Immunomodulatory effect of Sema4D on Th1/Th2 in asthmatic rats induced by RSV and intervention of Kechuanning oral liquid

AIM To explore the effect of semaphorin 4D (Sema4D) on the immune regulation of Th1/Th2 in asthmatic rats induced by respiratory syncytial virus (RSV) and the effect of Kechuanning (KCN) intervention on the asthmatic rats. METHODS Healthy SPF female Wistar rats (n=100) were randomly divided into 10 groups: normal group, model group, Sema4D group, Sema4D antibody group, low, middle and high doses of KCN groups, and Sema4D+low, middle and high doses of KCN groups. Except the rats in normal group, the other rats were treated with RSV combined with ovalbumin (OVA) to induce asthmatic model. The pathological changes of the lung tissue were observed by HE staining, and the levels of interleukin-4 (IL-4), IL-6, IL-8, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. RESULTS Inflammatory cell infiltration and inflammatory damage in lung tissues of the rats in model group, Sema4D group and Sema4D+low dose of KCN group were observed by HE staining, while these pathological changes were attenuated in Sema4D antibody group， low, middle and high doses of KCN groups, and Sema4D+middle and high doses of KCN groups. Compared with normal group, the levels of IL-4, IL-6, IL-8 and TNF-α in BALF of the rats in the other groups were significantly increased, and the level of IFN-γ was significantly lowered (P<0.05 or P<0.01). Compared with model group, the levels of IL-4, IL-6, IL-8 and TNF-α in Sema4D group were increased, and the content of IFN-γ was decreased (P<0.05 or P<0.01). Compared with model group, the levels of IL-4, IL-6, IL-8 and TNF-α in Sema4D antibody group, low, middle and high doses of KCN groups, and Sema4D+low, middel and high doses of KCN groups were significantly decreased (P<0.01), while the content of IFN-γ was significantly increased (P<0.05 or P<0.01). No significant difference among Sema4D antibody group, Sema4D+middle and high doses of KCN groups, and low, middle and high doses of KCN groups was observed (P>0.05). CONCLUSION Sema4D causes Th1/Th2 immune imbalance and aggravates asthma. Inhibition of Sema4D reduces the production of inflammatory factors and regulates the balance of Th1/Th2. KCN may attenuate RSV-induced immune inflammation of asthmatic rats by inhibiting Sema4D, so as to achieve the anti-asthma effect.     

Effects of neutrophil extracellular traps on acute lung injury in neonatal rats

AIMTo investigate the role of neutrophil extracellular traps (NETs) in neonatal rats with acute lung injury (ALI). METHODSThirty 7-day-old SD rats were randomly divided into normal saline control group, ALI group and ALI+deoxyribonuclease (Dnase) group (each n=10). The rats in ALI group were intraperitoneally injected with lipopolysaccharides (LPS) at 20 mg/kg, and the rats in ALI+Dnase group were intraperitoneally injected with Dnase at 5 mg/kg after LPS injection. After 6 h, the rats were anesthetized with chloral hydrate, bronchial alveolar lavage fluid (BALF) was collected, and the content of cell-free DNA (cf-DNA) in BALF was detected by fluorescence microarray. The right lung tissues were fixed in 4% paraformaldehyde, and the morphological structure of the lung tissues were observed by HE staining. The content of interleukin-6 (IL-6) and tumor necrosis factor-α （TNF-α） in the left lung homogenate was measured by ELISA. Immunofluorescence and Western blot were used to detect the production of citrullinated histone H3 (CitH3) and myeloperoxidase (MPO) in the rat lung tissues. RESULTSCompared with control group, the levels of cf-DNA, CitH3, MPO, IL-6 and TNF-α in lung tissues of neonatal rats in ALI group and ALI+Dnase group were all increased (P<0.05), and severe inflammatory infiltration in the lung tissues was observed. Compared with ALI group, the levels of cf-DNA, CitH3, MPO, IL-6 and TNF-α in ALI+Dnase group were decreased (P<0.05), and the inflammatory infiltration was attenuated. CONCLUSION In neonatal rats with ALI, the level of NETs is an important indicator of lung tissue injury, and NETs may be a new target for the treatment of neonatal ALI.     

Glyburide reduces PFOS-induced lung injury in young rats through inhibiting NLRP3 inflammasome

AIM: To explore the possible mechanism of NLR family Pyrin domain-containing protein 3 (NLRP3) inflammasome involved in perfluorooctane sulfonate (PFOS)-induced lung injury in young rats. METHODS: Twenty-eight SD rats (21-day-old) were randomly divided into control (C) group, PFOS (P) group, glyburide (G) group and glyburide + PFOS (GP) group. PFOS exposure model and glyburide protection model were established. The lung specimens were collected for HE staining. The levels of myeloperoxidase (MPO) in the lung tissues, interleukin-1β (IL-1β) and interleukin-18 (IL-18) in the bronchoalveolar lavage fluid (BALF) were measured by ELISA. The concentration of PFOS in serum was measured by high-performance liquid chromatography (HPLC). The protein expression of NLRP3, caspase-1 and apoptosis-associated speck-like protein containing CARD (ASC) in the lung tissues was determined by Wes-tern blot. RESULTS: HE staining of lung tissues showed that compared with the control rats, there were obvious inflammatory infiltration in trachea and alveolar interstitium of the rats in P group. Glyburide reduced the inflammatory responses significantly. ELISA results showed that the level of MPO in the lung tissues of the rats in P group was higher than those in other 3 groups (P<0.05). The levels of IL-1β and IL-18 in the BALF of the rats in P group were significantly higher than those in control group and GP group (P<0.05). The results of Western blot showed that the protein levels of NLRP3, caspase-1 and ASC in P group were significantly higher than those in control group and GP group (P<0.01). Immunohistochemical staining results showed that compared with the other 3 groups, the expression of NLRP3 in P group was significantly increased (P<0.01). CONCLUSION: PFOS exposure may lead to lung injury in rats by activating NLRP3 inflammasome and then triggering inflammation, releasing inflammatory factors such as IL-1β. Glyburide specifically inhibits the assembly of NLRP3 inflammasome, suppresses the inflammatory responses and reduces the toxicity of PFOS in lung.     

Development and study of experimental C57BL/6 mouse lupus nephritis model induced by pristane

AIM To establish a C57BL/6 mouse lupus nephritis model induced by pristane and to explore the pathogenic mechanism of this model. METHODS Thirty C57BL/6 mice were randomly divided into 2 groups. The mice in model group were intraperitoneally injected with 0.5 mL pristane, while the mice in control group were injected with 0.5 mL normal saline. The state of health and survival of the animals were monitored during the whole experiment. The activation of macrophages, granulocytes, dendritic cells and B cells in spleen and the markers on B cells were detected by flow cytometry on the 10th day after injection. The serum levels of antinuclear antibodies (ANA) and anti-double strand DNA antibodies (anti-dsDNA) were measured every month after injection by indirect immunofluorescence assay, and proteinuria was checked mouthy by Albustix test strips. Eight months after injection, all mice were killed, and the kidneys were slided and stained with HE or PE-labeled IgG to analyze the expression of interferon-γ (IFN-γ) and interleukin-4 (IL-4). The evidence of glomerulonephritis was histopathologically observed, and the ultrastructural changes of the glomerulus were determined by transmission electron microscopy. RESULTS Ten days after the pristane injection, the populations of macrophages, granulocytes, dendritic cells and B cells were increased significantly compared with control mice (P<0.05). The expression of B7-1, B7-2 and MHC-II on the B cells was also increased (P<0.05). The serum levels of ANA and anti-dsDNA were increased from 3 to 8 months (P<0.05). Meanwhile, part of the mice showed different degrees of ascites, emaciation or even death, part of the mice had proteinuria 4 months later, and 8 months later, all of the model mice had proteinuria, with 1~20 g/L of urine protein. At the 8th month, a large number of heterotopic lymphoid tissues appeared in the abdominal cavity. The deposition of immune complexes in glomeruli and mesangial areas was serious. The expression of IFN-γ in the renal tissues was increased. Pathological damages such as lymphocyte infiltration, tissue necrosis and deposition of electron dense substances were obvious in the model mice. In control group, autoantibodies and proteinuria was not detected under the same experimental condition. Meanwhile, no evidence of glomerulonephritis in the control mice was observed. CONCLUSION The C57BL/6 mouse model of lupus nephritis induced by pristane is established successfully.     

SUMO-specific protease 3 aggravates CaPO4-induced mouse abdominal aortic aneurysm formation by promoting macrophage polarization

AIMTo investigate the role of SUMO-specific protease 3 (SENP3) in macrophage polarization and calcium phosphate (CaPO₄)-induced abdominal aortic aneurysm (AAA) formation in mice. METHODS（1） Bone marrow-derived monocytes (BMDMs) in Senp3^flox/flox (wild-type, WT) mice and Senp3^flox/flox; Lyz2-Cre (monocyte-specific SENP3 knockout, i.e. conditioned knockout, cKO) mice were isolated and induced for M1 and M2 polarization. The mRNA and protein expression level of SENP3 were detected by RT-qPCR, Western blot and immunocytofluorescence, and the differential distribution of M1/M2 BMDMs from WT and cKO mice was analyzed. （2） CaPO₄ was administrated to induce AAA model in 8~12-week-old male WT and cKO mice. The AAA incidence, survival rate and maximal aortic diameter were analyzed between the 2 groups. Aortic aneurysm tissues were collected for pathological analysis, and the expression levels of SENP3, interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), IL-6 and matrix metalloproteinases-9 (MMP-9) were measured by RT-qPCR and Western blot. Dihydroethidium staining in situ in frozen sections was used to analyze the production of reactive oxygen species (ROS). （3） To explore the potential mechanisms, Western blot and co-immunoprecipitation were used to verify the de-SUMO modification of mitogen-activated protein kinase kinase 7 (MKK7) induced by SENP3. Besides, BMDMs were transfected with Flag-MKK7 wild type (Flag-MKK7 WT) and SUMO-modified site K18 mutant (Flag-MKK7 K18R mutant), and then M1 polarization of the cells was induced. The protein levels of p-JNK and MMP-9 in the 2 groups were determined by Western blot. RESULTS(1) SENP3 expression was up-regulated in M1 polarized macrophages (P<0.01), but was down-regulated in M2 polarized macrophages (P<0.01). The expression of SENP3 was decreased during the transformation of M1 to M2 in the macrophages (P<0.01), but was significantly up-regulated during the opposite process (P<0.01). Besides, more M1 macrophages and less M2 macrophages after induction were observed in the BMDMs from cKO mice than those from WT mice. (2) SENP3 expression was up-regulated in AAA tissues (P<0.05). The AAA incidence of cKO mice was significantly reduced after CaPO₄ induction (P<0.01), the survival rate was significantly improved (P<0.05), and maximal aortic diameter was significantly reduced in cKO group (P<0.01). The levels of IL-1β, IL-6 and TNFα, and the production of ROS were significantly down-regulated (P<0.01), meanwhile MMP-9 expression was also down-regulated in cKO mice (P<0.05). (3) the SUMO2/3 modification of MKK7 was reduced during M1 polarization, and MKK7 interaction with SENP3 was enhanced. Significantly up-regulated protein level of p-JNK and MMP-9 were verified in the M1 macrophages transfected with Flag-MKK7 K18R mutant (P<0.05). CONCLUSION SENP3 activates the MAPK/JNK pathway via de-SUMOylation of MKK7, regulates the M1/M2 polarization of macrophages and promotes the protein level of MMP-9, thus aggravating AAA formation.     

Effects of eosinophils deletion on sPLA2-X in bronchial asthma model mice

AIM To investigate the association between soluble phospholipase A2-X（sPLA2-X） and eosinophils in bronchial asthma， and to provide new insight and strategies for the treatment of bronchial asthma. METHODS Female Babl/c mice （n=48） of SPF grade and 6~8 weeks old were divided into 4 groups （with 12 in each group： healthy control group，asthma control group， eosinophil deletion group， and asthma /eosinophil deletion isotype control group）. The mouse model of bronchial asthma was constructed. The mice in healthy control group were intraperitoneally injected with saline on days 0， 7， and 14. The mice in other groups were intraperitoneally injected with 50 μg OVA and 2 mg aluminum hydroxide gel（soluble in 200 μL saline.On the 21st d and 26 th d， eosinophil deletion antibody （anti-CCR3） and isotype control were intraperitoneally injected and intranasally respectively， and then the lungs function test was conducted within 48 h after the end of nebulization.Half of the mice in each group were subjected to whole lung lavage， the remaining half were used for lung tissue section with HE staining， the whole blood was used to measure serum IgE， the supernatant of broncho alveolar lavage fluid （BALF） was used to measure cytokines， and total number of cells in the bronchoalveolar lavage fluid was analyzed for cell classification and flow cytometry. RESULTS （1）Compared with asthma control group，the airway and alveolar inflammatory responses in asthma/eosinophil deletion group was significantly alleviated.（2） Compared with asthma control group， anti-CCR3 successfully deleted eosinophils， and the percentage of eosinophils in bronchoalveolar lavage fluid in asthma/eosinophil deletion group was significantly reduced （P<0.05）.（3） The airway hyperresponsiveness in asthma/eosinophil deletion group was significantly decreased as compared with asthma control group（P<0.05）.（4） The levels of sPLA2-X in the serum and BALF was significantly reduced in asthma/eosinophil deletion group as compared with asthma control group（P<0.05）.（5）Compared with asthma control group，the levels of IL-4， IL-5， and IL-13 in the BALF of the mice in asthma/eosinophil deletion group were significantly reduced， and the serum level of IgE was also decreased （P<0.05）. CONCLUSION Eosinophils in bronchial asthma are importantly associated with sPLA2-X.     

Procaine regulates CXCR7 and affects AKT/STAT3 signaling pathway to inhibit viability,migration and invasion of bladder cancer cells

AIM To investigate the effects of procaine (PCA) and CXC chemokine receptor 7 (CXCR7) on the viability, migration and invasion of bladder cancer cells and its potential mechanism. METHODS Human bladder cancer RT4 cells were treated with PCA at different concentrations， and were divided into PBS group (without PCA treatment), PCA group (treated with 4 mmol/L PCA), siRNA negative control (si-Con) group (transfected with si-Con), CX?CR7 siRNA (si-CXCR7) group (transfected with si-CXCR7), PCA+pcDNA group (treated with 4 mmol/L PCA and transfected with pcDNA) and PCA+pcDNA-CXCR7 group (treated with 4 mmol/L PCA and transfected with pcDNA-CX?CR7). The siRNA and pcDNA were transfected into the RT4 cells by liposome method. The mRNA expression of CX?CR7 in the RT4 cells was detected by RT-qPCR. The cell viability was measured by CCK-8 assay. The invasion and migration abilities of the cells were detected by Transwell assays. The protein levels of CXCR7, AKT, STAT3, p-AKT and p-STAT3 were determined by Western blot . RESULTS Compared with PBS group, the viability, migration ability and invasion ability of the RT4 cells treated with PCA at different concentrations were significantly decreased (P<0.05), and the expression of CXCR7 at mRNA and protein levels in PCA group was also significantly decreased (P<0.05). Compared with si-Con group, the expression of CXCR7 at mRNA and protein levels in si-CXCR7 group was significantly decreased, and the viability, migration ability and invasion ability of the cells were also significantly decreased (P<0.05). Compared with PCA+pcDNA group, the expression of CXCR7 at mRNA and protein levels in PCA+pcDNA-CXCR7 group was significantly increased, and the viability, migration ability and invasion ability of the cells were also significantly increased (P<0.05). Compared with PBS group, the protein levels of p-AKT and p-STAT3 in PCA group were significantly decreased(P<0.05). Compared with PCA+pcDNA group, the protein levels of p-AKT and p-STAT3 in PCA+pcDNA-CX?CR7 group were significantly increased (P<0.05). No significant difference in the protein levels of AKT and STAT3 among the groups was observed. CONCLUSION Treatment with PCA inhibits the viability, migration and invasion of bladder cancer cells by inhibiting the expression of CXCR7. Over-expression of CXCR7 reverses this effect of PCA. Its mechanism may be related to AKT/STAT3 signaling pathway.     

Changes of nitric oxide synthase and nitric oxide in lung of smoking rats

AIM:To observe the changes of iNOS and eNOS in lung tissue and NO in bronchoalveolar lavage fluid (BALF) in smoking rats.METHODS:80 Wistar rats were divided into control, smoking group, L-NIL group and L-NAME group (rats were exposed to smoke and injected (i.p.) with selective iNOS inhibitor L-NIL or NOS inhibitor L-NAME). iNOS and eNOS protein levels in whole lung were detected by immunohistochemical staining, and NOS mRNA was quantified using RT-PCR. In addition, NO₂^-/NO₃^- was determined using Griess assay.RESULTS:The expression of iNOS mRNA and protein in smoking rats increased, the expression of eNOS mRNA and eNOS protein decreased, and the total cell count and the level of NO₂^-/NO₃^-in BALF increased(P＜0.05). In vivo, L-NIL reduced the total cell count and NO₂^-/NO₃^- in BALF (P＜0.05), while L-NAME had no effect on them.CONCLUSION:Cigarette smoke increased expression of iNOS mRNA and protein and decreased expression of eNOS mRNA and protein. The large amount of NO generated by iNOS may amplify inflammation in lung tissue.     

Effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE-/- mice

AIM To observe the effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE^-/- mice. METHODS Thirty-two ApoE^-/- mice were randomly divided into model group, high-dose (60 mg/kg) tanshinone ⅡA group, low-dose (30 mg/kg) tanshinone ⅡA group and simvastatin group, and C57BL/6J mice (n=8) were used as normal control group. The mice in normal control group were given the basic feeding, while the others were given high-fat diet. The mice in tanshinone ⅡA groups and simvastatin group were given corresponding drugs. The mice in normal control group and model group were intraperitoneally injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by automatic biochemistry analyzer. The liver tissues were stained with HE and oil red O. The contents of reactive oxygen species (ROS) and glutathione (GSH) in liver tissues of the mice were measured by commercially available kits. The liver glutathione peroxidase 4 (GPX4) and p53 were detected by immunohistochemical method. The protein and mRNA expression levels of ferroptosis-related factors GPX4, xCT/SLC7A11, p53 and ferritin heavy chain 1 (FTH1) were determined by Wes automatic Western blot quantitative analysis system and RT-qPCR. RESULTS Compared with normal control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.05 or P<0.01), and HDL-C did not change significantly. The fat vacuoles were clearly visible in liver tissue. The content of ROS in liver tissue was increased significantly,and GSH was decreased significantly (P<0.01). The mRNA and protein expression levels of p53 were increased significantly, and GPX4, xCT/SLC7A11 and FTH1 were decreased significantly (P<0.05 or P<0.01). Compared with model group, tanshinone ⅡA significantly decreased the serum levels of TC, TG and LDL-C (P<0.05 or P<0.01), and HDL-C did not change significantly. High-dose and low-dose tanshinoneⅡA also significantly decreased the degree of steatosis, and the size of lipid droplets. The content of ROS in liver tissues was decreased significantly, and GSH was increased significantly (P<0.01). The mRNA and protein expression levels of GPX4, xCT/SLC7A11 and FTH1 were increased significantly, and p53 were decreased significantly (P<0.05 or P<0.01). CONCLUSION Tanshinone ⅡA reduces liver lipid deposition and lipid peroxidation damage in ApoE^-/- mice, which may be related to the intervention of ferroptosis-related proteins in the liver cells.     

Effect of inhibiting lncRNA LINC01503 on viability,migration and invasion of lung cancer cells by targeted regulation of miR-335-5p

AIM To investigate the effects of long non-coding RNA (lncRNA) LINC01503 on the viability, migration and invasion of lung cancer cells and its mechanism. METHODS Human lung carcinoma H1299 cells were divided into si-NC group (transfected with si-NC), si-LINC01503 group (transfected with si-LINC01503), pcDNA group (transfected with pcDNA), pcDNA-LINC01503 group (transfected with pcDNA-LINC01503), miR-NC group (transfected with miR-NC), miR-335-5p group (transfected with miR-335-5p mimics), si-LINC01503+anti-miR-NC group (co-transfected with si-LINC01503 and anti-miR-NC), si-LINC01503+anti-miR-335-5p group (co-transfected with si-LINC01503 and anti-miR-335-5p), miR-NC+WT-LINC01503 group (co-transfected with miR-NC and WT-LINC01503), miR-NC+MUT-LINC01503 group (co-transfected with miR-NC and MUT-LINC01503), miR-335-5p+WT-LINC01503 group (co-transfected with miR-335-5p and WT-LINC01503) and miR-335-5p+MUT-LINC01503 group (co-transfected with miR-335-5p and MUT-LINC01503). The expression of miR-335-5p and LINC01503 was detected by RT-qPCR. Western blot was used to detect protein expression. MTT assay was used to detect cell viability. Transwell assay was used to detect the migration and invasion abilities. Dual-luciferase reporter assay was used to confirm the targeted relationship between LINC01503 and miR-335-5p. RESULTS Compared with normal tissues, the expression of LINC01503 was significantly increased in the lung cancer tissues, and the expression of miR-335-5p was significantly decreased (P<0.05). Compared with stage I/II , the expression level of LINC01503 in the lung cancer tissues of stage III/IV was significantly increased, and the expression of miR-335-5p was significantly decreased (P<0.05). The patients with high expression of LINC01503 had lower short-term survival rates than those with low expression of LINC01503 (P<0.05). Compared with normal human bronchial epithelial cell line BEAS-2B, the expression of miR-335-5p in lung cancer cell lines H1299, A549 and SPC-A-1 were significantly decreased, and the expression of LINC01503 was significantly increased (P<0.05). Over-expression of miR-335-5p and inhibition of LINC01503 expression inhibited the viability, migration and invasion of H1299 cells, and inhibited the protein expression of cyclin D1, matrix metalloproteinase-2 （MMP-2） and MMP-9 (P<0.05). LINC01503 targeted and regulated miR-335-5p expression, and interfering with miR-335-5p expression reversed the inhibitory effect of inhibiting LINC01503 expression on the viability, migration and invasion of H1299 cells. CONCLUSION Inhibition of lncRNA LINC01503 inhibits the viability, migration and invasion of lung cancer cells. The mechanism may be related to the targeted regulation of miR-335-5p.     

Activation of PPARs is involved in effect of bezafibrate on diabetic hepatopathy in mice

AIM: To investigate the effects of bezafibrate (BEZ) on diabetic hepatopathy in mice. METHODS: Diabetic hepatopathy model was established by a long-high-energy diet combined with streptozotocin (40 mg·kg^-1·d^-1× 5 d, ip), and then bezafibrate (75 mg·kg^-1·d^-1, ig) was supplemented for 4 weeks. Fasting blood glucose (FBG) was detected every week. The structure of liver was observed by HE staining, and the liver function was measured by observing the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Total cholesterol (TC), triglyceride (TG), insulin and HbA1c were determined by commercial kits, and then HOMA insulin resistance index (HOMA-IR) was calculated. The expression of peroxisome proliferator-activated receports (PPARs) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTS: After 7 days of treatment with streptozotocin, the FBG level of the mice exceeded 11.1 mmol/L. At the end of the experiment, the structural deformation of the hepatocytes (containing abundant fat vacuoles, infiltrated by inflammatory cells, etc.) was observed, with the increases in insulin, HbA1c, HOMA-IR, TC, TG, ALT and AST in diabetic mice (P<0.01). Meanwhile, the expression of PPARα, PPARβ and PPARγ at mRNA and protein levels was decreased (P<0.01). Bezafibrate treatment markedly attenuated the structural and functional damages of the liver in the diabetic mice (P<0.01), and reduced the levels of FBG, insulin, HbA1c, HOMA-IR, TC and TG (P<0.05). Bezafibrate also up-regulated the expression of PPARα, PPARβ and PPARγ (P<0.01). CONCLUSION: Bezafibrate attenuates hepatic injury in diabetic mice via the activation of PPARs-related signaling pathway.     

Mangiferin attenuates hypoxia/reoxygenation-induced injury of human myocardial cells

AIM To investigate the effect of mangiferin on hypoxia/reoxygenation (H/R)-induced injury of human myocardial cells and its mechanism. METHODS Human myocardial AC16 cells were divided into normal group, H/R group and H/R + mangiferin (50, 100 and 200 μmol/L) treatment groups. The mRNA and protein expression levels of Kelch-like epichlorohydrin-associated protein-1 (Keap-1), Bax, Bcl-2, caspase-3, caspase-9 and superoxide dismutase 2 (SOD2) were detected by RT-qPCR and Western blot, respectively. The protein expression of nuclear factor E2-related factor 2 (Nrf-2) in nucleus was determined by Western blot. The expression of microRNA-432-3p (miR-432-3p) was detected by RT-qPCR. The generation of reactive oxygen speciess (ROS) in the cells was measured by DCFH-DA probing. The cell viability was measured by CCK-8 assay. Apoptosis was analyzed by flow cytometry. RESULTS No significant difference in the expression of miR-432-3p and Keap-1 between normal group and H/R group was observed. Compared with normal group, the nuclear translocation of Nrf-2, the ROS level, and the mRNA and protein expression of Bax, caspase-3 and caspase-9 were significantly increased in H/R group （P<0.05）. The mRNA and protein expression of SOD2 and Bcl-2, and the cell viability significantly decreased in H/R group compared with normal group, while the apoptosis was increased significantly （P<0.05）. Treatment with mangiferin resulted in an increase in the miR-432-3p expression and a decrease in the ROS level, and the expression of Keap-1, Bax, caspase-3 and caspase-9 was also inhibited as compared with H/R group （P<0.05）. The Nrf-2 nuclear translocation, and the protein levels of SOD2 and Bcl-2 in mangiferin treatment groups were significantly increased as compared with H/R group （P<0.05）. The cell viability was increased significantly, and the apoptosis was decreased significantly in mangiferin treatment groups as compared with H/R group （P<0.05）. The effects of mangiferin in middle- and high-dose groups were better than those in low-dose group, and no significant difference between middle- and high-dose groups was found. CONCLUSION Mangiferin inhibits the decrease in myocardial cell viability and the apoptosis induced by H/R injury. The mechanism may be related to the up-regulation of Nrf-2 antioxidant stress effect via enhancing the expression of miR-432-3p.     

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.     

circRNA-42398 inhibits activation of hepatic stellate cells via TGF-?β1/Smads signaling pathway modulation

AIM To study the effect of mouse circular RNA-42398 (mmu_circ_42398) over-expression on the activation of hepatic stellate cells. METHODS Mouse hepatic stellate JS1 cells were cultured and randomly divided into control group, vector group and mmu_circ_42398 over-expression group.mmu_circ_42398 over-expression plasmid vector was constructed, and then transiently transfected into JS1 cells using Lipofectamine 2000. The cells were collected 48 h after transfection. Expression of mmu_circ_42398 was detected by RT-qPCR.The backsplice site of PCR products was verified by sequencing. The protein levels of α-smooth muscle actin (α-SMA), collagen type I （Col I), transforming growth factor β1(TGF-β1), Smad2, Smad3, p-Smad2 and p-Smad3 in the cells were determined by Western blot. RESULTS RT-qPCR results showed that the expression of mmu_circ_42398 was significantly increased after mmu_circ_42398 over-expression vector was transiently transfected into the JS1 cells (P<0.01). The protein expression levels of α-SMA and Col I were significantly decreased(P<0.01), and the phosphorylation levels of Smad2 and Smad3 were decreased significantly in mmu_circ_42398 over expression group (P<0.01). However, the protein expression levels of TGF-β1, Smad2 and Smad3 had no significant change (P>0.05). CONCLUSION mmu-circ-42398 inhibits the activation of hepatic stellate cells via TGF-β1/Smads signaling pathway modulation.     

Effect of resveratrol on autophagy and renal interstitial fibrosis in kidney of diabetic mice

AIMTo observe the effect of resveratrol (Res) on renal autophagy level and renal interstitial fibrosis in the mice with diabetes mellitus (DM), and to discuss the possible mechanism. METHODSThe wild-type C57BL/6 mice were randomly divided into 3 groups, including normal control (NC) group, DM group and Res group (8 in each group). The diabetic mouse model was established by injection of streptozotocin. After 8 weeks of successful replication of the diabetic model, Res was given to the mice in Res group by continuous gavage for 12 weeks, and then the mice in each group were sacrificed to detect the relevant biochemical parameters. The pathological changes of the kidney tissues were observed by HE staining and Masson staining. The levels of the proteins related to autophagy, epithelial-mesenchymal transition (EMT) and fibrosis were determined by Western blot. The mRNA expression of collagen type IV (Col IV), α-smooth muscle actin (α-SMA) and E-cadherin were detected by real-time PCR. RESULTSCompared with NC group, fasting blood glucose (FBG), kidney index (KI), serum creatinine, 24-hour urinary albumin excretion rate and 24-hour urine total protein were remarkably increased in DM group (P<0.05). The results of HE and Masson staining indicated that renal tissue presented fibrosis in DM group. The protein levels of E-cadherin, beclin-1, microtubule-associated protein 1 light chain 3-II (LC3-II) were reduced in DM group, while the levels of α-SMA, Col IV and Snail1 were increased (P<0.05). After intervention with Res for 12 weeks, all the relevant biochemical parameters and KI were reduced (P<0.05) except FBG (P>0.05), and renal fibrosis lesions were obviously alleviated. Compared with DM group, the protein levels of E-cadherin, beclin-1 and LC3-II were increased in Res group, but the protein expression levels of α-SMA, Col IV, Snail1 were reduced (P<0.05). Compared with DM group, the mRNA level of E-cadherin was increased in Res group , but the mRNA levels of Col IV and α-SMA were reduced (P<0.05). CONCLUSION Resveratrol significantly inhibits EMT and reduces renal interstitial fibrosis in diabetic mice, and its mechanism may be related to the promotion of renal autophagy.     

CHIP participates in high glucose-induced vascular endothelial cell injury

AIM To investigate the effects of carboxy terminus of heat shock protein 70-interacting protein (CHIP) on high glucose (HG)-induced vascular endothelial cell injury. METHODS Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with 5.5 mmol/L glucose (normal glucose， NG) or 25.5 mmol/L glucose (HG) for 24 h. Down-regulation of CHIP expression by RNA interference was conducted. Before the experiment, mannitol was used to eliminate the interference of osmotic pressure. Subsequently, the cells was divided into 4 groups: NG+siRNA NC group, NG+siRNA CHIP group, HG+siRNA NC group, and HG+siRNA CHIP group. Additionally, MTT assay and TUNEL staining were used to detect the viability and apoptosis. The level of endothelin-1 (ET-1) was measured by ELISA, and the level of reactive oxygen species (ROS) was detected by fluorescence probe dihydroethidium. The level of nitric oxide (NO), and the activity of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in the cells were detected by their respective kits. The mRNA expression of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) was detected by RT-qPCR. The protein levels of CHIP, NADPH oxidase (NOX) 2, NOX4, p38, p65, p-p38, p-p65, Bax and Bcl-2 were determined by Western blot. RESULTS Compared with NG+siRNA NC group, the cell viability was decreased, the apoptosis rate, the mRNA expression of IL-8 and MCP-1, and the level of ROS were increased (P<0.05), the activity of SOD was decreased (P<0.05), while the levels of ET-1, NO and iNOS and the protein levels of p-p38, p-p65 and Bax were increased in HG+siRNA NC group (P<0.05). Compared with HG+siRNA NC group, the inflammatory response, the oxidative stress, the apoptosis rate, and the protein levels of p-p38, p-p65 and Bax were significantly increased in HG+siRNA CHIP group (P<0.05). CONCLUSION Down-regulation of CHIP expression aggravates HG-induced vascular endothelial cell injury.     

Inhibitory effect of Wendan decoction on atherosclerotic plaque formation in ApoE-/- mice by regulating expression of membrane proteins involved in reverse cholesterol transport

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on reverse cholesterol transport. METHODS Eight-week-old apolipoprotein E gene knockout （ApoE^-/-） mice with high-fat diet and daily drug gavage were randomly divided into model group, simvastatin group, and low-, middle- and high-dose Wendan decoction groups, with 15 mice in each group. The C57BL/6 mice of the same age served as control group. The mice were weighed once every week. After 10 weeks, the mice were anesthetized with chloral hydrate. The serum were collected for lipid level examination. The atherosclerotic plaque buildup in aortic root and whole aorta was observed by HE staining and oil red O staining, respectively. The levels of proteins related to cholesterol transport, ATP-binding cassette transporter A1 （ABCA1） and caveolin-1 in the aorta, and scavenger receptor class B type I （SR-BI） and CD36 in the liver, were quantified by Western blot. RESULTS Wendan decoction at middle dose inhibited the increase in the body weight of ApoE^-/- mice fed with high-fat diet （P<0.05）. Wendan decoction at different doses significantly reduced the serum levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol in the ApoE^-/- mice （P<0.05 or P<0.01）, but had no effect on serum high-density lipoprotein cholesterol level （P>0.05）. Wendan decoction at different doses inhibited the formation of atherosclerotic plaques in whole aorta of the ApoE^-/- mice （P<0.05 or P<0.01）. Middle- and high-dose Wendan decoction significantly inhibited the formation of atherosclerotic plaques in the aortic root （P<0.05）. Bedsides, Wendan decoction at different doses increased the protein level of ABCA1 and decreased the protein level of caveolin-1 in the aorta of the ApoE^-/- mice （P<0.01）. Middle- and high-dose Wendan decoction increased the liver protein level of SR-BI in the ApoE^-/- mice （P<0.01）. However, Wendan decoction at different doses had no effect on the liver protein level of CD36 in the ApoE^-/- mice （P>0.05）. CONCLUSION Wendan decoction reduces the body weight, serum lipid levels and formation of atherosclerotic plaques in ApoE^-/- mice fed with high-fat diet, and its mechanism is related to up-regulation of ABCA1 protein level in the aorta and SR-BI protein level in the liver as well as down-regulation of caveolin-1 protein level in the aorta.