Regulatory role of CCN1 in lipopolysaccharide-induced mouse acute lung injury

AIM: To observe the cellular location and expression change of cysteine-rich protein 61 (CYR61/CCN1) in lung tissues of the mice with lipopolysaccharide (LPS) intratracheal instillation, and to clarify the regulatory role of CCN1 expression in mediating inflammatory response. METHODS: The expression change of CCN1 in the lung tissues in vivo was observed by the method of immunohistochemistry, and immunofluorescence was employed to certify the cellular location of CCN1 in bronchial epithelial cells. Bronchial epithelial 16HBE cells were cultured in vitro, and the expression of CCN1 under the condition of LPS stimulation was quantified by RT-qPCR and Western blot with or without specific inhi-bitors of ERK1/2, JNK, P38 and PI3K signaling pathways. The mRNA expression levels of interleukin-6 (IL-6), IL-8, transformrg growth factor-β (TGF-β) and vascular endothlial growth factor (VEGF) were measured by RT-qPCR under the condition of recombinant CCN1 exposure or transfection with CCN1-siRNA. RESULTS: The results of immunohistochemistry indicated that CCN1 was primarily located in bronchial epithelium. The results of immunofluorescence revealed that CCN1 was localized in the cytoplasm. The specific inhibitors of ERK1/2, JNK, P38 and PI3K signaling pathways reversed the up-regulation of CCN1 upon LPS stimulation. Exposure to recombinant CCN1 resulted in the up-regulation of IL-6, IL-8, TGF-β and VEGF, while LPS-related up-regulation of IL-6, IL-8, TGF-β and VEGF was blocked by silencing of CCN1. CONCLUSION: Airway epithelium-derived CCN1 is up-regulated under the condition of lung injury and the regulatory mechanism involves ERK1/2, JNK, P38 and PI3K signal transduction pathways. CCN1 acts as an inflammatory mediator in amplification of inflammatory response, laying theoretical basis for the potential molecular therapeutic target of acute lung injury.     

Sphingosine-1-phosphate receptor 2 attenuates influenza A virus-induced viral pneumonia through PI3K/AKT/eNOS pathway

AIM: To investigate the effects of sphingosine-1-phosphate receptor 2 (S1PR2) on influenza A virus-induced viral pneumonia.METHODS: The animal model of influenza A virus pneumonia was established by infecting wild-type C57BL/6 mice and S1pr2^-/- mice with influenza virus subtype FM1 mouse lung adaptable strain through nose drops. The pathological changes of the lung tissues of wild-type mice (model group), JTE-013 (S1PR2 effective antagonist)-challenged mice and S1pr2^-/- mice were observed, and the protein concentration, total cell number, and interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) levels were determined in the bronchoalveolar lavage fluid (BALF) at 4 d and 6 d after virus infection. The phosphorylation levels of AKT and eNOS in the lung tissues were determined by Western blot. RESULTS: Compared with the wild-type mice of control group, the influenza A virus pneumonia in JTE treatment group and S1pr2^-/- mice were more serious, and the protein concentration, total cell number and inflammatory cytokines in the BALF were remarkably increased. Moreover, the phosphorylation levels of AKT and eNOS, the downstream targets of PI3K, were significantly increased (P<0.01). CONCLUSION: S1PR2 mediates PI3K/AKT/eNOS signaling transduction pathway to regulate NO generation, and inhibit vascular permeability and inflammatory cytokine release, thus attenuating the viral pneumonia induced by influenza A virus.     

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.     

Dihydroartemisinin enhances radiotherapy efficiency in H22 cell tumor-bearing mice by regulating PI3K/AKT signaling pathway

AIM To study the effect of dihydroartemisinin （DHA） on the radiotherapy efficiency in hepatocellular carcinoma H22 cell tumor-bearing mice and the role of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （AKT） signaling pathway in this process. METHODS A model of H22 cell tumor-bearing mice was established. The mice was divided into model group, single radiotherapy group, 5-fluorouracil （5-FU） group, and low-, medium- and high-dose DHA groups. The body weight and tumor volume in each group were measured every other day. At the end of administration, blood was collected from the tail of the mice and the animals were killed by neck removal immediately. The synergistic effect of DHA on radiotherapy was determined, and tumor growth inhibitory rate was calculated. The degree of lymphocyte transformation and natural killer （NK） cell activity were measured by MTT, the serum levels of interleukin-2 （IL-2） and IL-4 were measured by ELISA, and the protein levels of PI3K, AKT and p-AKT were determined by Western blot. RESULTS The H22 cell tumor-bearing mouse model was successfully constructed. Compared with model group, the TGT₃ （tumor growth time to reach 3 times of volume） of single radiotherapy group was remarkably increased （P<0.05）, while tumor weight, lymphocyte transformation degree, NK cell activity, IL-2 and IL-4 levels, PI3K protein level and AKT phosphorylation level were remarkably decreased （P<0.05）. Compared with single radiotherapy group, TGT₃, EF （enhancement factor）, tumor inhibitory rate, lymphocyte transformation degree, NK cell activity, IL-2 level and IL-4 level were increased with the increase in DHA dose （P<0.05）, and the PI3K protein level and AKT phosphorylation level were decreased （P<0.05）. CONCLUSION DHA may enhance the immunity of tumor-bearing mice by inhibiting the activity of PI3K/AKT signaling pathway, thereby enhancing the efficacy of radiotherapy.     

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.     

Klebsiella pneumoniae capsular polysaccharide induces secretion of cytokines in bronchial epithelial cells via EGFR signaling pathway

AIM: To investigate the role of epidermal growth factor receptor (EGFR) in the secretion of inflammatory cytokines in human bronchial epithelial BEAS-2B cells induced by Klebsiella pneumoniae (KP) capsular polysaccharide (CPS). METHODS: KP was cultured in vitro, and the CPS was extracted. The BEAS-2B cells were stimulated with CPS at different concentrations, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in the supernatant were measured by ELISA. The phosphorylation level of EGFR was detected by Western blot at different time points after stimulation. After pretreatment of the BEAS-2B cells with EGFR inhibitor AG1478, the phosphorylation level of ERK was detected by Western blot, the nuclear translocation of P65 was detected by indirect immunofluorescence, and the levels of TNF-α and IL-8 in the supernatant of the cells were measured. Finally, the levels of TNF-α and IL-8 in the culture supernatant of CPS-stimulated cells were detected by ELISA after pretreated with ERK inhibitor PD98059 and NF-κB inhibitor PDTC. RESULTS: Exposure to CPS at 10 mg/L for 12 h significantly induced the BEAS-2B cells to secret TNF-α and IL-8. The phosphorylation levels of EGFR and ERK and the nuclear translocation of p65 in the BEAS-2B cells were significantly increased after CPS stimulation (P<0.05). The phosphorylation level of ERK and the nuclear translocation of p65 were significantly reduced in the cells pretreated with EGFR inhibitor AG1478. Furthermore, the levels of TNF-α and IL-8 in the supernatant were significantly decreased after pretreated with the inhibitors of EGFR, ERK and NF-κB. CONCLUSION: Klebsiella pneumonia capsular polysaccharide activates the ERK and NF-κB signaling pathways via EGFR, and then induced the secretion of inflammatory cytokines TNF-α and IL-8 in the bronchial epithelial cells, indicating that EGFR may be a key factor in the inflammatory response induced by KP infection.     

Adipolin/CTRP12 protects against LPS-induced ARDS by up-regulating alveolar epithelial sodium channel in mice

AIM: To investigate the effect of adipolin/CTRP12 in LPS-induced acute respiratory distress syndrome(ARDS) and its potential regulation on alveolar epithelial sodium channel(ENaC) in mice. METHODS: C57BL/6J mice(n=40) were randomly divided into control group, LPS group, adipolin group and wortmannin(PI3K inhibitor) group with 10 mice in each group using random number table. The pathological changes of the lung tissues were evaluated by HE staining. The alveolar fluid clearance(AFC) was measured by Evans blue-marked albumin, and the concentrations of total protein in bronchoalveolar lavage fluid(BALF) were assessed by bicinchoninic acid(BCA) method. In BALF, the levels of IL-1β and TNF-α were determined by ELISA, and the activity of myeloperoxidase(MPO) was detected by an MPO assay kit. The total cell counts and polymorphonuclear neutrophil(PMN) counts in the BALF were analyzed by Giemsa staining. The mRNA levels of α-ENaC were assessed by qPCR, while the protein levels of α-ENaC and p-Akt were determined by Western blot. RESULTS: Compared with control group, the classic ARDS pathological changes were observed in the mice in LPS group, manifesting by severe pathological lung injury(P<0.05), increases in W/D weight ratio, total protein levels, cell counts, MPO activitiy, and IL-1β and TNF-α levels in the BALF, and decrease in AFC(P<0.05), accompanied by down-regulated levels of α-ENaC and p-Akt in the lung tissues(P<0.05). The deteriorating effects triggered by LPS were significantly reversed by administration of adipolin. However, PI3K inhibitor wortmannin canceled the beneficial effects of adipolin on LPS-induced ARDS, as evidenced by aggravated lung injury, increased levels of W/D weight ratio, protein levels, cell counts, MPO activity, and IL-1β and TNF-α levels in the BALF(P<0.05), and decreased levels of AFC, α-ENaC and p-Akt in the lung tissues. CONCLUSION: Adipolin protects against LPS-induced ARDS in the mice by up-regulating α-ENaC and enhancing AFC via PI3K/Akt signal pathway.     

Nontypeable Hemophilus influenzae induces inflammatory response of bronchial epithelial cells via NF-κB activation and histone deacetylase activity reduction

AIM: To investigate the effect of nontypeable Hemophilus influenzae (NTHi) on the inflammatory response of bronchial epithelial cells, and to preliminarily explore its mechanism. METHODS: The human normal bronchial epithelial BEAS-2B cells were cultured, and the expression levels of interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant were measured by ELISA and RT-qPCR after infection with NTHi at MOI=10. The levels of IκBα and the phosphoacetylation of histones were determined by Western blot, and then the expression of histone deacetylase (HDAC) and the enzyme activity of HDAC were detected. The binding activity of NF-κB and IL-8 was measured by electrophoretic mobility shift assay and chromatin immunoprecipitation. Finally, the expression levels of GM-CSF and IL-8 were measured after pretreatment with NF-κB and HDAC inhibitors. RESULTS: The expression levels of IL-8 and GM-CSF in the culture supernatant of BEAS-2B cells were significantly increased (P<0.05) after infection with NTHi at MOI=10. In addition, infection with NTHi significantly down-regulated the expression of cytoplasmic IκBα and enhanced the DNA-binding activity of NF-κB. The phosphoacetylation of histones H3 and H4 and the binding of IL-8 and RNA polymerase II were also significantly increased after infection with NTHi. The expression level and enzyme activity of intracellular HDAC were also significantly reduced (P<0.05) after infection with NTHi. The expression levels of GM-CSF and IL-8 were significantly reduced after pretreatment with NF-κB inhibitor (P<0.05), while the secretion of IL-8 was significantly increased after pretreatment with HDAC inhibitor (P<0.05). CONCLUSION: NTHi inhibits the expression and activity of HDAC by activating the NF-κB signaling pathway, and promotes the secretion of IL-8 and GM-CSF in bronchial epithelial cells, thereby aggravating the inflammatory response.     

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.     

CYP450 epoxygenase/EET pathway attenuates adipose inflammation of obese mice through HIF-1α

AIM To investigate the effects of cytochrome P450 （CYP450） epoxygenase/epoxyeicosatrienoic acid （EET） pathway on insulin resistance in obese mice, and to explore the possible mechanisms. METHODS High-fat diet-induced obesity model was established in C57BL/6Cnc mice, and the obese mice were randomly divided into 3 groups, including obesity group （treated with saline; n=10）, EET group （treated with 11,12-EET; n=10） and EET inhibitor 14,15-epoxyeicosa-5（Z）-enoic acid （EEZE） group （n=10）. Normal C57BL/6Cnc mice （n=10） treated with saline served as control. Protein expression of CYP2J2 （one of CYP450 epoxygenases） and hypoxia-inducible factor-1α （HIF-1α） was measured by Western blot. Vessel-like structure was detected by immunofluorescence staining. The serum levels of insulin, tumor necrosis factor-α （TNF-α）, interleukin （IL）-1β, IL-6 and monocyte chemoattractant protein-1 （MCP-1） were measured by ELISA. RESULTS In obese mice, homeostasis model assessment of insulin resistance （HOMA-IR） values were increased, the protein level of CYP2J2 was reduced, and the protein level of HIF-1α was increased in adipose tissues as compared with the controls （P<0.05）. The serum levels of MCP-1, IL-1β, IL-6 and TNF-α were also significantly increased in obese mice （P<0.05）. After treatment with 11, 12-EET, the HOMA-IR values were decreased compared with vehicle-treated obese mice, HIF-1α expression levels were decreased in the adipose tissue, and the serum levels of MCP-1, IL-1β, IL-6 and TNF-α were reduced （P<0.05）. Immunohistochemical results of adipose tissue from vehicle-treated obese mice showed a marked decrease in vessel-like structures （CD31-positive） compared with normal control mice （P<0.05）. EET treatment significantly increased the newly formed vessel-like structures in the visceral adipose tissues of obese mice as compared with vehicle-treated obese mice （P<0.05）. CONCLUSION High-fat diet-induced obesity and insulin resistance are closely related to the CYP450 pathway. Exogenous EETs effectively decrease obesity-induced insulin resistance possibly through pro-angiogenesis and attenuation of hypoxia and inflammation.     

Inhibitory effects of paeonol on keratinocyte viability,cytokines secretion stimulated by IL-17A via STAT3 signaling pathway

AIM To observe the effect of paeonol on interleukin-17A （IL-17A）-induced human keratinocyte viability, cytokine secretion, and related signal transduction pathways. METHODS In vitro HaCaT cells stimulated by IL-17A （200 μg/L） were co-cultured with paeonol （200 mg/L and 100 mg/L） for 24 h. The cell viability was measured by CCK-8 assay. The Th1/Th2/Th17 cytokine （including IL-6, etc.） levels were measured by cytometric bead array assay. The IL-23 level was measured by ELISA. The mRNA expression of IL-23, IL-6, CXCL2, CXCL8, CCL20 and STAT3 was detected by real-time PCR, and Western blot was used to determine the protein levels of STAT3 and ERK1/2. RESULTS Paeonol significantly inhibited IL-17A-induced HaCaT cell viability （P<0.05）, as well as reduced IL-6 level. Meanwhile, paeonol decreased mRNA levels of IL-23, CXCL2, CXCL8, and CCL20. Paeonol also inhibited the expression of STAT3 at mRNA and protein levels. However, no significant effect of paeonol on ERK1/2 protein expression was observed. CONCLUSION Paeonol inhibits HaCaT cell viability and cytokine secretion induced by IL-17A, and its mechanism might be related to STAT3 singaling pathway.     

Effects of arsenic trioxide on T-bet/GATA3 signal pathway in MRL/lpr mice

AIM: To investigate the effect of arsenic trioxide (ATO) on T-bet/GATA3 signal pathway in MRL/lpr mice.METHODS: MRL/lpr mice and C57BL/6J mice at the age of 20 weeks were chosen and then divided in 2 different sub-groups, respectively. The mice in 2 sub-groups received ATO (0.4 mg·kg^-1·d^-1) and sodium chloride (NS, volume weight-determined) by intraperitoneal injection respectively for 2 months. Afterward, the spleens were isolated from the MRL/lpr and C57BL/6J mice under pathogen-free condition and the suspensions were prepared. The mRNA level of T-bet, GATA3, IFN-γ,IL-4 and the mRNA ratio of T-bet/GATA3 were detected by RT-qPCR. The protein expression of T-bet and GATA3 was determined by Western blot. The serum levels of IFN-γ and IL-4 were measured by ELISA.RESULTS: The mRNA and protein levels of T-bet, IFN-γ and the mRNA ratio of T-bet/GATA3 in NS group of MRL/lpr mice were higher than those in NS group of C57BL/6J mice (P<0.05). However, the GATA3 and IL-4 were lower in NS group of MRL/lpr mice in both mRNA and protein level (P<0.05). In MRL/lpr mice, the mRNA and protein levels of T-bet, IFN-γ and the mRNA ratio of T-bet/GATA3 were lower in ATO group compared with NS group (P<0.05), no difference was found in GATA3 and IL-4. No difference of the indexes mentioned above between ATO group and NS group in C57BL/6J mice was observed.CONCLUSION: ATO may affect the signaling pathway of T-bet/GATA3 to down-regulate the mRNA expression and the protein secretion of IFN-γ by decreasing the expression of T-bet in MRL/lpr mice.     

Effect of protease inhibitor bortezomib on treatment of rheumatoid arthritis by affecting IL-33/ST2 signaling pathway

AIM To investigate the effect of bortezomib, a protease inhibitor, on the treatment of rheumatoid arthritis （RA） and it mechanism, based on interleukin-33 （IL-33）/suppression of tumorigenicity 2 （ST2） signaling pathway. METHODS A total of 40 Wistar rats were randomly divided into 4 groups： control group, model group, and low- and high-dose bortezomib groups, with 10 rats in each group. In addition to control group, the rats in other groups were used to construct RA model. Bortezomib was given intraperitoneally at 0.2 mg/kg and 0.5 mg/kg in low- and high-dose bortezomib groups, respectively, while the rats in control group and model group were injected with the same amount of saline, once a day for 21 d. The general situation of the rats in each group was observed, the swelling degree of the foot was calculated, and the inflammation score was evaluated. HE staining was used to observe the pathological changes of ankle joint. The automatic biochemical analyzer was used to detect blood hemoglobin content, the total number of platelets （PLT）, serum creatinine （SCr） level and blood urea nitrogen （BUN） level. The serum levels of IL-6, tumor necrosis factor-α （TNF-α）, IL-33 and ST2 were measured by ELISA. The protein expression of IL-33 and ST2 in ankle tissues of each group was determined by Western blot. RESULTS On the 7th, 14th and 21th days after modeling, compared with control group, the degree of paw swelling in model group was significantly increased （P<0.05）. Compared with model group, the swelling degree of paw in low- and high-dose groups was decreased （P<0.05）. At the end of administration, compared with control group, the synovial cells in model group were increased and in disorder, with a lot of inflammatory exudates in the articular cavity, and the inflammatory score, the levels of PLT, SCr and BUN, the serum levels of IL-6, TNF-α, IL-33 and ST2, and the protein expression of IL-33 and ST2 in ankle tissues were significantly increased （P<0.05）. Compared with model group, the inflammatory exudates in the articular cavity of the rats in low- and high-dose bortezomib groups were decreased, and the inflammatory score, the levels of PLT, SCr and BUN, the serum levels of IL-6, TNF-α, IL-33 and ST2, and the protein expression of IL-33 and ST2 in ankle tissues were decreased （P<0.05）. CONCLUSION Bortezomib may reduce the inflammation and swelling of the joints in RA rats by regulating the IL-33/ST2 signaling pathway.     

Effects of IL-6 and AG490 on growth of diffuse large B-cell lymphoma and Burkitt lymphoma transplanted into nude mice

AIM: To observe the effects of interleukin-6 (IL-6) and AG490 on diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) transplanted into nude mice, and to explore the effects of STAT3 activation on growth of these kinds of lymphoma in nude mice and its related mechanisms. METHODS: The nude mouse models with DLBCL and BL were established by transplantation with OCI-LY8 cells and Raji cells, respectively, and were divided into 3 groups:control group, IL-6 group and AG490 group. The body weight of mice and tumor size were measured. Western blot and immunohistochemical staining were used to detect the protein levels of p-STAT3, survivin and vascular endothelial growth factor (VEGF), and real-time PCR was used to detect the mRNA expression of survivin and VEGF. RESULTS: The tumorigenic rate of 2 kinds of tumor cell lines in nude mice was 83.3% (25/30) totally. The tumorigenicity of OCI-LY8 cells (66.7%, 10/15) was significantly lower than that of Raji cells (100%, 15/15) (P<0.05). The tumor size and body weight on days 9 and 10 in IL-6 group increased as compared with the control group, and the total difference value of tumor size between day 1 and day 10 in IL-6 group was obviously larger than that in control group (P<0.05). The positive protein of p-STAT3 was found in the nucleus, while the positive expression of survivin and VEGF was found in the cytoplasm. As compared with control group, the expression of survivin and VEGF was significantly increased (P<0.05), while the protein level of p-STAT3 was not significantly increased in IL-6 group of DLBCL. The protein levels of p-STAT3 and VEGF were significantly decreased (P<0.05), while the expression of survivin did not significantly decreased in AG490 group of DLBCL. The p-STAT3 and VEGF levels significantly increased (P<0.05) in IL-6 group of BL, while the levels of 3 kinds of proteins significantly deceased (P<0.05) in AG490 group of BL, as compared with control group. No statistical difference of mRNA expression of survivin and VEGF among IL-6, AG490 and control groups was observed. CONCLUSION: IL-6 and AG490 affect the growth of DLBCL and BL through activation of STAT3 pathway. The activated STAT3 participates in pathogenesis and progress of DLBCL and BL by up-regulating the expression of survivin and VEGF.     

Inflammation driven activation of PI3K/Akt/Sp1 signaling pathway and endogenous H2S production aggravate intestinal injury in severe acute pancreatitis

AIM:To investigate the potential role of endogenous hydrogen sulphide (H₂S) in severe acute pancreatitis (SAP). METHODS:A rat model of SAP was used to evaluate the role of H₂S on intestinal motility by counting the number of fecal pellets and the effect of H₂S on the expression of inflammation-related molecule in intestine was investigated. The colonic muscle cells (CMCs) were treated with plasma of SAP rats, tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6), and the expression of cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), Sp1 and PI3K/Akt related proteins at mRNA and protein levels were determined by RT-qPCR, Western blot and immunohistochemical staining,respectively. The PI3K inhibitor LY294002 and the siRNA-Sp1 were used to suppress the activity of PI3K/Akt/Sp1 signaling pathway. RESULTS:H₂S facilitated an inhibitory effect on the intestinal motility and enhanced the inflammatory responses in SAP (P<0.05). The expression of CSE and CBS in CMCs was significantly increased after treatment with TNF-α or IL-6 (P<0.05). Blockage of the PI3K/Akt/Sp1 signaling pathway remarkably inhibited the synthesis of CSE and CBS in CMCs(P<0.05). CONCLUSION:Inflammation driven activation of PI3K/Akt/Sp1 signaling pathway and endogenous production of H₂S play a vital role in the pathogenesis of SAP.     

Effects of cholecystokinin octapeptide on expression of IL-1β, IL-6, IL-4 and IL-10 in lipopolysaccharide-attacked mice

AIM:To observe the effects of cholecystokinin octapeptide (CCK-8) on the expression of proinflammatory cytokines IL-1β, IL-6 and anti-inflammatory cytokines IL-10, IL-4 in LPS- attacked mice. METHODS:Kunming mice were randomly assigned and injected intraperitoneally with LPS alone or/and CCK-8 at different time points. The expression of IL-1β, IL-6, IL-10 and IL-4 in the serum and lung tissues were assayed by ELISA and RT-PCR. RESULTS:The expression of IL-1β, IL-6, IL-10 and IL-4 were upregulated in LPS-attacked mice. Pre-treatment of CCK-8 decreased both IL-1β and IL-6 expression and augmented IL-10 and IL-4 expression in LPS-attacked mice. CONCLUSIONS:CCK-8 exerts an anti-inflammatory effect by inhibiting the expression of IL-1β, IL-6 and increasing the expression of IL-10, IL-4 in LPS-attacked mice, which could alleviate the inflammatory response in lung tissue.     

Cordyceps militaris polysaccharide promotes reverse cholesterol transport in CETP-tg mice

AIM To verify whether Cordyceps militaris polysaccharide （CMPS） has the effect of promoting reverse cholesterol transport （RCT） in vitro and in vivo, and to explore the underlying mechanism. METHODS For in vivo experiments, RCT efficiency was detected in cholesterol ester transporter transgene （CETP-tg） mice by isotope tracer technique, and the plasma lipid levels were measured by enzyme method. For in vitro experiments, the residual lipid content after cholesterol efflux in RAW264.7 macrophage-derived foam cells was tested by oil red O staining and total cholesterol （TC） kit. Western blot was used to analyze the protein expression of the molecules involved in cholesterol transport, uptake and transformation in the foam cells and mice liver. RESULTS After 4 weeks of intragastric administration of CMPS, the concentrations of TC, low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C） in the plasma of CETP-tg mice were reduced by 24%, 23% and 22%, respectively. RCT efficiency of CETP-tg mice was accelerated and the appearance of ³H-cholesterol tracer in plasma, bile, intestine and feces was significantly increased in CMPS group. Meanwhile, the expression levels of cholesterol receptors scavenger receptor B1 （SR-B1） and LDL receptor （LDLR）, and cholesterol converting rate-limiting enzyme cholesterol 7α-hydroxylase A1 （CYP7A1） were upregulated by 105%, 71% and 58% in the liver of CMPS group, respectively. The results of in vitro experiments showed that CMPS preincubation promoted cholesterol efflux, decreased intracellular lipid and TC levels, and up-regulated the expression of peroxisome proliferator-activated receptor γ （PPARγ）-liver X receptor α （LXRα）-ATP-binding cassette transporter A1 （ABCA1）/ABCG1 signaling pathway related proteins in macrophage-derived foam cells. CONCLUSION CMPS promotes excess cholesterol efflux from peripheral macrophage-derived foam cells and accelerates its discharge through liver pathway. PPARγ-LXRα-ABCA1/ABCG1 pathway may be involved in the mechanism.     

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.