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.     

Adipophilin induces expression of inflammatory factors through ERK1/2-AP-1 signaling pathway

AIM: To observe the effects of adipose differentiation-related protein (adipophilin) on the expression of inflammatory factors in RAW264.7 macrophage and to clarify the related mechanism. METHODS: The cell models with high expression and low expression of adipophilin were constructed by transfecting PA317 packaging cells with stable high or low expression adipophilin retroviral vectors into the RAW264.7 cells. The concentrations of IL-6, MCP-1 and TNF-α in the cell culture medium were detected by ELISA. The protein levels of AP-1, p-AP-1, ERK1/2 and p-ERK1/2 were measured by Western blot. The protein levels of adipophilin, p-ERK1/2 and p-AP-1 and the releases of the inflammatory factors in the RAW264.7 cells treated with or without ERK1/2 inhibitor PD98059 or AP-1 inhibitor curcumin were determined. RESULTS: The RAW264.7 cells with high expression of adipophilin had higher levels of IL-6, MCP-1 and TNF-α, and higher protein levels of p-AP-1 and p-ERK1/2 than those in the cells with low expression of adipophilin. ERK1/2 inhibitor had no significant effect on the expression of adipophilin, but the protein expression of ERK1/2 and AP-1 was significantly inhibited (P<0.05). The administration of AP-1 inhibitor curcumin had no significant effect on the protein expression of adipophilin and ERK1/2, but the protein expression of AP-1 was significantly inhibited (P<0.05). At the same time, the releases of inflammatory factors IL-6, MCP-1 and TNF-α were significantly decreased. CONCLUSION: Adipophilin may regulate the expression of inflammatory factors through ERK1/2-AP-1 pathway in RAW264.7 macrophages.     

Expression of STC1 in colorectal cancer and its significance in tumor microenvironment

AIM:To explore the expression of stanniocalcin 1 (STC1) in colorectal cancer tissues and to analyze its relationship with prognosis, further to investigate the correlation between STC1 and tumor microenvironment. METHODS:The data of the STC1 mRNA expression were accessed by the UALCAN database (http://ualcan.path.uab.edu/index.html) and Oncomine database (https://www.oncomine.org). RT-qPCR and Western blot were used to detect the expression of STC1 in the clinical samples. OncoLnc (http://www.oncolnc.org/) analytical tool was adopted to evaluate the correlation of STC1 level and the prognosis of colorectal cancer. CoCl₂ was used to establish the hypoxia status and lipopolysaccharide (LPS) was utilized to establish inflammatory condition in the colorectal cancer HT-29 cells, respectively, and then STC1 expression was examined by RT-qPCR and Western blot. RESULTS:STC1 was over-expressed in the colorectal cancer tissues, and its high expression was positively correlated with poor prognosis of the colorectal cancer patients (P<0.01). In colorectal cancer HT-29 cells, treatment of CoCl₂ up-regulated the expression of STC1. Under the condition of inflammation by stimulating with LPS, the expression of STC1 was significantly increased. In the colorectal cancer, STC1 level was positively correlated with hypoxia-inducible factor-1α or inflammatory molecules expression, and the levels of tumor-infiltrating immune cells in colorectal cancer tissue. CONCLUSION:STC1 is over-expressed in the colorectal tumor tissue, and the high level of STC1 is an important prognosis indicator for colorectal cancer. Furthermore, STC1 is closely correlated with tumor microenvironment, especially with the tumor hypoxia and tumor immune regulation of colorectal cancer.     

Protective effect of C1q/tumor necrosis factor-related protein 3 on vascular endothelium in rats with hyperuricemia

AIM To study whether C1q/tumor necrosis factor （TNF）-related protein 3 （CTRP3）protect vascular endothelium in rats with hyperuricemia and its potential mechanisms. METHODS An animal model of hyperuricemia was established by using male SD rats drinking 10% fructose water （n=10）. The rats drinking normal water served as normal controls （n=10）. After 12 weeks, the rats were given a single injection with Ad-CTRP3 or Ad-GFP. The experiment was ended at 14th day after transfection.The serum levels of uric acid and nitric oxide （NO） were evaluated. The serum contents of TNF-α and interleukin-6 （IL-6） were measured by ELISA. HE staining and TUNEL assay were used to assess the morphological changes of intima and apoptosis of endothelial cells in thoracic aorta, respectively. The mRNA levels of endothelial nitric oxide synthase （eNOS）, TNF-α and IL-6 were detected by RT-qPCR. The protein levels of CTRP3 and Toll-like receptor 4 （TLR4） were determined by Western blot. RESULTS Compared with normal control group, the rats with hyperuricemia showed lower CTRP3 and higher TLR4 protein levels in the thoracic aorta （P<0.05）. Hyperuricemic rats had higher serum contents of uric acid, TNF-α and IL-6 （P<0.05）. Also, the intima structure disturbance of thoracic aorta, increased apoptotic rate, higher mRNA levels of TNF-α and IL-6 as well as lower mRNA levels of eNOS were observed （P<0.05）. By contrast, CTRP3 over-expression decreased TLR4 protein levels, reduced inflammatory cytokines, and obviously improved the morphology and function of thoracic aorta in the rats with hyperuricemia. CONCLUSION CTRP3 protect vascular endothelium in rats with hyperuricemia maybe via down-regulation of TLR4- mediated inflammatory signaling pathway.     

Molecular mechanisms of deubiquitinase OTUD7B in regulating tumor microenvironment and progression

Ubiquitination and deubiquitination are important mechanisms for regulating cell signal transduction. They regulate target proteins precisely and participate in physiological and pathological processes such as growth, development, immune response and cancer. OTUD7B (OTU domain-containing 7B) is a deubiquitinase that hydrolyzes K11-, K48- and K63-linked polyubiquitin chains, regulates intracellular signal transduction and mediates cell survival and proliferation. The abnormal expression of OTUD7B in various malignant tumors affects tumor progression and immune microenvironment. Here, we reviewed OTUD7B-mediated cellular signal transduction and its role in malignant tumors, in order to provide clues for the discovery of new targets of anti-cancer therapy targeting ubiquitin system.     

Interferon-γ up-regulates programmed death ligand 1 in human gastric cancer cells

AIM:To study the expression of programmed death ligand 1 (PD-L1) in human gastric cancer cells with or without the stimulation of interferon-γ (IFN-γ). METHODS:The protein levels of PD-L1 in 4 different human gastric cancer cell lines AGS, BGC823, MGC803 and SGC7901 with or without IFN-γ treatment were analyzed by flow cytometry. The mRNA expression of PD-L1 in those cell lines was also detected by real-time PCR. RESULTS:Flow cytometry analysis showed that the rates of PD-L1 surface expression in the 4 human gastric cancer cell lines AGS, BGC823, MGC803 and SGC7901 were (1.567±0.109)%, (2.640±0.577)%, (1.760±0.236)% and (16.030±1.289)%, respectively. After the 4 gastric cancer cell lines were treated with IFN-γ at different concentrations or for different time, the PD-L1 surface expression increased at different levels with significant differences between groups. Real-time PCR also indicated that IFN-γ up-regulated PD-L1 expression at mRNA level. CONCLUSION: PD-L1 surface expression is found in human gastric cancer cell lines AGS, BGC823, MGC803 and SGC7901. IFN-γ up-regulates the expression of PD-L1. SGC7901 cell line, which is from metastatic lymph nodes, expresses the highest protein level of PD-L1 among the 4 cell lines, indicating that PD-L1 expression may be related to lymph node metastasis, not to differentiation grade. IFN-γ may mediate the tumor immune escape so that it should be carefully applied in the treatment of gastric cancer.     

Effects of B7-1 gene expression on the biological characteristics of hepatocarcinoma cells

AIM:Modification of tumor cells with B7-1 (CD80) costimulatory adhesion molecules has been proposed as a means to develop therapeutic cancer vaccines for use in human immunotherapy.METHODS:Glycosyl-phosphatidylinositol (GPI)-anchored B7-1 was transfered into QK10341 cell membranes with the plasmid of pcDNA3.1(+)/GPI-B7-1 by lipofectamine transfection. Then the GPI-B7-1 protein was isolated and purified from QK10341 cells. The SKOV3 cells incubated with GPI-B7-1 protein resulted in stable incorporation of B7-1 on SKOV3 cell membranes. Expression of B7-1 in tumors by protein transfer created an immunogenic tumor cell that induced antitumor immunity. The growth curve of T cells, the change of Fas (CD95) expression on cell membranes, and level of cytokines secreted from CTL were determined by MTT, FCM, and ELISA, respectively. The cytotoxicity alteration of the CTL was also studied.RESULTS:Compared with SKOV3 cells, B7-1-SKOV3 cells more effectively induced the proliferation of effector lymphocytes and the generation of specific lytic activity (P<0.01). The level of cytokine secretion also increased.CONCLUSION:The costimulation signal of B7-1 is required for the activation and proliferation of T lymphocytes. The B7-1 expression in SKOV3 tumor cells can increase their immunogenicity and induce more effective T lymphocytes activation.     

miR-92b-5p attenuates renal injury and inflammatory response in diabetic nephropathy rats by targeting HMGB1

AIM To investigate the effect of microRNA-92b-5p （miR-92b-5p） on renal injury and inflammatory response in diabetic nephropathy （DN） rats and its mechanism. METHODS The rats were divided into control group, DN group, lentiviral negative control （LV-NC） group, LV-miR-92b group, LV-high mobility group protein B1 （LV-HMGB1） group and miR-92b+HMGB1 group, with 15 rats in each group. After fasting for 12 h, the model rats were intraperitoneally injected with streptozotocin at dose of 60 mg/kg, and the control rats were intraperitoneally injected with an equal volume of citrate buffer. Three days later, the rats in each treatment group were intravenously injected with 100 μL LV-NC, LV-miR-92b and LV-HMGB1 （1×10¹¹ U/L） twice a week for 8 consecutive weeks. Urinary protein, blood glucose, blood urea nitrogen and serum creatinine were detected by an automatic biochemical analyzer. The expression of miR-92b-5p and HMGB1 mRNA was detected by RT-qPCR. The targeting relationship between miR-92b-5p and HMGB1 was verified by dual-luciferase reporter assay. HMGB1 expression in kidney tissue was detected by Western blot. The kidney damage was observed by HE staining. The apoptosis was detected by flow cytometry. The levels of interleukin-6 （IL-6）, IL-1β and tumor necrosis factor-α （TNF-α） in renal tissues were detected by ELISA. RESULTS In DN model rats, miR-92b-5p was down-regulated, while HMGB1 was highly expressed. There was a binding site between miR-92b-5p and HMGB1 3'-untranslated region. High expression of miR-92b-5p inhibited the luciferase activity of the wild-type HMGB1 plasmid （P<0.01）, but had no effect on the luciferase activity of the mutant HMGB1 plasmid. Compared with DN group, urinary protein, blood glucose, serum creatinine and blood urea nitrogen in LV-miR-92b group were significantly reduced （P<0.01）. The degree of hyperplasia, swelling and inflammatory cell infiltration of glomerular mesangium and basement membrane tubules, the apoptosis rate of renal tissues, and the content of IL-6, IL-1β and TNF-α in renal tissues were significantly decreased （P<0.01）. Co-transfection of LV-HMGB1 significantly reversed the effect of miR-92b-5p on DN rats. CONCLUSION miR-92b-5p reduces renal injury and inflammatory response in DN rats by targeting HMGB1 and down-regulating its expression.     

Effect of recombinant plasmids encoding IL-1RII and IL-1RAcP on rat experimental autoimmune myocarditis and possible mechanism

AIM To evaluate the effects of recombinant plasmids encoding interleukin-1 type II receptor （IL-1RII） and interleukin-1 receptor accessory protein （IL-1RAcP） on rat experimental autoimmune myocarditis （EAM） and the possible mechanism. METHODS The recombinant plasmids pCAGGS-IL-1RII and pCAGGS-IL-1RAcP were constructed, and pCAGGS-SP （signal peptide） served as the control plasmid. Male Lewis rats （n=29） were divided into 4 groups： control group （rats without immunization or injection, n=5）, EAM+SP group （immunized rats injected with pCAGGS-SP, n=9）, EAM+IL-1RII group （immunized rats injected with pCAGGS-IL-1RII, n=8） and EAM+IL-1RII+IL-1RAcP group （immunized rats injected with pCAGGS-IL-1RII and pCAGGS-IL-1RAcP, n=7）. The rats were immunized to induce EAM on day 0, and injected with recombinant plasmids by hydrodynamics-based delivery on day 6. Echocardiography was performed, and the rats were killed on day 17. The ratio of heart weight to body weight （HW/BW） was evaluated, and the histopathological changes of the myocardial tissues were observed by HE staining. The mRNA expression of atrial natriuretic peptide （ANP）, brain natriuretic peptide （BNP） and inflammatory factors in the myocardial tissues was detected by RT-qPCR. Recombinant plasmids pUC19-IL-1RII-actin and pUC19-IL-1RAcP-tub were transfected into Cos7 cells, and the culture supernatants were collected and added to lipopolysaccharide （LPS）-induced H9c2 cells. The expression of inflammatory genes were detected by RT-qPCR. Recombinant plasmids pEGFP-IL-1RII-actin and pEGFP-IL-1RAcP-tub were transfected into the Cos7 cells to identify the formation of IL-1RII/IL-1RAcP heterodimer by co-immunoprecipitation （Co-IP）. RESULTS Compared with EAM+SP group, injection with plasmids effectively attenuated EAM in EAM+IL-1RII group and EAM+IL-1RII+IL-1RAcP group, as indicated by the decreases in HW/BW, left ventricular end-systolic diameter, and myocardial expression of ANP, BNP, TNF-α, IL-2, IFN-γ and TGF-β, and the increase in expression of IL-4 in the hearts. In LPS-induced H9c2 cells, compared with LPS group, the levels of TGF-β and IL-6 in the culture supernatants were significantly decreased （P<0.01）, and the level of IL-10 was significantly increased （P<0.05） in LPS+IL-1RII group and LPS+IL-1RII+IL-1RAcP group. Compared with LPS+IL-1RII group, the expression of TNF-α and IL-2 was significantly decreased （P<0.05）, and the expression of IL-13 was significantly increased in LPS+IL-1RII+IL-1RAcP group （P<0.01）. The formation of IL-1RII/IL-1RAcP heterodimer was detected by Co-IP. CONCLUSION Plasmids encoding IL-1RII and IL-1RAcP effectively attenuate EAM, and the possible mechanism may be related to the inhibition of inflammatory factor expression and the formation of IL-1RII/IL-1RAcP heterodimer.     

Isorhamnetin inhibits ovalbumin-induced pulmonary inflammation in asthmatic mice

AIM To investigate the effect of isorhamnetin on pulmonary inflammation in asthmatic mice and to analyze its primary mechanism. METHODS BALB/c mice were randomly divided into normal control group, asthma model group, isorhamnetin low-dose （50 mg/kg） treatment group and isorhamnetin high-dose （150 mg/kg） treatment group. Ovalbumin （OVA） was used to establish a mouse asthma model. HE staining and PAS staining were used to observe the pathological changes of lung tissue. The contents of cysteinyl leukotriene1 （CysLT1）, cystelinyl leukotriene receptor 1 （CysLTR1）, interleukin-4 （IL-4）, IL-5, IL-13 and tumor necrosis factor-α （TNF-α） in bronchoalveolar lavage fluid （BALF） were measured by ELISA. The expression of nuclear factor of activated T cells 4（NFATc4） in lung tissue was detected by immunohistochemical staining. The protein expression of NFATc4, intercellular cell adhesion molecule-1 （ICAM-1） and vascular cell adhesion molecule-1 （VCAM-1） was determined by Western blot. RESULTS Isorhamnetin improved histopathological changes in OVA-induced asthma model mice, reduced the contents of CysLT1, CysLTR1, IL-4, IL-5, IL-13 and TNF-α in BALF, and reduced NFATc4, ICAM-1 and VCAM-1 expression in lung tissue （P<0.05）. CONCLUSION Isorhamnetin inhibits the inflammatory response of lung tissue in asthmatic model mice, and the mechanism may be related to the inhibition of the calcineurin/NFATc4 signaling pathway.     

Effects of Ku70 on protein expression of HTLV-1 in HTLV-1 positive T cells

AIM:To explore the effects of Ku70 on the protein expression of human T-lymphotrophic virus 1 (HTLV-1) in HTLV-1 positive T cells. METHODS:The expression level of Ku70 in HTLV-1 positive T cells was exa-mined by Western blot. The siRNA targeting Ku70 was constructed and the effect of the siRNA on knockdown of Ku70 expression was determined by Western blot. After knockdown of Ku70 expression in the HTLV-1 positive T cells by siRNA, the expression of HTLV-1-related proteins at mRNA and protein levels was examined by real-time PCR and Western blot, and the expression levels of interferons and proinflammatory cytokines were examined by real-time PCR. RESULTS:The HTLV-1 positive T cells, including MT2, MT4 and C8199 cells, displayed a higher expression level of Ku70. The protein expression of HTLV-1 was increased in Ku70-silencing MT2 cells and MT4 cells. After knockdown of Ku70 expression in the MT2 cells and MT4 cells, the production of interferon (IFN)-α, IFN-γ and tumor necrosis factor-α was reduced.CONCLUSION:The HTLV-1 positive T cells have a higher expression level of Ku70. In HTLV-1 positive T cells, Ku70 promotes the production of interferons and proinflammatory cytokines and inhibits HTLV-1-related protein expression.     

Curcumin inhibits Porphyromonas gingivalis LPS-induced inflammatory response in human gingival fibroblasts by up-regulating miR-124

AIM To investigate the effects of curcumin （Cur） on the inflammatory response of human gingival fibroblasts （HGFs） induced by Porphyromonas gingivalis （Pg） lipopolysaccharide （LPS） and the role of microRNA-124 （miR-124） in this process. METHODS The HGFs were divided into control group, LPS group （10 mg/L LPS） and LPS+Cur （20, 40 and 80 μmol/L） groups （10 mg/L LPS+corresponding dose of Cur）. After treatment for 24 h, CCK-8 assay was used to measure the cell viability. ELISA was used to measure the levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in the supernatant. The level of miR-124 in the cells was detected by RT-qPCR. The protein levels of nuclear factor kappa B （NF-κB） p-p65 in cytoplasm and nucleus were determined by Western blot, and the nuclear translocation of NF-κB p-p65 was evaluated by laser confocal microscopy. After transfection with mimic-NC or miR-124 mimic, the expression of miR-124 and NF-κB p-p65 protein in the cytoplasm and nucleus of the cells were also detected. RESULTS The cell viability, the level of miR-124 in the cells and NF-κB p-p65 protein level in cytoplasm of LPS group were lower than those in control group （P<0.05）, while the levels of IL-1β and TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were higher than those in control group （P<0.05）. The cell viability, the level of miR-124 in cells and NF-κB p-p65 protein level in the cytoplasm of LPS+Cur （40 and 80 μmol/L） groups were higher than those in LPS group （P<0.05）, while the level of TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were lower than those in LPS group （P<0.05）. The level of IL-1β in the supernatant of LPS+80 μmol/L Cur group was lower than that in LPS group （P<0.05）. The levels of miR-124 and NF-κB p-p65 protein level in the cytoplasm of miR-124 mimic group were higher than those in LPS group and mimic-NC group （P<0.05）, while the level of NF-κB p-p65 proteinlevel in the nucleus was lower than that in LPS group and mimic-NC group （P<0.05）. CONCLUSION Curcumin inhibits the inflammatory response of HGFs induced by Pg LPS, which may be achieved by up-regulating miR-124 and then inhibiting the nuclear translocation of NF-κB p-p65.     

Aβ1-40 induces inflammation and phenotypic switching via activation of MAPKs signaling pathway in vascular smooth muscle cells

AIM To investigate the effects of amyolid β-protein 1-40 （Aβ1-40） on inflammation, viability, migration and phenotypic switching in vascular smooth muscle cells （VSMCs）, and to analyze the underlying mechanisms. METHODS The VSMCs were treated with Aβ1-40 at different concentration gradients for appropriate time. CCK-8 and Transwell assays were performed to evaluate the viability and migration ability of VSMCs. The levels of inflammatory factors including interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α）, phenotypic switching-related proteins including α?smooth muscle actin （α?SMA）, osteopontin （OPN） and Krüppel?like factor 4 （KLF4）, and mitogen-activated protein kinases （MAPKs） signaling pathway-related proteins including p-p38 MAPK, p-ERK1/2 and p-JNK were determined by Western bolt. RESULTS After Aβ1-40 treatment, the levels of inflammatory factors IL-1β and TNF-α in the VSMCs were significantly increased （P<0.05）, and the expression of phenotypic switching-related proteins was altered, as indicated by down-regulation of α?SMA and up-regulation of OPN and KLF4 （P<0.05）. Treatment with Aβ1-40 within a certain concentration range promoted the viability and migration of the VSMCs. In addition, the protein levels of p-p38 MAPK, p-ERK1/2 and p-JNK were significantly increased by Aβ1-40 treatment （P<0.05）. Furthermore, pretreatment with specific inhibitors of MAPKs pathway significantly reduced the levels of IL-1β and TNF-α （P<0.05）, and inhibited the phenotypic switching, as indicated by up-regulation of α?SMA and down-regulation of OPN and KLF4 （P<0.05）. CONCLUSION Treatment with Aβ1-40 induces the inflammation and phenotypic switching in VSMCs via activation of MAPKs signaling pathway.     

Differential expression of nesfatin-1 in intestinal tissues of premature infants with necrotizing enterocolitis and and its mechanism

AIM To analyze the expression of nesfatin-1 in intestinal tissues of premature infants with necrotizing enterocolitis （NEC）, and to explore the effect of nesfatin-1 on lipopolysacharide （LPS）-induced enterocytes and its mechanism. METHODS The intestinal tissues were obtained from infants who underwent intestinal surgery for NEC in our hospital from 2017 to 2019. The mRNA expression of nesfatin-1 in the tissue samples of NEC were evaluated by RT-qPCR. Human fetal normal colon epithelial HCoEpiC cells and human colon cancer Caco-2 cells were used as research objects. The effect of nesfatin-1 on the secretion of cytokines was measured by ELISA. Western blot was used to analyze the protein expression of nesfatin-1 and Toll-like receptor 4 （TLR4）, NLRP3, AIM2, caspase-1 and ASC, and co-immunoprecipitation assay were conducted to explore the relation between nesfatin-1 and TLR4. RESULTS The expression of nesfatin-1 in NEC preterm infants was significantly lower than that in the healthy group （P<0.01）. Compared with control group, the expression of nesfatin-1 in HCo Epic cells and HT-29 cells induced by LPS was decreased （P<0.01）, while the transfection of nesfatin-1 reversed the stimulation of LPS, and the over-expression of nesfatin-1 decreased the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6）, and increased the level of IL-10 （P<0.05）. In addition, nesfatin-1 over-expression inhibited the expression of NLRP3, AIM2, caspase-1 and ASC. The expression of TLR4 in NEC tissue samples was significantly higher than that in healthy infants （P<0.05）. Pearson correlation analysis showed that there was a significant negative correlation between nesfatin-1 and TLR4 （r=-0.816, P<0.01）. TLR4 was found to co-precipitate with nesfatin-1. CONCLUSION Nesfatin-1 protects intestinal cells from LPS induced inflammation by targeting TLR4, which may be a potential target of anti-NEC therapy.     

Secoisolariciresinol diglucoside attenuates renal inflammatory injury of mice induced by chronic intermittent hypoxia via inhibiting TXNIP and activating NLRP3 inflammasome

AIM To investigate the effectof flax lignan/secoisolariciresinol diglucoside （SDG） on the inflammatory damage of kidney induced by chronic intermittent hypoxia （CIH）. METHODS C57BL/6N mice were divided into normal （control） group, model （CIH） group and treatment （SDG） group. The changes of the body weight was recorded. Hematoxylin-eosin （HE） staining was used to observe the morphological alterations in the renal tissues. The levels of serum creatinine and blood urea nitrogen were measured by a biochemical analyzer. Hydroxylamine and thiobarbituric acid methods were used to detect the activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the renal tissues. The protein levels of thioredoxin-interacting protein （TXNIP） and nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） were detected by immunohistochemical staining, while those of tumor necrosis factor-α （TNF-α）, interleukin-6 （IL-6） and IL-1β were measured by ELISA. The protein levels of TXNIP, NLRP3, apoptosis-associated speck-like protein containing a CARD （ASC）, caspase-1, IL-1β and IL-18 in the renal tissues were also determined by Western blot. RESULTS No significant difference in the body weight and kidney index among the 3 groups was observed （P>0.05）. HE staining showed the swollen epithelial cells of renal tubules with vesicular degeneration, and irregular glomerular morphological change in CIH group, while SDG treatment attenuated the above changes. Compared with control group, the levels of serum creatinine, TNF-α, IL-6 and IL-1β were significantly increased in CIH group （P<0.05）. The significantly increased expression levels of NLRP3 and TXNIP in the cytoplasm of renal tubular epithelial cells in CIH group were detected by immunohistochemical staining. Compared with control group, the activity of SOD was decreased, the content of MDA was increased in CIH group, and the protein expression levels of TXNIP, NLRP3, ASC, caspase-1, IL-1β and IL-18 were up-regulated and then decreased after SDG treatment （P<0.05）. CONCLUSION SDG attenuates the renal inflammatory damage of the mice induced by CIH, and its mechanism may be associated with the inhibition of oxidative stress and activation of NLRP3 inflammasome.     

Bi-directional regulation of histamine in tumor immunity

Histamine is one of the important mediators of many diseases such as hypersensitivity, gastric ulcer, and inflammat ion.It regulates the local immune response in tumor tissues by multiple mechanisms with ambivalent and double-faced effects, which have been uncovered partially by thorough molecular immunological analyses. Histamine has differential ef ects on the growth of tumor cell depending on selective reaction with its receptors, and on the secretion of variety of cytokines from local activated immune cells in a reciprocal manner by shifting TH1/TH2 polarization towards predominance of TH2. In this review, we summa rized recent data suggesting that endogenous histamine should be an important correlated factor involved in bi-directional regulat ion both to tumor tissue and to infiltrating immune cells.     

A study on the mechanism of doxorubicin promoting IL-1β and collagen type I secretion in cardiac fibroblasts

AIM To observe the effect of adriamycin/doxorubicin （DOX） on the production of inflammatory cytokines and collagen in cardiac fibroblasts and its mechanism. METHODS Neonatal SD rat cardiac fibroblasts were isolated, cultured, and identified by immunofluorescence staining with monoclonal antibodies against vimentin observed under a confocal laser-scanning microscope. The Cell Counting Kit-8 assay was used to detect the toxicity of DOX on cardiac fibroblasts, and flow cytometry with annexin V-FITC/PI double staining was used to detect apoptosis. ELISA was used to detect the release of inflammatory factors in the supernatant of cultured cells. Immunofluorescence labeling assay was used to detected α-smooth muscle actin （α-SMA） expression and mitochondrial reactive oxygen species （mROS） in the cells. Western blot was used to detect the expression of nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3） inflammasome-related proteins in cardiac fibroblasts. RESULTS （1） Compared with the control group, DOX inhibited the proliferation of cardiac fibroblasts （P<0.05）, but had no significant effect on apoptosis （P>0.05）. （2） Treatment with DOX promotes the release of proinflammatory factors interleukin-1β （IL-1β） and IL-6 in cardiac fibroblasts （P<0.05）. （3） The expression of α-SMA, collagen type I and transforming growth factor-β in DOX treatment group increased significantly compared with control group （P<0.05）. （4） Compared with the control group, the levels of mROS, cellular NLRP3 and cleaved caspase-1 in cardiac fibroblasts increased significantly after DOX treatment. CONCLUSION Doxorubicin promotes cardiac fibroblasts to secrete IL-1β and collagen type I by promoting mROS production and activating NLRP3 inflammasome.