This study was designed to investigate the role of ring finger protein 20(RNF20)in host innate immunity against RNA viruses.Dual luciferase reporter assay was employed to examine the effects of RNF20 overexpression on Sendai virus(SeV)infection-induced activation of interferon a4(Ifna4)gene promoter in 293T human embryonic kidney epithelial cells.Rnf20 myeloid conditional knockout mouse model was constructed(Rnf2OF/F;Lyz2-Cre),and the frequency of myeloid subsets in the bone marrow,spleen,blood of Rnf20F/F;Lyz2-Cre mice and littermate Rnf20F/F mice were detected by flow cytometry.After bone marrow-derived macrophages(BMDMs)were cultured and subjected to the infection of SeV and vesicular stomatitis virus(VSV),Western blot was used to detect the phosphorylation of transcription factor interferon regulatory factor 3(IRF3)and nuclear factor-κB(NF-κB).ELISA was used to detect the production of IFN-β,TNF-α and IL-6,and Bulk RNA-sequencing was employed to explore transcriptional changes.Furthermore,M1 and M2 macrophage polarization was induced by LPS and IL-4,respectively,to confirm the changes revealed by transcriptome analysis.Data showed that RNF20 overexpression showed no significant effect on SeV infection-induced activation of Ifna4 gene promoter in 293T cells.There is almost no difference in the development of myeloid subsets between Rnf20F/F;Lyz2-Cre and Rnf2OF/F mice.After RNA viral infection of BMDMs from Rnf20F/F;Lyz2-Creand Rnf201 mice,the phosphorylation of IRF3 and NF-κB p65 and the expression of IFN-[3,TNF-α and IL-6 were comparable between the two groups,while the expression levels of several genes related to macrophage polarization were significantly different.The loss of RNF20 showed the tendency of hindering M1 macrophage polarization while promoting M2 macrophage polarization.In conclusion,RNF20 does not affect RNA viral infection-induced activation of IRF3 or NF-κB pathways,but it might get involved in the resolution of inflammation afterwards.

This study aims to explore the role of mouse GSDME in tumor progression and its mechanisms.Wide type and GSDME knockout mice were used to establish tumor models and the tumor growth was monitored;CRISPR-Cas9 was employed to knockout GSDME in MC38 cells.Bone marrow-derived macrophage and dendritic cells were culture in vitro and adoptively transferred into tumor-bearing mice,respectively.Western blotting was conducted to detect protein expression in cells;flow cytometry was used to analysis the immune cell number,CD8+T cell function,and Cleaved Caspase-3 expression in TAM and TADC cells.According to the above experiments,we found that GSDME deficiency promoted tumor progression in mice.GSDME knockout reduced the number of CD8+T cells and decreased IFN-γ and Perforin in CD8+T cells in tumors.GSDME was mainly expressed in macrophages and dendritic cells,and adoptive transferring GSDME deficient macrophages or dendritic cells did not affect tumor progression.However,GSDME deficient mice reduced the ratio of Cleaved Caspase-3 positive macrophages and dendritic cells in tumors,and Cleaved Caspase-3 positive macrophages expressed more iNOS and MHCII,while Cleaved Caspase-3 positive dendritic cells expressed more CD80 and CD86.In general,GSDME increases Cleaved Caspase-3 positive macrophages and dendritic cells that promote CD8+T cell function,thereby inhibiting tumor progression.

This study was designed to investigate the effects of TNFSF14 on mitochondrial function in ischemia/reperfusion-induced acute kidney injury(I/R-AKI)and its mechanism.TNFSF14-/-and TNFSF14+/+mice underwent renal ischemia-reperfusion operation to establish I/R-AKI models,and their histopathology changes were compared by using Periodic Acid-Schiff stain,transmission electron microscopy.Immunohistochemistry(IHC)was used to detect levels of TNFSF14,HVEM,LT[3R,mitochondrial activity related proteins(Dpr1 and Mfn2)and inflammatory cells infiltration in kidney tissues of mice and relevant patients.In vitro cell experiments,immunofluorescence and immunoblotting were used to observe the effects of exogenous recombinant TNFSF14 factor on the damage and mitochondrial activity of renal tubular epithelial cell line HK-2 cells.Data showed that the expression of TNFSF14 and its receptors were significantly increased in kidney tissues of I/R-AKI mice and clinical human renal tissues of acute tubular injury.Compared with the sham group,I/R mice showed significantly higher levels of renal tubular injury score,inflammatory cells infiltration,cell apoptosis,mitochondrial damage,and Drp1 expression,while knocking out the TNFSF14 gene,the above indicators were significantly reduced.In vitro,exogenous TNFSF14 stimulation could aggravate the hypoxia-induced apoptosis and the decrease of mitochondrial membrane potential in HK-2 cells,while increasing phosphorylation of Drp1 at Se616 to promote its transfer from cytoplasm to mitochondria,leading to an abnormal increase in mitochondrial fission.In conclusions,TNFSF14 may mediate the pathological process of I/R-AKI by promoting mitochondrial damage.

This study aims to enhance the activation level and efficiency of second-generation chimeric antigen receptor-T(CAR-T)cells in killing tumor cells by modifying the lymphocyte-specific protein tyrosine kinase(LCK)structure of second-generation CAR,so as to lay the foundation for the preparation of novel second-generation CAR-T cells.Using genetic engineering techniques,lentiviral vectors for CD137-LCK2 CAR and CD137-PYAP2 CAR targeting human epidermal growth factor receptor 2(HER2)were constructed,and CD137-LCK2 and CD137-PYAP2 CAR-T cells were prepared by lentiviral packaging and infection of activated T cells.The differences between them in terms of CAR expression level,activating molecule CD 137 expression,phenotypic distribution,cytokines secretion,and killing effect on HER2-expressing tumor cells were detected and compared using flow cytometry,enzyme-linked immunosorbent assay,and cytotoxicity assay,respectively.Compared with the second-generation CD137 CAR-T cells,the structurally modified CD137-LCK2 and CD137-PYAP2 CAR-T cells targeting HER2 can increase the expression of activating molecule CD 137,increase the secretion of cytokine IFN-γ,and enhance the killing ability of target antigens;at the same time,these cells can be maintained in the memory state and can rapidly activate proliferation and differentiation after stimulation by antigen.In conclusion,CD137-LCK2 and CD137-PYAP2 CAR-T cells targeting HER2 are expected to activate and exert anti-tumor effects more effectively than second-generation CD 137 CAR-T cells.

This study was performed to explore the effect of flagellin+rapamycin on the growth and metastasis of 4T1 breast cancer in tumor-bearing mice,and their regulatory effect on several immunocytes.4T1 cell line was applied to establish an breast cancer model in Balb/c mice,which then injected with flagellin+rapamycin,and the volume and inhibition rate of tumor were recorded.MTT assay was used to detect the inhibitory effect of flagellin+rapamycin against 4T1 cells.Data showed that the combination of flagellin and rapamycin had the best inhibition effect on 4T1 cells,and the same results were found in animal model experiments.Flow cytometry result indicated that flagellin+rapamycin significantly down-regulated the levels of CD11b+Gr-1+myeloid-derived suppressor cells(MDSCs)and CD11b+F4/80+tumor-associated macrophages(TAMs)of tumor-bearing mice.Furthermore,flagellin+rapamycin alleviates the metastasis of 4T1 cancer cells into liver and lung.Taken together,flagellin+rapamycin can inhibit proliferation and metastasis of 4T1 cells,thus exert antitumor effects in mice,which may related with its regulatory effects on immunocytes in tumor microenvironment.

This study was designed to investigate the effet of phillyrin(PHI)on sciatic nerve injury in diabetic peripheral neuropathy(DPN)rats based on the high mobility group protein box-1(HMGB1)/advanced glycation end product receptor(RAGE)signal pathway.DPN rat model was established by high fat and high sugar diet combined with injection of streptozotocin(STZ)solution,and the rats were randomly grouped into model group,phillyrin low dose(PHI-L,50 mg/kg)group,phillyrin medium dose(PHI-M,100 mg/kg)group,phillyrin high dose(PHI-H,200 mg/kg)group,and positive drug(mecobalamin,250 μg/kg)group,while another rats with normal diet were treated as the control group.Each group consisted of 10 rats.The conduction velocity of sciatic nerve was measured by BL-420S biological function experiment system;fasting blood glucose(FBG)level was detected by blood glucose meter;the levels of serum HbAlc,IL-6 and TNF-α were measured with ELISA kits;the ultrastructure of sciatic nerve was observed with electron microscope;the levels of ROS,SOD,MDA and MBP in sciatic nerve were detected with commercial kits;the mRNA and protein levels of HMGB1 and RAGE in sciatic nerve were detected by RT-qPCR and Westem blotting.Compared with model group,pathological injury of rats in PHI-M group,PHI-H group and positive drug group was significantly alleviated,the sciatic nerve conduction velocity and MBP/SOD levels were significantly recovered,the levels of FBG,HbAlc,IL-6,TNF-α,and the mRNA and protein levels of ROS,MDA,HMGB1 and RAGE were decreased significantly(P＜0.05).In conclusion,PHI can reduce the inflammatory reaction,reduce DPN in rats,thus plays a therapeutic role,and the mechanism may be related to the inhibition of HMGB1/RAGE signal pathway.

This study was designed to investigate the effect of Sijunzi decoction(SJZD)on the expression of inflammatory factors and autophagic proteins in the hippocampus of senescence-accelerated mouse prone 8(SAMP8)mice and its mechanism.SAMR1 mice were used as control group,and 32 SAMP8 mice were divided into model group,donepezil group,SJZD low and SJZD high dose treatment groups.Y-maze experiment was performed to detect changes in mouse memory function;the expression of NF-κB was detected by immunohistochemistry;the levels of TNF-α,IL-1β,IL-6 were detected by ELISA;the expression of A[3,caspse-1,beclin-1 and LC3-Ⅱproteins in hippocampal tissue were detected by Western blotting.Compared with the control group,the model group mice showed a decrease in total number of entries and alternations,an elevation in the levels of TNF-α,IL-1β,IL-6,Aβ protein and caspse-1 protein,and downregulation in the expression of beclin-1 and LC3-Ⅱ proteins.Both donepezil and SJZD(low-dose group and high-dose group)can reverse these changes in model mice.In conclusion,the mechanism of SJZD in treating Alzheimer's disease may relate to the correction of central hippocampal inflammatory factors and autophagy dysfunction.

To investigate the effect of mitochondrial division inhibitor 1(Mdivi-1)on imiquimod(IMQ)-induced psoriasis-like skin inflammation in mice and its mechanism,female 8-week-old C57BU6 mice were recruited and randomly divided into control group,IMQ model group,IMQ+Mdivi-1 experiment group.IMQ was used to induce the psoriasis-like skin inflammation model in mice.The mice in the experiment group were injected intraperitoneally(i.p.)with Mdivi-1,and the mice in the control group and model group were injected with the same volume of solvent.The mice were sacrificed on the 7th day for sampling.Psoriasis area and severity index(PASI)score was used to evaluate the severity of skin lesions in each group;the reactive oxygen species(R0S)content in skin tissue was detected by fluorescence staining of frozen section;HE staining was used to observe the histomorphologic change of skin lesions;immunohistochemical staining was used to detect the expression of dynamin-related protein 1(Drp1)in the skin of mice;Western blot was used to detect the protein levels of Drp1,NLRP3 and IL-1β in the skin tissues of mice in each group;and the expressions of IL-17A and IL-18 in mouse serum were detected by ELISA.Data showed that the model group had typical psoriatic lesions such as erythema,scale and thickening,and the Mdivi-1 group demonstrated obvious reduction of the lesions.The PASI score of the experiment group was significantly lower than that of the model group.HE staining indicated that the epidermal thickness of the back skin in the treatment group was significantly lower than that in the model group,and Munro microabscess was significantly reduced.R0S fluorescence staining indicated that ROS content in the experiment group was significantly lower than that in the model group;immunohistochemical results showed that the expression of Drp1 protein in the experiment group was significantly lower than that in the model group;Western blot results showed that the expression levels of Drp1,NLRP3 and IL-1 β in the experiment group were significantly lower than those in the model group;ELISA results indicated that the expressions of IL-17A and IL-18 in serum of mice in the experiment group were lower than those in the model group.Taken together,Mdivi-1 can reduce mitochondrial damage and ROS production by inhibiting the expression of Drp1,thereby reducing the production of NLRP3 inflammasome,down-regulating IL-1 β,IL-18 and IL-17A,and alleviating the IMQ-induced psoriasis-like skin inflammation in mice.

This study was performed to explore the therapeutic effect of Xiezhuo Jiedu Formula on ulcerative colitis rats and its effects on β-catenin/FOSL2/ARID5A signaling pathway and macrophage polarization.Rats of ulcerative colitis was induced and divide into control group,model group,positive group(intervention with sulfasalazine),and low,medium and high-dose groups(intervention with Xiezhuo Jiedu Fang).After 14 days of intervention,the disease activity index(DAI)and colon mucosa damage index(CDMI)were calculated and used to evaluate the state of rats.HE staining was used to observe lesion tissue;ELISA was used to detect of serum levels of TNF-α and IL-6;flow cytometry was used to detect peripheral blood M1 and M2 macrophage content;RT-PCR was used to detect the mRNA expression of iNOS,CD206,and β-Catenin/FOSL2/ARID5A;immunohistochemistry was used to detect β-Catenin/FOSL2/ARID5A signaling pathway protein expression in colon tissue.Data showed that DAI score,CMDI score,and serum TNF-α and IL-6 in the low,medium,and high dose groups were significantly lower than the model group(P＜0.05).HE staining showed that the colon tissue damage and inflammatory infiltration in the low,medium,and high dose groups were slighter than those in the model group(P＜0.05).The rate of M1 type macrophages and iNOS mRNA in the low,medium,and high dose groups were significantly lower than those in the model group,while the rate of M2 type macrophages and CD206 mRNA were significantly higher than those in the model group(P＜0.05).The mRNA and protein expressions of β-catenin and FOSL2 in colon tissue of low,medium,and high dose groups were significantly higher than those of the model group,while the mRNA level and protein expression of ARID5A were significantly lower than that of the model group(P＜0.05).Taken together,Xiezhuo Jiedu formula can effectively alleviate the clinical symptoms,reduce inflammatory response,downregulate β-catenin/FOSL2/ARID5A expression,regulate macrophage polarization and promote disease recovery of ulcerative colitis in rats.

The purpose of this study was to investigate the role of miR-4281 in immune thrombocytopenic purpura(ITP)and its underlying mechanism.Differentially expressed miRNAs in ITP were searched by using GEO database.Newly diagnosed ITP patients,treatment-effective ITP patients and healthy subjects were collected.Venous blood was obtained and peripheral blood mononuclear cells(PBMCs)and CD4+T cells were separated.Real-time quantitative PCR was used to detect the expression of miR-4281 and other factors in cells.Flow cytometry was conducted to analyze the rate of CD4+CD25+FOXP3+Tregs and Th17 cells.Pearson correlation coefficient method was used to test the relationship between miR-4281 expression level and various factors.Bioinformatics analysis revealed an abnormal expression of miR-4281 in ITP.Compared with healthy subjects and treatment-effective ITP patients,the expression level of miR-4281 in newly diagnosed ITP was decreased.The expression level of miR-4281 in PBMCs of ITP patients was positively correlated with Tregs content and Treg/Th17,while negatively correlated with Th17 cell content.miR-4281 can promote the differentiation of Tregs cells and inhibit the differentiation of Th17.Furthermore,the expression level of miR-4281 is positively correlated with the expression level of FOXP3.Taken together,the expression level of miR-4281 is reduced in ITP.This decrease can inhibit the expression of FOXP3,cause Treg/Th17 immune imbalance,and promote the progression of ITP.