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 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.     

PCDH10 inhibits proliferation of breast cancer MDA-MB-231 cells by NF-κB/cyclin D1 signaling pathway

AIM To investigate the inhibitory effect of protocadherin 10 （PCDH10） on proliferation of human breast cancer cells and its possible mechanism. METHODS RT-PCR was used to measure the mRNA expression levels of PCDH10 in 4 human breast cancer cell lines and normal mammary epithelial MCF-10A cells. The breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed by recombinant lentivirus （pLV-PCDH10） infection， and blank control （blank） group and negative control （pLV-NC） group were also set up. The cell proliferation ability was detected using methyl thiazolyl tetrazolium （MTT） and colony formation experiments. The cell cycle was detected by flow cytometry. Western blot was used to determine the expression of cyclin D1， cyclin-dependent kinase 4 （CDK4）， nucear factor-κB p65 subunit （NF-κB p65） and NF-κB inhibitor α （IκBα）. RESULTS The mRNA expression levels of PCDH10 in 4 breast cancer cell lines were lower than that in normal mammary epithelial MCF-10A cells （P<0.05）. A breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed successfully. Compared with negative control group， PCDH10 overexpression significantly inhibited cell proliferation， induced cell cycle arrest at G₁ phase， down-regulated the expression of cyclin D1 and CDK4， and decreased phosphorylation of NF-κB p65 and IκBα（P<0.05）. CONCLUSION PCDH10 inhibits the proliferation and blocks cell cycle progression of breast cancer cells by targeting NF-κB/cyclin D1 signaling pathway.     

Scutellarin affects LPS-induced oxidative stress and apoptosis of glomerular epithelial cells by up-regulating miR-7-5p

AIM To investigate the effect of scutellarin （SCU） on oxidative stress and apoptosis induced by lipopolysaccharide （LPS） in human glomerular epithelial cells and its mechanism. METHODS Human glomerular epithelial cells were cultured in vitro， and were treated with LPS （1.0 mg/L） to establish a cell injury model. The cells were divided into normal control （NC） group， LPS group， NC+SCU group， LPS+SCU group， LPS+miR-NC group， LPS+microRNA-7-5p （miR-7-5p） group， LPS+SCU+anti-miR-NC group and LPS+SCU+anti-miR-7-5p group. Cell viability was detected by CCK-8 assay. Apoptosis was detected by flow cytometry. The intracellular malondialdehyde （MDA） content and superoxide dismutase （SOD） activity， and lactate dehydrogenase （LDH） activity in the cell culture supernatant were determined by kit. RT-qPCR was used to detect the expression level of miR-7-5p. RESULTS Compared with NC group， the cell viability， miR-7-5p expression and SOD activity in LPS group were significantly reduced， and the apoptotic rate， MDA content and LDH activity were significantly increased （P<0.05）. Compared with LPS group， the cell viability， miR-7-5p expression and SOD activity in LPS+SCU group were significantly increased， and the apoptotic rate， MDA content and LDH activity were significantly reduced （P<0.05）. Compared with LPS+miR-NC group， the cell viability and SOD activity in LPS+miR-7-5p group were significantly increased， and the apoptotic rate， MDA content and LDH activity were significantly reduced （P<0.05）. Compared with LPS+SCU+anti-miR-NC group， the cell viability and SOD activity in LPS+SCU+anti-miR-7-5p group were significantly reduced， and the apoptotic rate， MDA content and LDH activity were significantly increased （P<0.05）. CONCLUSION Scutellarin inhibits LPS-induced oxidative stress damage and apoptosis in glomerular epithelial cells via up-regulating miR-7-5p expression.     

Protective effect of curcumin on MRL/lpr mice with lupus nephritis and its mechanism

AIM To observe the effect of curcumin （Cur） on lupus nephritis （LN） and its possible mechanism. METHODS Thirty 10-week-old MRL/lpr lupus mice were randomly divided into MRL/lpr group， Cur-L and Cur-H group with 10 mice in each group， and C57BL/6 mice （n=10） served as normal control （NC） group. The mice in Cur-L group and Cur-H group were given intragastric administration of Cur at 100 and 200 mg·kg^-1·d^-1 for 12 weeks， respectively， and the same volume of normal saline was given to the mice in NC group and MRL/lpr group. The urine protein was detected， and the morphological changes of the renal tissue were observed by HE staining after treatment. The levels of serum creatinine （SCr）， blood urea nitrogen （BUN） and serum anti-double-stranded DNA （dsDNA）， and interleukin-1β （IL-1β）， IL-6 and tumor necrosis factor-α （TNF-α） levels in serum and renal tissues were detected. The protein levels of p-IκB， NF-κB， NLRP3 and caspase-1 in the renal tissues were determined by Western blot. RESULTS Compared with MRL/lpr group， the content of urine protein in Cur groups was significantly reduced， and the renal injury was relieved. The SCr， BUN， serum anti-dsDNA， and the serum and renal levels of IL-1， IL-6 and TNF-α were all significantly reduced， and the protein levels of p-IκB， NF-κB， NLRP and caspase-1 in the renal tissue were significantly decreased （P<0.05）. CONCLUSION Cur has a certain protective effect on the kidney of MRL/lpr mice， and its mechanism may be related to the inhibition of NF-κB and NLRP3 signaling pathways.     

Inhibitory effect of HET0016 on LPS-induced proliferation and migration of microglia

AIM To study the effects of HET0016 on the proliferation and migration of microglia stimulated by lipopolysaccharide (LPS). METHODS Primary microglia from neonatal SD rats were isolated, purified and cultured. CCK-8 assay was performed to detect the effect of HET0016 on the viability of microglia after treatment with LPS. The levels of 20-hydroxyeicosatetraenoic acid （20-HETE）, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were measured by ELISA. The proportion of S phase was evaluated by flow cytometry. The cell migration ability was detected by Transwell assay and scratch wound healing assay. The protein expression of NF-κB p50 and p65 was determined by Western blot. RESULTS LPS induced the increases in the proliferation and migration of microglia and the release of inflammatory cytokines (P<0.05). Compared with LPS group, HET0016 inhibited the cell proliferation and migration (P<0.05), decreased the levels of TNF-α and IL-1β (P<0.05), and reduced the expression of NF-κB p50 and p65 (P<0.05). CONCLUSION HET0016 has inhibitory effects on the proliferation and migration of microglia induced by LPS, and reduces the release of inflammatory cytokines. The mechanism may be related to NF-κB signaling pathway.     

Effects of simvastatin on proliferation of esophageal cancer cells through NF-κB p65 pathway

AIM To investigate the effects of simvastatin （SIM） on the proliferation of esophageal cancer Eca109 cells through NF-κB p65 pathway. METHODS Esophageal cancer Eca109 cells were cultured in vitro and treated with SIM at different concentrations （2 ， 4， 8， 16 and 32 μmol/L）. The proliferation of Eca109 cells was measured by MTT assay and plate colony formation assay. The proliferation-related protein levels of cyclin D1， c-Myc， NF-κB p65 （p65） and IκB-α in the esophageal cancer Eca109 cells were determined by Western blot. ELISA was used to detect the levels of tumor necrosis factor α （TNF-α） and interleukin-6 （IL-6） in the culture supernatant of Eca109 cells. RESULTS Compared with control group， the inhibitory effects of SIM at 2 ， 4 ， 8 and 16 μmol/L on the viability of Eca109 cells were increased in a concentration-dependent and time-dependent manners （P<0.05）. Simultaneously， the inhibitory rates of SIM at 16 and 32 μmol/L on the viability of Eca109 cells showed no significant difference （P>0.05）. The inhibitory rates of SIM at 16 μmol/L on the viability of esophageal cancer cells was 50.61% at 48 h， which was closed to half of the inhibitory dose IC₅₀， and it was used as the optimum concentration and time for follow-up experiments. Compared with control group， the plate colony formation rate of Eca109 cells， the protein levels of cyclinD1， c-Myc， nuclear p65， TNF-α and IL-6 in 8 and 16 μmol/L groups were decreased， while the levels of cytosolic p65 and IκB-α proteins were increased （P<0.05）. No significant difference of plate colony formation rate in Eca109 cells， and the protein levels of cyclin D1， c-Myc， nuclear and cytoplasmic p65， IκB-α， TNF-α and IL-6 between 16 μmol/L SIM group and 32 μmol/L SIM group was observed （P>0.05）. CONCLUSION Simvastatin inhibits the proliferation of esophageal cancer Eca109 cells， and its mechanism may be related to up-regulation of IκB-α, down-regulation of cyclinD1 and c-myc, inhibition of p65 nuclear translocation, and the expression of TNF-α and IL-6 downstream of NF-κB p65 pathway.     

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.     

Activation of NF-κB-iNOS/COX-2 signaling pathways is involved in impaired endothelium-dependent relaxation under high glucose condition

AIM To investigate the role of nuclear factor-κB （NF-κB）-inducible nitric oxide synthase （iNOS）/cyclooxygenase-2 （COX-2） signaling pathways in impaired endothelium-dependent relaxation under high glucose （HG） condition. METHODS The endothelium-dependent relaxation induced by acetylcholine （ACh） in phenylephrine-precontracted rat thoracic aortic ring was performed in the absence or presence of different inhibitors under HG （55 mmol/L glucose） condition, and then the maximal relaxation effect of ACh （E_max） and the negative logarithm of ACh concentration for inducing 50% of E_max （pD₂） were calculated. The structure of aorta was observed by HE staining and electron microscopy. The mRNA and protein expression was detected by RT-qPCR and Western blot, respectively. RESULTS The structure of endothelial cells was interrupted by HG. Meanwhile, ACh-induced vasodilatation was also impaired, in which the E_max and pD₂ were both decreased significantly （P<0.01）, accompanied by the up-regulation of NF-κB p65, iNOS and COX-2 expression at mRNA and protein levels （P<0.01）. The NF-κB inhibitor PDTC, iNOS inhibitor SMT, and COX-2 inhibitor meloxicam restored the ACh-induced vasodilatation under HG condition, in which the E_max and pD₂ were increased significantly （P<0.01）. Moreover, PDTC attenuated the pathological damage of endothelial structure, and down-regulated the expression levels of NF-κB p65, iNOS and COX-2 induced by HG （P<0.01）. CONCLUSION The activation of NF-κB-iNOS/COX-2 signaling pathways is involved in the impaired endothelium-dependent relaxation under HG condition.     

Effects of Huanglian-jiedu decoction on TLR4/NF-κB signaling pathway and goblet cells of mucosa in rats with allergic rhinitis

AIM To investigate the effect of Huanglian-jiedu decoction （HLJD） on goblet cells and Toll-like receptor 4 （TLR4）/nuclear factor （NF）-κB signaling pathway in allergic rhinitis rats. METHODS The rat model of allergic rhinitis was made by ovalbumin. The model rats were divided into model group, low-, medium- and high-dose HLJD groups, and positive control drug group. The rats in low-, medium- and high-dose HLJD groups were given different doses（5, 10 and 20 g/kg, respectively） of crude drug by intragastric administration, the rats in positive control group was given fluticasone propionate nasal spray （50 μg per side）, and the rats in control group and model group were given normal saline, once per day for 10 days. The behaviors were observed and scored after modeling and treatment, the weight of nasal secretion was measured after the treatment. The goblet cells in nasal mucosa were observed by periodic acid-Schiff （PAS） staining. The morphological changes of nasal mucosa were observed by hematoxylin-eosin （HE） staining. The interleukin （IL）-4 and IL-5 levels in nasal mucosa were measured by ELISA. The mRNA levels of mouse calcium-activated chloride channel 3 （mCLCA3） and mucin 5AC （MUC5AC） were detected by RT-qPCR. The protein expression of TLR4 and NF-κB p65 was determined by Western blot. RESULTS After modeling, compared with control group, the behavioral scores in model group, low-, medium- and high-dose HLJD groups and positive control group were increased （P<0.05）. The eosinophils, neutrophils and lymphocytes in nasal mucosa were seriously infiltrated, inflammatory infiltration was obvious, and obvious small vessel dilatation and interstitial edema in model group were observed. With the increase in the dosage of HLJD, the lymphatic infiltration was obviously relieved, but the eosinophil and neutrophil infiltration still existed, and the inflammatory infiltration was relieved. Compared with control group, the behavioral score, nasal secretion, the relative proportion of goblet cells in the mucosa, the IL-4 and IL-5 levels, the mRNA levels of mCLCA3 and MUC5AC, and the protein expression of TLR4 and NF-κB p65 in the mucosa of model group were increased （P<0.05）. Compared with model group, the behavioral score, nasal secretion, the relative proportion of goblet cells in the mucosa, the IL-4 and IL-5 levels, the mRNA levels of mCLCA3 and MUC5AC, and the protein expression of TLR4 in the mucosa of low-, medium- and high-dose HLJD groups and positive control group were increased （P<0.05）, and the protein expression of NF-κB p65 in the mucosa of medium- and high-dose HLJD groups and positive control group was decreased （P<0.05）. CONCLUSION Huanglian-jiedu decoction reduces the relative proportion of goblet cells in the nasal mucosa of rats with allergic rhinitis, which may be achieved by inhibiting TLR4/NF-κB signaling pathway.     

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.     

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

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

Mechanism of Fuzilizhong decoction alleviating inflammatory damage of rats with non-alcoholic fatty liver disease

AIM To observe the effect of Fuzilizhong decoction on the inflammatory damage of non-alcoholic fatty liver disease （NAFLD） rats and to explore its mechanism. METHODS SPF male SD rats were randomly divided into 6 groups： control group, model group, high dose （20 mg·kg^-1·d^-1）, middle dose （10 mg·kg^-1·d^-1）, low dose （5 mg·kg^-1·d^-1） Fuzilizhong decoction group and Yishanfu （30 mg·kg^-1·d^-1）group, 8 rats in each group. A NAFLD rat modelwas established by intragastric administration of fat emulsion for 4 weeks. Then the drug was given for 4 weeks in each treatment group. HE staining was performed to observe the histopathological changes of the rat liver.The serum levels of interleukin-2（IL-2）, IL-6 and tumor necrosis factor-α（TNF-α） were measured by ELISA. The expression of toll like receptor 4（TLR4） and NF-κB p65 in liver tissues at mRNA and protein levels was determined by RT-qPCR and Western bolt,respectively. RESULTS Compared with control group, the inflammatory damage of liver tissue was more serious, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression TLR4 and NF-κB p65 in liver tissues were significantly increased in model group（P<0.05）. However, compared with model group, the liver pathological changes in each treatment group were significantly relieved, the serum levels of IL-2, IL-6 and TNF-α, the mRNA expression of TLR4 and NF-κB p65 in liver tissues were significantly reduced（P<0.05）.In addition, the changes of TLR4 and p-NF-κB p65 protein levels in liver tissue were consistent with the changes of TLR4 and NF-κB p65 mRNA. CONCLUSION Fuzilizhong decoction attenuates the inflammatory damages of NAFLD in rats by inhibiting TLR4/NF-κB p65 signaling pathway.     

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

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

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.     

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.     

Niflumic acid inhibits the proliferation,migration and invasion of glioma U87 cells

AIM To investigate the effect of niflumic acid （NFA） on human glioma U87 cells and to clarify the potential mechanism. METHODS The U87 cells were cultured in vitro and divided into blank control group， and 50， 100 and 200 μmol/L NFA groups. MTT assay was performed to determine the viability of cells in various groups. Migration and invasion abilities were measured by real-time cell analysis （RTCA）. RESULTS The results of MTT assay showed that compared with blank control group， the viability of U87 cells was increased after treatment with NFA for 12 h （P<0.05 or P<0.01）， while the viability was significantly decreased after treatment with NFA for 24 and 48 h （P<0.05 or P<0.01） in a concentration-dependent manner. The results of RTCA showed that compared with control group， the cell migration and invasion abilities were inhibited in 100 and 200 μmol/L NFA groups （P<0.05 or P<0.01） and the inhibitory effects were more obvious in 200 μmol/L NFA group （P<0.01）. CONCLUSION NFA inhibits the viability， migration and invasion of human glioma U87 cells.     

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.     

Minimally modified low-density lipoprotein up-regulates endothelin receptors in mouse mesenteric artery through p38 MAPK pathway

AIMTo investigate whether minimally modified low-density lipoprotein (mmLDL) affects the quantity and activity of endothelin （ET） type A (ET_A) and type B (ET_B) receptors in mouse mesenteric artery by activating p38 mitogen-activated protein kinase (MAPK) inflammatory pathway. METHODSThe KM mice were divided into normal saline (NS) group (injection of NS via caudal vein), mmLDL group (injection of mmLDL via caudal vein), LDL group (injection of LDL via caudal vein), mmLDL+SB 203580 group (injection of mmLDL via caudal vein and intraperitoneal injection of p38 MAPK pathway specific inhibitor SB 203580) and mmLDL+DMSO group (injection of mmLDL via caudal vein and intraperitoneal injection of DMSO). Mesenteric artery ring segment vasoconstriction dose-response curves affected by sarafotoxin 6c (S6c) and ET-1 were recorded by the myography system. The mRNA levels of ET_B receptor, ET_A receptor and interleukin-6 (IL-6) were detected by RT-qPCR. The protein levels of ET_B receptor, ET_A receptor, IL-6, p38 MAPK, p-p38 MAPK, NF-κB and p-NF-κB were determined by Western blot. The serum concentration of IL-6 was measured by ELISA. RESULTSThe contractile responses of the blood vessel segments to S6c and ET-1 were significantly increased by mmLDL (P<0.01). The mRNA and protein expression levels of ET_A receptor, ET_B receptor, and IL-6 significantly increased (P<0.01). The protein levels of p-p38 MAPK and p-NF-κB were significantly increased (P<0.01). The serum level of IL-6 was significantly increased (P<0.01). These effects of mmLDL were inhibited by p38 MAPK inhibitor SB 203580. CONCLUSION mmLDL increses the serum concentration of IL-6, up-regulates the expression of IL-6, ET_A receptor and ET_B receptor in mouse mesenteric artery, and enhances the vasoconstriction function medi?ated by ET_A and ET_B receptors, which is related to the activation of p38 MAPK inflammatory pathway and downstream NF-κB pathway.