other three groups (P<0.01). Compared to ethanol and control groups, use of yellow wine and red wine significantly reduced the atherosclerosis lesion area (P<0.01). However, no significant discrepancy between the yellow wine group and red wine group was found. Compared to control group, the expression of MMP-2 in yellow wine group, red wine group and ethanol group decreased by 26.3%, 27.6% (P<0.01) and 5.7% (P>0.05), respectively. The activity of MMP-2 in yellow wine group, red wine group and ethanol group decreased by 31.7%, 32.5% (P<0.01) and 6.7% (P>0.05), respectively. CONCLUSION: Yellow wine and red wine inhibit the expression of MMP-2 and improve the pathologic changes of atherosclerosis, indicating that they have benefic effects on cardiovascular system.     

Effects of Xiaoyaosan on imiquimod induced psoriatic lesions and depression neurotransmitters in mouse model

AIM: To observe the effect of Xiaoyaosan decoction on the psoriatic lesions and depression neurotransmitters induced by imiquimod in mice. METHODS: BALB/c male mice were randomly divided into control group, model group, methotrexate group and Xiaoyaosan high, medium and low dose groups, 6 mice in each group. Imiquimod (IMQ, 5%) was used on the back of the animals to induce psoriasis-like lesions in the mice. The psoriasis area and seve-rity index (PASI) were evaluated for daily scoring. The sugar water preference experiment was conducted to explore the behavioral differences in the mice. The morphological changes and epidermal thickness of the lesions were observed under light microscopy. Immunohistochemical method was used to detect the expression of CD3 on T lymphocyte surface. The expression of Ki67 in the skin lesions was detected by immunofluorescence. The contents of monoamine neurotransmitters such as adrenaline (AD), gamma-aminobutylic acid (GABA), glutamate (Glu), dopamine (DA) and their metabolites in the hippocampus and hypothalamus of mice were detected by high performance liquid chromatography-mass spectrometry (HPLC-MS). RESULTS: Compared with model group, the back skin lesions of Xiaoyaosan each dose group and methotrexate group were significantly improved, and the PASI score and epidermal thickness were both lower than those in model group (P<0.05). The expression levels of Ki67 and CD3⁺ T cells in Xiaoyaosan group and methotrexate group were lower than those in model group (P<0.05). Compared with model group, the body mass change range of Xiaoyaosan high-dose group and blank control group was significantly smaller than that in model group (P<0.05). The sugar water preference rate in blank control group was significantly higher than that in model group (P<0.01). Compared with model group, the sugar water preference rate in methotrexate and Xiaoyaosan groups showed a certain increase trend, but no statistical diffe-rence was observed. Compared model group, the levels of 3, 4-Dihydroxypheny-lacetic acid (DOPAC), AD, GLU and GABA levels in the mouse hippocampus in blank control group were decreased significantly (P<0.01), while the levels of DA and homovanillic acid (HVA) had no significant difference (P>0.05). No significant difference of DA, DOPAC, HVA and GLU levels in the mouse hypothalamus was observed between blank control group and model group (P>0.05), while the content of AD and GABA in the mouse hypothalamus in blank control group was lower than that in model group. The AD content of the hypothalamus in high-dose Xiaoyaosan group was significantly higher than that in model group (P<0.01), and the HVA content of the hypothalamus in low-dose Xiaoyaosan group was significantly higher than that in model group (P<0.01). PASI score was negatively correlated with the content of DOPAC, AD, GLU and GABA in the hippocampus and the content of AD, GLU and GABA in the hypothalamus, those were, the more severe the back skin lesion was, the lower the expression of depression-related neurotransmitters were, indicating the aggravation of depression in the mice. CONCLUSION: Xiaoyaosan improves the skin lesions induced by imiquimod in the mice with psoriasis, improves the behavior of depression in the mice with psoriasis, and up-regulates the expression of depression-related monoamine neurotransmitters. The expression of depression-related neurotransmitters is negatively correlated with the skin lesions induced by imiqumod in the mice with psoriasis. The degree of depression is increased with the aggravation of psoriatic lesions.     

Changes of lung tissue and structure on bone marrow mesenchymal stem cells transplantation to treat the experimental pulmonary arterial hypertension in rats

AIM: To investigate the changes of lung tissue and structure on bone marrow mesenchymal stem cells (MMSCs) transplantation to treat the pulmonary arterial hypertension (PAH) in rats.METHODS: MMSCs cells were collected from bone marrow of SD rat’s femoral’s tibial bones, mix cultured with Hoechst 33342 fluorescence dye in vitro. Sixty healthy male Sprague-Dawley rats (weighing 180 g±5 g) were randomly divided into 3 groups: normal control group (group C), MMSCs transplanted group (group M), pulmonary model group (group H). The rats of the two latter groups were given a single subcutaneous monocrotaline (50 mg/kg) to induce the model of PAH. The rats of normal control group were injected respectively a single subcutaneous isotonic Na chloride (6 mL/kg). The three groups were fed in the same condition. After 3 weeks, 5×109 cells/L MMSCs with l mL phosphate-buffered saline were infused into the rats respectively in group M by sublingual vein and 1 mL L-DMEM was administered in group H. Nothing was given to group C. The viability, right ventricle systolic pressure (RVSP), right ventricle hypertrophy index, the microstructure and ultrastructural changes of small pulmonary vascular were observed after 28 d.RESULTS: The viability of administrated MMSCs 28 d after PAH was 100% in transplanted group, and it nearly completely prevented the increase in right ventricular systolic pressure (RVSP) compared to PAH alone ［(32.20±2.32)mmHg vs (48.30±1.56)mmHg, respectively, P<0.05］, right ventricle hypertrophy index also degraded (38.80%±3.24% vs 45.10%±3.43%, respectively, P<0.05). The pulmonary arteries remodeling and ultrastructural changes of lung, such as blood gas barrier, chondriosome, osmiophilic lamellar body and so on, were improved significantly in MMSCs transplanted group. The changes were similar to the results of normal control group, but they were better than that in pulmonary arterial hypertension model group, which had conspicuous significance of statistics. MMSCs labeled with Hoechst 33342 were observed that they permanent planted and differentiated into plentiful collateral circulations in lung observed by fluorescence microscope. The pulmonary arteries remodeling were attenuated effectively in MMSCs transplanted group. Pulmonary fibrosis and tumorous were not observed in rats of MMSCs transplanted group.CONCLUSION: These results indicate that MMSCs reduce and even reverse the progression of pulmonary arterial hypertension (PAH) induced by crotaline. It restores the structure and function of microvasculature with marked improvement of tissue and structure changes of the lung.     

Effects of traditional Chinese medicine Dan-shao-hua-xian capsule on expression of miR-200s in rat fibrotic livers

AIM: To investigate the effect of Dan-shao-hua-xian (DSHX) capsule on the expression of the family of microRNA-200 (miR-200s) in rat fibrotic livers. METHODS: Forty male Wistar rats weighing 180 g to 220 g were divided into 5 groups (control group, two model groups and two interference groups). The rats in model groups and interference groups were induced by hypodermic injection of CCl4 for 4 weeks and 8 weeks. The rats in interference groups were also treated with DSHX capsule (0.5 g/kg) once daily for 4 weeks and 8 weeks at the same time. The liver index and serum activity of ALT and AST were analyzed. The liver fibrosis was observed under microscope. Additionally, the expression of miR-200a, -200b, -200c, -141 and -429 was determined by quantitative real-time PCR. RESULTS: The liver index, and serum activity of ALT and AST in model groups and 4-week interference group were obviously higher than those in normal control group. The apparent liver fibrosis was observed in 8-week model group. The expression of miR-200a,-200b, -200c, -141 and -429 in the liver of 8-week model groups was obviously higher than that in control group. CONCLUSION: In the process of liver fibrosis induced by CCl4, the obvious changes of miR-200s may play an important role in the development of liver fibrosis. The miR-200s might be the potential target that DSHX capsule inhibits the process of liver fibrosis.     

Effect of forsythiaside A on immune function in rats with ulcerative colitis

AIM To investigate the effect of forsythiaside A (FA) on immune function in rats with ulcerative colitis and its related mechanism. METHODS Healthy SD rats were randomly divided into 5 groups: control group (no treatment, normal feeding), model group (establishment of rat ulcerative colitis model), and low, medium and high doses of FA groups (treatment of the model rats with FA at 5 mg/kg, 20 mg/kg and 80 mg/kg, respectively). The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in rat colon tissues were measured by colorimetry, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2) and IL-4 were detected by ELISA. The spleen index and thymus index, the percentages of CD3⁺, CD4⁺ and CD8⁺ T-lymphocytes in peripheral blood mononuclear cells （PBMC）, the serum IgA and IgG levels, and the serum complement C3 and C4 levels were also determined. RESULTS The colon tissues of the rats in model group showed obvious inflammation and ulceration, indicating that the animal model was successfully established. Compared with model group, the colonic inflammation and ulceration were significantly attenuated in FA groups, among which the high dose had the best effect. Compared with control group, the spleen index and thymus index of the rars in model group were decreased (P<0.05), MDA content in colon tissues was increased (P<0.05), and SOD activity in colon tissues was decreased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were decreased (P<0.05), while the serum levels of IgA, IgG, TNF-α, and IL-2 were increased in model group as compared with control group. Furthermore, the spleen index and thymus index of the rats in FA groups were increased (P<0.05), the MDA content in the colon tissues was decreased (P<0.05), and the SOD activity in the colon tissues was increased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were increased (P<0.05), while serum IgA, IgG, TNF-α and IL-2 levels were decreased in FA groups as compared with model group (P<0.05). CONCLUSION Forsythiaside A effectively attenuates the colonic lesions in rats with ulcerative colitis, and its mechanism may be related to reinforcement of oxygen free radical scavenging power, alleviation of inflammatory response, and enhancement of immune function.     

Role of hydrogen sulfide in acute lung injury induced by ischemia-reperfusion of hind limbs in rats

AIM: To explore the role of endogenous and exogenous hydrogen sulfide (H₂S) in acute lung injury (ALI) induced by ischmia-reperfusion (IR) of hind limbs in rats.METHODS: A Sprague-Dawley rat model of acute lung injury was induced by ischemia of the hind limbs for 4 h and reperfusion for another 4 h. The rats (n=120) were randomly divided into 4 groups: control, IR, NaHS (H₂S donor)+IR, and propargylglycine +IR. The animals were sacrificed after reperfusion. Lung weight/body weight ratio (LW/BW) was measured and calculated. Morphological changes of the lung tissues were observed. The concentrations of H₂S, nitric oxide (NO) and carbon monoxide (CO) in plasma were tested. The content of malondialdehyde (MDA), the activity of CSE, inducible nitric oxide synthase (iNOS) and hemeoxygenase (HO) in the lungs were determined. The polymorpho-nuclear neutrophils(PMN) and protein content in bronchoalveolar lavage fluid(BALF) were also measured. The correlation of H₂S content with the above indices was analyzed.RESULTS: Compared with control group, severe injuries of the lung tissues, raised LW/BW, MDA concentration, PMN and protein contents in BALF were observed in IR group. Limb IR also made a drop in the concentration of plasma H₂S and the activity of lung CSE, while the activity of iNOS and HO in the lung tissues and the levels of plasma NO and CO increased. Administration of NaHS before IR attenuated the changes induced by IR, while pre-administration of PPG exacerbated the IR injuries and increased the plasma NO level and lung iNOS activity. The H₂S content was positively correlated with CSE activity, CO content and HO-1 activity (P<0.01), and negatively correlated with the other indices (P<0.01).CONCLUSION: Down-regulation of H₂S/CSE is involved in the pathogenesis of acute lung injury induced by IR. Endogenous and exogenous H₂S protects against lung injuries. The anti-injury effects of H₂S are related with its anti-oxidative activity to attenuate the inflammatory over-reactions in the lung induced by PMN. Down-regulation of NO/iNOS system and up-regulation of CO/HO-1 system by H₂S are also involved in the process of anti-injury to ALI.     

Protective role of endogenous hydrogen sulfide in cholecystokinin octapeptid against acute lung injury induced by lipopolysaccharide

AIM: To explore the role of endogenous hydrogen sulfide (H₂S) in the mechanism of cholecystokinin octapeptide (CCK-8) to alleviate acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: Eighty-four Sprague-Dawley rats were randomly divided into seven groups: control, LPS (instilled intratracheally to reproduce the model of ALI), NaHS (H₂S donor) +LPS, propargylglycine ［inhibitor of cysathionine-γ-lyase (CSE), PPG］+LPS, CCK-8+LPS, PPG+CCK-8+LPS and CCK-8 group. Animals were sacrificed at 4 h and 8 h after agent instillation. The wet and dry ratio (W/D) of the lung weight was measured and calculated. Morphological changes of lung tissues were observed. H₂S concentration in plasma, malondialdehyde (MDA) content, myeloperoxidase (MPO) and CSE activities in the lung were determined. Furthermore, the level of P-selectin of lung tissue was measured by radioimmunoassay, the CSE mRNA expression in the lung was detected by RT-PCR, and the protein content in bronchoalveolar lavage fluid (BALF) was detected. RESULTS: Compared with control, severe injury of lung tissues and increase in W/D, protein content in BALF, MDA content, MPO activity and P-selectin level in the lung were observed in rats treated with LPS. LPS also lead to a drop in plasma H₂S concentration, lung CSE activity and CSE mRNA expression. Administration of NaHS before LPS could attenuate the changes induced by LPS, while H₂S concentration, CSE activity and CSE mRNA expression were higher than those in LPS group. However, pre-treatment with PPG exacerbated the lung injury induced by LPS, H₂S concentration, CSE activity and CSE mRNA expression were lower than those in LPS and CCK-8 +LPS group, respectively. CONCLUSION: CCK-8 attenuates LPS-induced acute lung injury by means of anti-oxidation and inhibition of PMN adhesion and aggregation, both of which are mediated by endogenous H₂S.     

Effects of sodium ferulate on function of macrophages in the colonic tissue of colitis rats

AIM: To explore the effects of sodium ferulate (SF) on function of macrophages in colonic tissue of the colitis rats in vivo. METHODS: The immunological colitis model of rats was produced. SF was used intracolonically for 21 days. The contents of malondialdehyde (MDA), nitric oxide (NO), prostaglandin E₂ (PGE₂) and the activity of superoxide dismutase (SOD), interleukin-1 (IL-1), TNF-α, myelopexoxidase (MPO), and the expression level of NF-κB p65 in colonic tissue of the rats were detected. RESULTS: SF (200,400,800 mg/kg) decreased the elevated contents of MDA, NO, PGE₂, the activity of IL-1, TNF-α, MPO, and the expression level of NF-κB p65, while increased the reduced activity of SOD in colonic tissue of the colitis rats in a dose-depended manner. CONCLUSION: SF restrained the activity of activated colonic macrophages and relieved the colonic inflammation reaction in vivo in colitis rats, which may be related to the suppression of NF-κB activation.