摘要:目的: 研究蜕皮甾酮对非酒精性脂肪性肝病大鼠模型肿瘤坏死因子α(TNFα)与核因子κB(NFκB)表达的影响，并探索其可能的作用机制。 方法 :健康成年SD大鼠36只,随机分为正常对照组12只与实验组24只;正常对照组喂以普通基础饲料,实验组应用高脂饲料喂养。实验12周末时将造模成功的实验组大鼠随机分为模型组与蜕皮甾酮治疗组2个亚组,每组12只;正常对照组喂以普通基础饲料至16周,模型组继续应用改良高脂饲料喂养至16周,蜕皮甾酮治疗组大鼠在高脂饮食同时加用蜕皮甾酮灌胃。实验16周末时处死3组所有大鼠;检测肝脏指数,血清与肝组织生化指标及肝组织病理改变;ELISA法检测肝脏TNFα水平;免疫组化检测各组大鼠肝组织中核因子κB蛋白表达情况。 结果 :蜕皮甾酮治疗组血清胆固醇(TC)、丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)明显低于模型组(212±058比263±024,Plt;005;5336±1848比8460±3627,P＜005;14020±3595比24359±3638,P＜001);蜕皮甾酮治疗组与模型组相比肝组织丙二醛(MDA)水平降低明显(18454±1645比23928±2376,P＜001),超氧化物歧化酶(SOD)活力增加显著(942±052比518±043,P＜001),肝脏指数显著降低(435±037比504±046,P＜001)，肝组织脂肪变性程度和炎症活动度明显减轻(546±037比630±049,P＜001)。蜕皮甾酮治疗组与模型组相比TNFα与核因子κB水平明显减轻(4304±748比6156±727,2465±539比4504±746,P值均＜001)。 结论 :蜕皮甾酮具有改善高脂饮食诱发的非酒精性脂肪性肝病大鼠肝脏酶学功能,通过增加肝组织SOD的含量和减少MDA的含量来减轻肝组织氧化应激水平,减轻肝组织TNFα和核因子κB来减轻肝脏炎症,发挥防治非酒精性脂肪性肝病的作用。Abstract: Objective: To investigate the effect and possible mechanism of ecdysterone on the expression of tumor necrosis factoralpha (TNFα) and nuclear factor κ B (NFκB) in rats with nonalcoholic fatty liver disease of rats. Methods : A total of 36 male Sprague Dawley rats were randomly divided into two groups, who were fed with highfat diet (experimental group, n=24) and normal basic food (normal control, n=12) respectively. At the end of the 12th week, the experimental group was randomly divided into two subgroups: model group and ecdysterone group, each group contained 12 rats. From the 13th week, the rats in the normal control group and model group were lavaged with normal sodium, and the rats in the ecdysterone group were lavaged with ecdysterone at 10 mg·kg-1·d-1. At the end of the 16th week, all rats were weighed, narcotized, sacrificed, and the liver index, biochemical indicators in serum and liver tissues and the hepatic pathological changes were observed. The expression of TNFα was detected by ELISA and the expression of NFκB was measured by immunohistochemical staining. Results : At the end 16th week in ecdysterone group, the serum levels of cholesterol (TC), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were reduced markedly (212±058 vs 263±024 and 5336±1848 vs 8460±3627, both P＜005; 14020±3595 vs 24359±3638, P＜001); the tissue content of malondialdehyde (MDA) was decreased evidently (18454±1645 vs 23928±2376, P＜001), while the activity of superoxide dismutase (SOD) was enhanced notably (942±052 vs 518±043, P＜001); the liver index was decreased significantly in comparison with that inmodel group (435±037 vs 504±046, P＜001); the degree of fatty degeneration and inflammation were relieved dramatically (546±037 vs 630±049, P＜001). The expression of TNFα and the levels of NFκB were significantly lower (4304±748 vs 6156±727 and 2465±539 vs 4504±746, both P＜001) in ecdysterone group compared with model group. Conclusion : The effects of ecdysterone in preventing NAFLD in rats could be related to the increase of SOD content in hepatic tissue and the decrease of MDA content, tumor necrosis factorα and NFκB.
Objective To study the variety and the action of inflammatory cytokines and the relevant anti-inflammatory factors in acute pancreatitis (AP). Methods The authors observed the change of peripheral blood IL-6 and sTNFR in 41 patients with mild and severe AP in two groups on 1, 5, 14d after acute attack by ELISA. Results All cases recovered gradually in mild group (n＝22) after five days. Twelve patients improved gradually in severe group (n＝19) after 5-7 days. The level of sTNFR increased markedly in 2 groups at 1, 5, 14d(Ｐ＜0.001), and that of the severe group was markedly higher decreased gradually (Ｐ＜0.01). The level of IL-6 increased apparently only in severe group on 1d, 40.38 pg/ml∶12.4 pg/ml, (Ｐ＜0.001). The levels of IL-6 and sTNFR correlated respectively with severity of AP. Conclusion These results show that peripheral blood IL-6 and TNFα are useful index to supervise the severity and conversion and final results of AP.
ObjectiveTo investigate the efficacy and safety of recombinant human tumor necrosis factor-α receptor Ⅱ:IgG Fc fusion protein (rhTNFR:Fc) for treatment of active rheumatoid arthritis (RA). MethodsThis study included 86 patients with active rheumatoid arthritis treated between September 2011 and January 2013. They were divided into two groups randomly. Forty-three patients in the treatment group received rhTNFR:Fc (25 mg, twice a week) by subcutaneous injection and methotrexate (MTX) (10 mg, orally once a week), and the other 43 patients in the contrast group received MTX (10 mg, orally once a week), hydroxychloroquine (100 mg, orally twice daily), and leflunomide (10 mg, orally once daily). The clinical efficacy of the treatments 12 weeks later were compared between the two groups. American College of Rheumatology (ACR) 20, 50, and 70 evaluation criteria were used for efficacy evaluation. ResultsThe ACR 20, 50 and 70 effective rates in 4, 8 and 12 weeks after the treatment in the treatment group were significantly higher than those in the control group (P<0.05). The seven indicators including the duration of morning stiffness, joint tenderness index, joint swelling index, erythrocyte sedimentation rate, C-reactive protein, platelets and rheumatoid factors within 12 weeks after treatment were significantly improved in both the two groups, and the improvements in the treatment group were more significant (P<0.05). There was no significant difference in the incidence of adverse drug reactions between the two groups (P>0.05). ConclusionRhTNFR:Fc is effecive and safe in treating active RA.
Human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL) might be developed as a novel anti-tumor drug due to its selective cytotoxicity in tumor cells. The predicted Macaca mulatta TRAIL (mmTRAIL) is highly homologous to hTRAIL in nucleotide acid as well as amino acid sequence, suggesting that mmTRAIL might induce apoptosis of human cancer cells. However, the cytotoxicity of mmTRAIL in human cancer cells has not been investigated. In this paper, it is reported that the gene encoding mmTRAIL has been cloned by using reverse-transcriptase polymerase chain reaction (RT-PCR) from monkey peripheral blood mononuclear cells (PBMCs) in our laboratory. Subsequently, an expression plasmid was constructed by inserting mmTRAIL gene into pQE30 plasmid. After induction by addition of Isopropylβ-D-1-Thiogalactopyranoside (IPTG), mmTRAIL was expressed. MmTRAIL was recovered from supernatant of sonicated bacteria by Ni-NTA agarose affinity chromatography. SDS-PAGE and gel filtration chromatography demonstrated that mmTRAIL forms trimer in solution. In vitro assays indicated that mmTRAIL was cytotoxic to human COLO205 tumor cells but not to normal cells at low concentration of nanomole. In addition, antitumor effect of mmTRAIL was evaluated in mice bearing human COLO205 tumor xenografts. Intratumorally injected mmTRAIL significantly inhibited growth of tumor grafts. These results suggested that mmTRAIL was valuable as candidate drug for cancer-targeted therapy.
The aim of this article is to study the effect of tumor necrosis factor alpha (TNF-α) on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in keratoconus fibroblasts in vitro. Normal cornea and keratoconus fibroblasts were extracted using enzyme digestion method and were cultured in the medium containing TNF-α (0, 10 and 100 ng/mL). The expression of MMPs proteins in the supernatant of corneal fibroblasts and the expression of TIMPs in the normal cornea and keratoconus fibroblasts were detected by Western blot and real-time quantitative polymerase chain reaction respectively. The active form of MMP1 could be detected in the supernatant of keratoconus fibroblasts and upregulated by TNF-α. TNF-α could increase the protein expression of MMP2, MMP3, MMP9 in the supernatant of keratoconus fibroblasts and decrease the gene expression of TIMP1, TIMP2 in keratoconus fibroblasts. The increased MMPs and the decreased TIMPs can increase the degradation of the extracellular matrix. TNF-α may play an important role in the occurrence and development of keratoconus by regulating the expression of MMPs/TIMPs.
Objective To investigate effect of different resuscitation liquids and different resuscitation methods on contents of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) in early resuscitation process of rats with traumatic hemorrhagic shock. Methods Sixty-four healthy SD rats (450–550 g) were chosen and divided into 4 groups randomly and averagely: crystal liquid limited resuscitation group, colloidal liquid limited resuscitation group, 7.5% NaCl limited resuscitation group, and colloidal liquid non-limited resuscitation group. There were 16 rats in each group. All the experimental rats were weighed before intraperitoneal injection of pentobarbital sodium anesthesia. Animal model was established via Chaudry’s method. The rats were killed and the abdominal aorta bloods were drew on hour 2, 6, 12, and 24 after recovering from anesthesia. The contents of IL-8 and TNF-α in plasmas were detected by enzyme linked immunosorbent assay. Results The contents of IL-8 and TNF-α among three kinds of limited resuscitation groups on hour 6 after resuscitation were significantly higher than those on hour 2 after resuscitation (P<0.05) and reached the peaks, then began to decrease. On hour 12 after resuscitation, the contents of IL-8 and TNF-α were decreased continuously among three kinds of limited resuscitation groups (P<0.05). The contents of IL-8 and TNF-α in the colloidal liquid non-limited resuscitation group at each point time were significantly higher than those among three kinds of limited resuscitation groups (P<0.05), which in the crystal liquid resuscitation group were significantly lower than those in the other limited liquid resuscitation groups (P<0.05). Conclusions In process of liquid resuscitation of rats with traumatic hemorrhagic shock, limited resuscitation method is better than that of non-limited resuscitation method. Among three kinds of limited resuscitation methods, crystal resuscitation liquid is more effective than the other two resuscitation liquids in prohibiting releases of IL-8 and TNF-α in rats with traumatic hemorrhagic shock.
Objective To explore the effectiveness of the transforming growth factor-β1(TGF-β1) and tumor necrosis factor-α(TNF-α) inducing human bronchial epithelial(HBE) cells to optimize epithelia-mesenchymal transformation(EMT) model. Methods Blank control, TGF-β1 10 ng/ml, TNF-α 10 ng/ml, TGF-β1 10 ng/ml+TNF-α 10 ng/ml induced human epithelial cells for 24 hours. Then the change of morphological alteration were observed by applying CCK8, cells migration assay and Western blot technique. Results When TGF-β1 plus TNF-α induced human epithelial cells for 24 hours, most of HBE cells traits changed including morphological alteration from cobblestone to fusiform, connection between cells vanishing, intercellular space broadening. In the experiments of checking cell migration capacity by the vitro scratch test, the group spacing was 420.06±10.38 μm in the blank control group, 499.86±34.00 μm in the TGF-β1 10 ng/ml group, 514.93±10.56 μm in the TNF-α 10 ng/ml group, 569.68±33.58 μm in the TGF-β1 10 ng/ml+TNF-α10 ng/ml group. TGF-β1 cooperated with TNF-α led to scratch spacing narrowing significantly. Western blot analysis showed that expression of E-cadherin and Vimentin varied significantly in the TGF-β1+TNF-α group. Conclusion Inducing human bronchial epithelial cell by TGF-β1 cooperated with TNF-α optimizes EMT model.
Objective To investigate the effect of heme oxygenase 1 (HO-1) on the apoptosis of human degenerated nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α), and explore its possible molecular mechanism. Methods The intervertebral disc tissues were derived from patients with lumbar intervertebral disc herniation. Then, the NP cells were cultured in vitro and the third generation of NP cells were used for subsequent experiments. Cell counting kit 8 (CCK-8) method was used to observe the proliferative effect of TNF-α on the NP cells in vitro at the concentration of 10, 20, 50, 100, and 200 ng/mL. The most apropriate concentration was selected according to the result of CCK-8. The NP cells were cultured with basal medium (control group), TNF-α (TNF-α group), TNF-α and CoPP 10 μmol/L (CoPP group), and TNF-α and ZnPP 15 μmol/L (ZnPP group), respectively. After cultured, the cell poptosis was detected by Hoechst staining and flow cytometry; the expression of cleaved Caspase-3, epithelial membrane protein 1 (EMP-1), HO-1, and p-P65 proteins were detected by Western blot. In order to further explore the potential molecular mechanisms of HO-1 for cell apoptosis, the NP cells were cultured with TNF-α (TNF-α stimulated group), TNF-α and pyrrolidine dithiocarbamate (PDTC) 5 μmol/L (TNF-α+PDTC stimulated group), respectively. Then the cell apoptosis rate was measured by flow cytometry at 24 hours after cultured. Results The optimal concentration of TNF-α was 100 ng/mL. Hoechst staining showed that a few apoptotic cells could be observed in control group and CoPP group; the apoptosis-like nucleis were observed in TNF-α group and ZnPP group, which was the most significant in ZnPP group. Flow cytometry showed that the cell apoptosis rates of TNF-α group, CoPP group, and ZnPP group were significantly increased when compared with the control group (P<0.05). Compared with TNF-α group, the cell apoptosis rate in CoPP group decreased (P<0.05), while in ZnPP group it increased (P<0.05). Western blot showed that the expression of HO-1 protein in TNF-α group was decreased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were increased when compared with the control group (P<0.05). Compared with TNF-α group, the expression of HO-1 protein in CoPP group increased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were reduced (P<0.05); the expression of HO-1 protein in ZnPP group decreased (P<0.05), the expressions of cleaved Caspase-3 and EMP-1 proteins increased (P<0.05), and the expression of p-P65 protein was not significantly changed (P>0.05). Compared with TNF-α stimulated group, the cell apoptosis rate in TNF-α+PDTC stimulated group was significantly reduced (t=3.076, P=0.031). Conclusion HO-1 can inhibit the apoptosis of degerated NP cells induced by TNF-α, and its mechanism effect is by inhibiting the nuclear factor кB signaling pathway.
ObjectiveTo explore the serum concentrations of complement C1q tumor necrosis factor related protein 5 (CTRP5) in patients with acute exacerbations and stable stage of chronic obstructive pulmonary disease (COPD), and analyze the correlation of CTRP5 with high sensitivity C-reactive protein (hs-CRP) and FEV1/FVC and FEV1%pred.MethodsThirty hospitalized patients with acute exacerbation of COPD and 30 outpatients with stable COPD according with diagnostic criteria and inclusive criteria were sampled successively. At the same time 30 healthy volunteers were selected as normal control. All subjects were measured the concentrations of CTRP5 and hs-CRP in serum and lung function test was performed.ResultsThe serum CTRP5 and hs-CRP concentrations of the acute exacerbation group was higher than those in the stable group and the control group. The serum CTRP5 and hs-CRP concentrations of the stable group was also higher than those of the control group. The FEV1/FVC of the acute exacerbation group was lower than those of the stable group and the control group; and the FEV1/FVC of the stable group was lower than that of the control group. The FEV1%pred of three groups by analysis indicated the difference was statistically significant. Further pairwise comparisons demonstrated that the FEV1%pred of two COPD groups were lower than that of the control group but the FEV1%pred of the acute exacerbation group and stable group was not significantly different. The correlation analysis of the acute exacerbation group and the stable group demonstrated that the levels of serum CTRP5 and hs-CRP were postively correlated and the level of serum CTRP5 was negatively correlated with FEV1/FVC and FEV1%pred.ConclusionsThe level of CTRP5 in serum of COPD patients is increased. No matter in acute exacerbation or stable phase, the level of serum CTRP5 is positively correlated with hs-CRP and negatively correlated with FEV1/FVC and FEV1%pred, which suggests that CTRP5 is involved in the pathogenesis of COPD but the exact mechanism needs further study.
Objective To investigate the effects of one-lung ventilation time on the concentration of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the bronchoalveolar lavage fluid (BALF), serum inflammatory markers and early pulmonary infection after radical resection of esophageal cancer. Methods Ninety patients with thoracoscope and laparoscopic radical resection of esophageal carcinoma were chosen. According to the thoracoscope operation time, the patients were divided into 3 groups including a T1 (0.5–1.5 hours) group, a T2 (1.5–2.5 hours) group and a T3 (>2.5 hours) group. Immediately after the operation, the ventilated and collapsed BALF were taken. Enzyme-linked immunosorbent assay (ELISA) method was used to determine the concentration of IL-6 and tumour necrosis TNF-α. The concentrations of procalcitonin (PCT), C-reactive protein (CRP), and white blood cell (WBC) were measured on the first, third, fifth day after operation. The incidence of pulmonary infection was observed within 3 days after operation. Result The IL-6 values of the right collapsed lung in all groups were higher than those in the left ventilated lung. The TNF-α value of the right collapsed lung in the T2 group and T3 group was higher than that in the left ventilated lung (P<0.05). Compared with in the right collapsed lung, the TNF-α and IL-6 values gradually increased with the the duration of one-lung ventilation (P<0.05). Compared with the left ventilated lung groups, the IL-6 value increased gradually with the duration of one-lung ventilation time (P<0.05). The TNF-α value of the T3 group was higher than that of the T1 and T2 groups (P<0.05). The PCT value of the T3 group was higher than that of the T1 group and T2 group on the third, fifth day after operation (P<0.05). But there was no significant difference in CRP and WBC among the three groups at different time points. The incidence of pulmonary infection in the T3 group was significantly higher than that in the T1 group within 3 days after operation (P<0.05). Conclusion With the extension of one-lung ventilation time, the release of local and systemic inflammatory mediators is increased, and the probability of pulmonary infection is higher.