Alleviating effect of exenatide on ectopic lipid accumulation in skeletal muscle of ob/ob mice is independent on reducing body weight

AIM To investigate the alleviating effect of exenatide （Exe）， a glucagon-like peptide-1 （GLP-1） receptor agonist， on the ectopic lipid accumulation in skeletal muscle of ob/ob mice and its mechanism. METHODS Eight-week-old male ob/ob mice and their wild-type （WT） littermates were randomly divided into 3 groups， ob/ob group， ob/ob+Exe group and WT group， and treated with Exe at 24 nmol/kg or the same volume of saline intraperitoneally once daily for 4 weeks. The body weight， fasting blood glucose （FBG） and fat content were measured after the 4-week treatment. The oil red O staining and the quantification of triglyceride （TG） were performed on the skeletal muscle. The serum levels of TG， total cholesterol and free fatty acid （FFA） were also measured by ELISA. The expression levels of AMP-activated protein kinase （AMPK） and lipid metabolism-related proteins were determined by Western blot. Mouse myoblast C2C12 cells were used as an in vitro model to further investigate the effects of Exe. RESULTS As compared with the ob/ob mice treated with saline， 4-week Exe treatment did not reduce body weight， FBG， food intake and fat content in ob/ob mice （P>0.05）. However， serum FFA was decreased （P<0.05）. Oil red O staining and the quantification of TG showed that 4-week Exe treatment significantly attenuated the ectopic lipid accumulation in the skeletal muscle of ob/ob mice （P<0.05）. The results of Western blot showed that the levels of phosphorylated AMPK （p-AMPK） and lipolysis-related proteins were up-regulated， while the lipid synthesis-related proteins were down-regulated by Exe （P<0.05）. Treatment with Exe alleviated the lipid accumulation in the C2C12 cells induced by sodium palmate （P<0.05）， and the effects of Exe on the levels of p-AMPK and lipid metabolism-related proteins in the C2C12 cells were consistent with those in the ob/ob mice （P<0.05）. Treatment with Exe also up-regulated the protein expression of glucose transporter 4 and improved the ability of glucose uptake in the C2C12 cells （P<0.05）. CONCLUSION Short-term Exe treatment attenuates the ectopic lipid accumulation in skeletal muscle of ob/ob mice by up-regulating lipolysis-related proteins and down-regulating lipid synthesis-related proteins， which is independent on body weight loss.     

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.     

Geniposide attenuates cognitive dysfunction in sleep-deprived rats by inhibiting TLR4/NF-κB signaling pathway

AIM To investigate the effects of geniposide （Gen） on Toll like receptor 4/nuclear factor-κB （TLR4/NF-κB） signaling pathway and cognitive dysfunction in sleep deprived rats. METHODS Wistar rats （n=120） were randomly divided into normal control （NC） group， model （M） group， low-dose （5 g·kg^-1·d^-1） Gen （Gen-L） group， medium-dose （10 g·kg^-1·d^-1） Gen （Gen-M） group， high-dose （20 g·kg^-1·d^-1） Gen （Gen-H） group and Gen-H+LPS （0.4 mg·kg^-1·d^-1， tail vein injection） group. After 7 days of intervention， the sleep deprivation model of rats in M group， Gen-L， Gen-M， Gen-H and Gen-H+LPS group was established by improved small platform water environment. The escape latency of Morris water maze experiment and the behavior correct rate of Y maze experiment were measured. The serum levels of S100B and neuron-specific enolase （NSE）， and the levels of interleukin-1β （IL-1β）， IL-6 and tumor necrosis factor-α （TNF-α） in hippocampus were detected by ELISA. The mRNA levels of TLR4 and NF-κB p65 were detected by RT-qPCR， and the protein levels of TLR4 and NF-κB p65 were determined by Western blot. RESULTS Compared with NC group， the escape latency， the serum levels of S100B and NSE， the hippocampal levels of IL-1β， IL-6 and TNF-α， and the mRNA and protein expression of TLR4 and NF-κB p65 were increased significantly in M group （P<0.01）， and the behavior correct rate was decreased significantly （P<0.01）. Compared with M group， the escape latency， the hippocampal levels of IL-1β， IL-6 and TNF-α， and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were decreased significantly in Gen-L， Gen-M and Gen-H groups （P<0.01）， and the behavior correct rate was increased in turn （P<0.01）. Compared with Gen-H group， the escape latency， the serum levels of S100B and NSE， the hippocampal levels of IL-1β， IL-6 and TNF-α， and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were increased significantly in Gen-H+LPS group （P<0.01）， and the behavior correct rate was decreased significantly （P<0.01）. CONCLUSION Geniposide may inhibit the TLR4/NF-κB p65 signaling pathway to effectively improve cognitive function in sleep-deprived rats and reduce hippocampus inflammation.     

Effect of Tripterygium wilfordii multiglucoside on intestinal flora and immune function in IgA nephropathy rats based on C1GALT1/Cosmc pathway

AIM To investigate the effects of Triptergium wilfordii multiglucoside （TWM） on intestinal flora and immune function in IgA nephropathy （IgAN） rats based on core 1 β1，3-galactosyltransferase （C1GALT1） and its chaperone protein Cosmc （C1GALT1/Cosmc pathway）. METHODS The rat model of IgAN was established， and the animals were randomly divided into model group （IgAN group）， dexamethasone （Dex） group and TWM group. Normal rats served as normal control （NC） group. The levels of serum creatinine （SCr） and blood urea nitrogen （BUN）， 24-hour urinary total protein （24 h UTP） and the number of urinary red blood cells were measured by automatic biochemical analyzer. The levels of serum IgA1， and plasma tumor necrosis factor-α （TNF-α）， B-cell activating factor （Baff） and interleukin-17 （IL-17） were detected by ELISA. The level of galactose-deficient IgA1 （Gd-IgA1） was detected by Vicia villosa lectin affinity ELISA. The intestinal colony was cultured in selective bacterial medium. The ratio of CD4⁺ CD25⁺ regulatory T cells （Treg） to CD4⁺ T cells （Treg proportion） in peripheral blood mononuclear cells （PBMC） was detected by flow cytometry.Western blot was used to determine the protein expression of C1GALT1 and Cosmc in intestinal mucosa. RESULTS Compared with NC group， 24 h UTP， the number of urinary red blood cells， SCr， BUN， serum IgA1 and Gd-IgA1， the numbers of Enterobacteriaceae， Enterococcus and Bacteroides， and the levels of TNF-α， Baff and IL-17 in plasma in IgAN group were significantly increased （P<0.05）， while the numbers of Bifidobacteria and Lactobacilli， the Treg proportion in PBMC， and the protein expression levels of C1GALT1 and Cosmc in intestinal mucosa were significantly decreased （P<0.05）. Compared with IgAN group， 24 h UTP， the number of urinary red blood cells， SCr， BUN， serum IgA1 and Gd-IgA1， the numbers of Enterobacteriaceae， Enterococcus and Bacteroides， and the levels of TNF-α， Baff and IL-17 in plasma in Dex group and TWM group were significantly reduced （P<0.05）， and those in TWM group were lower than those in Dex group （P<0.05）. Moreover， the numbers of Bifidobacteria and Lactobacilli， the Treg proportion in PBMC， and the protein expression levels of C1GALT1 and Cosmc in intestinal mucosa were significantly elevated （P<0.05）， and those in TWM group were higher than those in Dex group （P<0.05）. CONCLUSION TWM reduces the abnormal glycosylation level of IgA in IgAN rats by promoting the activation of C1GALT1/Cosmc pathway， and attenuates the intestinal flora disorder and immune dysfunction in IgAN rats， thus exerting the therapeutic effect.     

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.     

Role and potential mechanism of fecal microbiota transplantation in treatment of chronic hepatitis B

AIM To investigate the effect of fecal microbiota transplantation （FMT） on the treatment of chronic hepatitis B （CHB） and the potential mechanism. METHODS Fifty C57BL/6J mice （6~8 weeks old） were divided into 5 groups： control group， CHB group， entecavir （ETV） group， comprehensive treatment （ETV+FMT， EFMT） group， and blocker （TAK-242+ETV+FMT， EFMT-TAK） group. The mice in each group were given corresponding treatment. The general condition of the mice was observed daily， and fecal specimens were kept every 10 d. The mice were sacrificed after 12 weeks， and the liver tissues and blood samples were collected. HE staining was used for histological scoring. Serum hepatitis B surface antigen （HBsAg） and interleukin-18 （IL-18） levels were measured by ELISA. Toll-like receptor 4 （TLR4） expression was detected by flow cytometry. Intestinal flora diversity was analyzed by high-throughput sequencing. RESULTS （1） Compared with control group， the body weight of the mice in CHB group was significantly reduced （P<0.05）. The body weight loss of the mice in ETV group， EFMT group and EFMT-TAK group was reversed to some extent as compared with CHB group （P<0.05）. （2） The histological score of the mice in CHB group was significantly higher than that in control group （P<0.05）. The score in ETV group was lower than that in CHB group （P<0.05）. The scores in EFMT group and EFMT-TAK group were lower than that in ETV group （P<0.05）， and that in EFMT-TAK group had a further downward trend compared with EFMT group （P<0.05）. （3） Compared with control group， the serum level of HBsAg in the CHB mice was significantly increased （P<0.05） and decreased after ETV treatment （P<0.05）. The HBsAg level in both EFMT group and EFMT-TAK group was significantly lower than that in ETV group （P<0.05）. （4） The IL-18 level in CHB group was significantly higher than that in control group （P<0.05）. After ETV treatment， the IL-18 level was decreased （P<0.05）， and that in both EFMT group and EFMT-TAK group was decreased more than that in ETV group （P<0.05）. （5） TLR4 expression in CHB group was higher than that in control group （P<0.05）， that in ETV group was lower than CHB group （P<0.05）， and that in EFMT group was further decreased （P<0.05）. （6） The heat map analysis at the class level showed that the abundances of Gammaproteobacteria， Deltaproteobacteria and Negativicutes in CHB group were significantly higher than those in control group， and those of Deltaproteobacteria and Negativicutes in EFMT group were close to those in control group. The heat map analysis at the family level indicated that the abundances of Burkholderiaceae， Desulfovibrionaceae and Veillonellaceae in CHB group were significantly higher than those in control group， while those in ETV group and EFMT group gradually approached normal levels. The α diversity index in CHB group was significantly decreased， while the diversity in ETV group was increased， that in EFMT group was further increased， and that in EFMT-TAK group was the highest. CONCLUSION FMT plays an active role in the treatment of CHB. The mechanism may be related to reducing the level of IL-18 and improving the structure and diversity of intestinal flora. The TLR4 signaling pathway is involved.     

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.     

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.     

RXRα agonist bexarotene inhibits atherosclerosis in ApoE-/- mice by regulating TGF-β1/Smad2 signaling pathway

AIM To observe the effect of retinoid X receptor α （RXRα） agonist bexarotene （Bex） on the proliferation of transforming growth factor β1 （TGF-β1）-induced vascular smooth muscle cells （VSMCs） and atherosclerosis in apolipoprotein E knockout （ApoE^-/-） mice， and to explore the underlying mechanism. METHODS Ten C57BL/6 mice were selected as normal control group， and 30 ApoE^-/- mice were randomly divided into 3 groups： ApoE^-/- group， ApoE^-/-+Bex5 （5 mg·kg^-1·d^-1 Bex） group and ApoE^-/-+Bex10 （10 mg·kg^-1·d^-1 Bex） group. Bex was intragastrically given once a day for 8 weeks. The levels of triglyceride （TG） and total cholesterol （TC） were determined by oxidase method， and select masking method was used to determine serum levels of low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C）. The protein levels of TGF-β1， p-Smad2 and Smad2 were determined by Western blot. HE staining was used to observe the intima of the thoracic aorta. The VSMCs were cultured with tissue patch method， and the proliferation of VSMCs was measured by BrdU incorporation method. RESULTS The serum levels of TG， TC and LDL-C， and the expression of TGF-β1 and p-Smad2 in thoracic aorta in ApoE^-/- group were significantly higher than those in C57BL/6 group （P<0.01）. Bex increased p-Smad2 protein level in thoracic aorta in a dose-dependent manner， inhibited the intimal plaque formation and vascular medial proliferation， and decreased the plaque area in ApoE^-/- mice （P<0.01）. No significant difference in serum levels of TG， TC， HDL-C and LDL-C， and TGF-β1 and Smad2 expression in thoracic aorta among ApoE^-/- group， ApoE^-/-+Bex5 group and ApoE^-/-+Bex10 group was observed. TGF-β1 （0.1~10 μg/L） promoted the proliferation of VSMCs， while Bex （10^-9~10^-7 mol/L） inhibited TGF-β1 （5 μg/L）-induced proliferation of VSMCs in a concentration-dependent manner. Bex （10^-7 mol/L） synergistically promoted the protein level of p-Smad2 in VSMCs induced by TGF-β1 （P<0.01）， but inhibited TGF-β1-induced nuclear translocation of p-Smad2. CONCLUSION RXRα agonist Bex inhibits the formation of atherosclerosis in ApoE^-/- mice， and its mechanism may be related to the regulation of TGF-β1/Smad2 pathway.     

Curcumin attenuates rat lung ischemia/reperfusion injury by interfering with autophagy

AIM To investigate the role of curcumin （CUR） in lung ischemia/reperfusion （I/R） injury （LIRI） and its relationship with autophagy. METHODS 40 SD rats were randomly divided into sham operation （sham） group， I/R group， solvent （DMSO） group， CUR group and CUR+rapamycin （CUR-Rap） group. The rats were intraperitoneally injected with normal saline， DMSO， CUR or CUR+Rap before operation. After the rat LIRI model was established， the lung tissues were taken to measure W/D， TLW， IAR， and the contents of SOD and MDA were also measured to indicate the oxidative stress level. Light and electron microscopes were used to observed the morphology and ultrastrucure of lung tissues. The expression levels of autophagy-related proteins were determined by Western blot to evaluate autophagy levels. RESULTS Compared with sham group， wet weight/dry weight （W/D）， total lung water （TLW）， injured alveoli rate（IAR） and malondialdehyde （MDA） content in all other groups were increased， superoxide dismutase （SOD） activity was decreased， the levels of autophagy were increased （P<0.05）， and lung tissue injury and cell ultrastructural damage were aggravated in CUR group. Compared with DMSO group， W/D， TLW and IAR and MDA content were decreased， SOD activity was decreased， autophagy levels were also decreased （P<0.05）， and lung tissue and cell ultrastructural damage were attenuated. Compared with CUR group， W/D， TLW， IAR and MDA content were increased， SOD activity declined， the autophagy levels were increased （P<0.05）， and damage of lung tissues and cells were more serious in CUR-Rap group. CONCLUSION Curcumin attenuates the lung I/R injury in rats， and its mechanism may be related to the reduction of oxidative stress and the inhibition of autophagy.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC， pcDNA-FEZF1-AS1， anti-miR-NC， anti-miR-363-3p， miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1， Ki67 and cleaved caspase-3. RESULTS Compared with control group， the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）， and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group， the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）， the apoptotic rate was significantly reduced （P<0.05）， the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）， and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group， the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）， the apoptotic rate was significantly reduced （P<0.05）， the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）， and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group， the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）， the apoptotic rate was significantly increased （P<0.05）， the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）， and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

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.     

Inhibitory effect of Wendan decoction on formation of foam cells induced by ox-LDL

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on formation of foam cells. METHODS The optimal concentrations of Wendan decoction without cytotoxity to cells were selected by MTT assay. After Wendan decoction treatment， the formation of foam cells was examined by oil red O staining. The cholesterol efflux， cholesterol level， free cholesterol level and cholesterol esterification rate were analyzed using cholesterol efflux assay， total cholesterol assay and free cholesterol assay. The expression levels of macrophage membrane proteins， including CD36， scavenger receptor class A （SR-A）， ATP-binding cassette transporter A1 （ABCA1） and scavenger receptor class B type I （SR-BI）， were quantified by Western blot. RESULTS The optimal concentrations of Wendan decoction without cytotoxity to the cells were 0~6 g/L. Wendan decoction at the concentrations of 1.5， 3 and 6 g/L were selected for the experiments. Wendan decoction at these concentrations inhibited the formation of foam cells induced by oxidized low-density lipoprotein （ox-LDL）， and reduced the accumulation of intracellular lipids in a concentration-dependent manner （P<0.05 or P<0.01）. Wendan decoction also reduced intracellular total cholesterol level， cholesterol ester level and cholesterol esterification rate （P<0.05 or P<0.01）， promoted efflux of intracellular cholesterol （P<0.01）， and decreased the protein level of CD36 in THP-1 cell-derived macrophages （P<0.01） in a concentration-dependent manner. Wendan decoction at the concentration of 6 g/L significantly reduced the protein level of SR-A in THP-1 cell-derived macrophages （P<0.05）. At the concentrations of 3 and 6 g/L， Wendan decoction significantly increased the protein levels of ABCA1 and SR-BI in THP-1 cell-derived macrophages （P<0.05 or P<0.01）. CONCLUSION Wendan decoction significantly inhibits ox-LDL-induced formation of foam cells by reducing cholesterol deposition and promoting cholesterol efflux， and its mechanism may be related to the down-regulation of CD36 and SR-A and the up-regulation of ABCA1 and SR-BI.     

Effects of oxidative stress and inflammatory response on kidney injury in mice with hyperthyroidism

AIM To explore the effects of oxidative stress and inflammatory response on kidney injury induced by hyperthyroidism in mice. METHODS Forty male Kunming mice were randomly divided into control group （n=20） and L-thyroxine （T4） group （n=20）. The mice in T4 group were intraperitoneally injected with T4 diluent at a dose of 1 mg/kg to induce hyperthyroidism， and those in control group were injected with normal saline of the same volume. After 7 weeks， the mice were weighed and dissected， the kidneys were removed and weighed， and the length of tibia was also measured. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the kidney tissues were detected. The pathological changes of the kidney tissues were observed by HE staining. The levels of 4-hydroxynonenal （4-HNE）-modified proteins， interleukin-1 receptor-associated kinase 1 （IRAK1） and tumor necrosis factor receptor-related factor 6 （TRAF6） were determined by Western blot and immunohistochemistry. RESULTS Compared with control group， the body weight of the mice was decreased， while the kidney size and weight were increased significantly in T4 group. In addition， the ratios of kidney weight/body weight and kidney weight/tibia length were also increased （P<0.05）. In T4 group， the renal tubules were enlarged， and the epithelial cells of renal tubules were swollen and exfoliated， with vacuolar degeneration. Furthermore， reduced SOD activity， and increased MDA content and 4-HNE-modified proteins were found in T4 group， all of which were related to oxidative stress （P<0.05）. The levels of inflammation-related proteins IRAK1 and TRAF6 were significantly increased in T4 group （P<0.05）. CONCLUSION Excessive T4 may lead to kidney hypertrophy and injury in mice， and the mechanism may be related to oxidative stress and inflammatory response.     

Effects of tripterine on viability and apoptosis of keloid fibroblasts by regulating GINS2

AIM To explore the effects of tripterine on the viability and apoptosis of keloid fibroblasts （KFB） and its molecular mechanism. METHODS The KFB were treated with tripterine at low， medium and high doses. The cell viability and apoptosis were measured by CCK-8 assay and flow cytometry， respectively. RT-qPCR and Western blot were applied to determine the expression of GINS complex subunit 2 （GINS2）. The KFB were divided into si-NC group， si-GINS2 group， Exp+pcDNA group and Exp+pcDNA-GINS2 group， and the changes of cell viability and apoptosis were measured using the above methods. RESULTS After treatment with tripterine， the viability of KFB was decreased， the apoptotic rate was increased， and GINS2 expression was decreased in a dose-dependent manner （P<0.05）. After knockdown of GINS2， the viability of KFB was decreased， and the apoptotic rate was increased （P<0.05）. Over-expression of GINS2 partially reversed the effect of tripterine on the viability and apoptosis of KFB （P<0.05）. CONCLUSION Tripterine inhibits KFB viability and promotes the apoptosis by down-regulating GINS2.     

Expression of histone chaperone ASF1B in prostate cancer cells and its effect on cell viability in vitro

AIM To investigate the expression of histone chaperone anti-silencing function 1B （ASF1B） in prostate cancer cells and its effect on cell viability in vitro. METHODS Human prostate cancer PC-3 cells were used， and knockdown of ASF1B was conducted by small interfering RNA （siRNA） transfection into the cells. The cells were divided into control group， siRNA negative control vector （mock） group and siRNA-ASF1B group. The viability of the PC-3 cells treated with ASF1B-siRNA for 12， 24 and 48 h was measured by MTT assay. Flow cytometry was used to analyze cell apoptosis and cell cycle distribution. The mRNA expression of apoptosis-related molecules was detected by RT-qPCR， and the expression levels of MAPK/JNK/ERK signaling pathway-related proteins were determined by Western blot. RESULTS The protein level of ASF1B in the normal cells （benign prostatic hyperplasia） was significantly lower than that in the PC-3 cells （P<0.01）. Compared with control group and mock group， the protein expression level of ASF1B in the PC-3 cells transfected with siRNA-ASF1B plasmid and the viability of the PC-3 cells were significantly decreased （P<0.01）. The apoptotic rate of the PC-3 cells transfected with siRNA-ASF1B plasmid was significantly higher than that in control group （P<0.01）， and the cell cycle was arrested at G₁ phase. The mRNA levels of p53， caspase-3， Bax and PARP-1 in the PC-3 cells transfected with siRNA-ASF1B plasmid were up-regulated compared with those in control group and Mack group （P<0.01）. In addition， the protein levels of MAP2K4 and p-JNK in the PC-3 cells in siRNA-ASF1B group were significantly higher than those in mock group （P<0.01）， while the protein level of p-ERK was significantly lower than that in mock group （P<0.01）. CONCLUSION ASF1B silencing induces G₁ arrest and promotes apoptosis of PC-3 cells. Activating MAPK/JNK/ERK signaling pathway may be a possible contributor to the anti-prostate cancer effect of siRNA-ASF1B.     

Effect of continuous renal replacement therapy on mRNA expression of autophagy-related molecules and prognosis in patients with acute kidney injury

AIM To explore the effect of continuous renal replacement therapy （CRRT） on mRNA expression of autophagy-related molecules and the prognosis in the patients with acute kidney injury （AKI）. METHODS A total of 174 patients from our hospital who were diagnosed to have AKI and underwent CRRT between February 2015 and March 2018 were involved in this study. The plasma levels of interleukin-1β （IL-1β） and interleukin-6 （IL-6）， the serum creatinine （SCr） level， and the mRNA expression levels of autophagy-related molecules， including microtubule-associated protein 1 light chain 3-II （LC3-II）， autophagy-related protein 5 （Atg5） and beclin-1， in the monocytes from peripheral blood were compared before and after CRRT. According to the survival of AKI patients after 4 weeks of CRRT， the enrolled patients were divided into death group （n=43） and survival group （n=131）， and the mRNA expression levels of the above molecules were compared between the 2 groups. RESULTS After CRRT treatment， the plasma levels of IL-1β and IL-6， the level of SCr， and the mRNA expression levels of LC3-II， Atg5 and beclin-1 in the monocytes were significantly lower than those before CRRT treatment （P<0.05）. The mRNA expression levels of LC3-II， Atg5 and beclin-1 in death group were significantly higher than those in survival group （P<0.05）. The positive correlation between SCr and IL-1β， IL-6， LC3-II or beclin-1 was observed （P<0.05）， and no correlation between SCr and Atg5 was found （P>0.05）. CONCLUSION CRRT decreases the mRNA expression levels of autophagy-related molecules in the patients with AKI and reduces the autophagy activity， which is protective for the patients. These autophagy-related molecules may be applied as a potential markers to predict the prognosis of CRRT.     

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.