Receptor-interacting protein kinase 1 (RIPK1) plays an essential role in regulating the necroptosis and apoptosis in cerebral ischemia-reperfusion (I/R) injury. However, the regulation of RIPK1 kinase activity after cerebral I/R injury remains largely unknown. In this study, we found the downregulation of protein arginine methyltransferase 1 (PRMT1) was induced by cerebral I/R injury, which negatively correlated with the activation of RIPK1. Mechanistically, we proved that PRMT1 directly interacted with RIPK1 and catalyzed its asymmetric dimethylarginine, which then blocked RIPK1 homodimerization and suppressed its kinase activity. Moreover, pharmacological inhibition or genetic ablation of PRMT1 aggravated I/R injury by promoting RIPK1-mediated necroptosis and apoptosis, while PRMT1 overexpression protected against I/R injury by suppressing RIPK1 activation. Our findings revealed the molecular regulation of RIPK1 activation and demonstrated PRMT1 would be a potential therapeutic target for the treatment of ischemic stroke.

AIM:To elucidate the intervention and mechanism of 4'-hydroxychalcone(4-HC)in colitis mice through the regulation of Th17/Treg homeostasis.METHODS:Using a dextran sodium sulfate(DSS)-induced colitis model in mice,we meticulously observed the pathological characteristics of colon tissue via HE staining.Additionally,we employed immunohistochemical analysis and Western blot techniques to assess the expression levels of proteins associated with the JAK/STAT signaling pathway,as well as the specific content of tight junction proteins such as ZO-1 and occludin.The differentiation of Th17 and Treg cells was analyzed through flow cytometry.RESULTS:Compared to the normal group,the DSS group exhibited a consistent decline in body weight,coupled with symptoms of diarrhea and hematochezia,an increase in the DAI score,and a notable reduction in colon length.In contrast,the body weight of the 4-HC group dis-played an upward trend following an initial decrease,with improvements in diarrhea and hematochezia symptoms,a reduc-tion in the DAI score,and a restoration of colon length relative to the model group.The integrity of colon tissue in the 4-HC group was significantly better than that in the DSS group,evidenced by a marked increase in the number of goblet cells and an enhancement in crypt integrity,while the average histology score showed a decrease.Western blot analysis re-vealed substantial increase in ZO-1 and occludin expression after 4-HC treatment.Flow cytometry results indicated a dra-matic decrease in the differentiation rate of Th17 cells in spleen lymphocytes and mesenteric lymph nodes,while the dif-ferentiation rate of Treg cells was significantly elevated.Immunohistochemical and Western blot analyses demonstrated that 4-HC markedly reduced the phosphorylation of STAT1 and STAT3,while up-regulating the phosphorylation of STAT6,suggesting that 4-HC modulates CD4+T cell activity through the JAK-STAT pathway.CONCLUSION:The 4-HC may enhance the course of DSS-induced colitis in mice,alleviate colonic tissue damage,and modulate the balance be-tween Th17 and Treg cells,potentially involving the JAK/STAT signaling pathway.

Objective:To investigate the role of Platycoside E(PE)in a mouse model of bleomycin(BLM)-induced pulmonary fibrosis by targeting the JAK/STAT signaling pathway to suppress macrophage M2 polarization.Methods:Forty BALB/c mice were randomly assigned to five experimental groups:blank control group,model group,pirfenidone(PDF)experimental group,PE high-dose group and PE low-dose experimental group,each with eight mice.After adaptive feeding,except for blank control group,all mice were used in the model of BLM nasal drip-induced pulmonary fibrosis.HE and Masson staining were employed to examine pathological alterations of lung tissue in mice;ELISA to detect concentrations of IL-10,IL-4,IL-17A and TNF-α in mice serum;expressions of CD206 and CD11b in lung tissue were detected by immunofluorescence.Western blot to detect protein expressions of JAK1,p-JAK1,STAT6 and p-STAT6 in lung tissues.Results:Compared with blank control group,tissues in model group showed distorted structure and thickened alveolar walls,fibrotic foci were formed,and alveolar inflammatory fraction and collagen volume fraction were significantly increased(P<0.01).ELISA showed that concentrations of IL-4,IL-10,IL-6 and TNF-α in serum were significantly reduced compared to those of model group.Immunofluorescence showed that fluorescence intensity of CD11b and CD206 treated by PE were decreased significantly.Western blot showed that expressions of JAK1,p-JAK,STAT6 and p-STAT6 proteins were significantly elevated in lung tissues of model mice.Following PE treatment,levels of the above proteins were diminished.Conclusion:PE can effectively improve lung fibrosis induced by BLM in mice,the mechanism may be related to the inhibition of JAK/STAT pathway to block macrophage M2 polarization.

AIM:To elucidate the intervention and mechanism of 4'-hydroxychalcone(4-HC)in colitis mice through the regulation of Th17/Treg homeostasis.METHODS:Using a dextran sodium sulfate(DSS)-induced colitis model in mice,we meticulously observed the pathological characteristics of colon tissue via HE staining.Additionally,we employed immunohistochemical analysis and Western blot techniques to assess the expression levels of proteins associated with the JAK/STAT signaling pathway,as well as the specific content of tight junction proteins such as ZO-1 and occludin.The differentiation of Th17 and Treg cells was analyzed through flow cytometry.RESULTS:Compared to the normal group,the DSS group exhibited a consistent decline in body weight,coupled with symptoms of diarrhea and hematochezia,an increase in the DAI score,and a notable reduction in colon length.In contrast,the body weight of the 4-HC group dis-played an upward trend following an initial decrease,with improvements in diarrhea and hematochezia symptoms,a reduc-tion in the DAI score,and a restoration of colon length relative to the model group.The integrity of colon tissue in the 4-HC group was significantly better than that in the DSS group,evidenced by a marked increase in the number of goblet cells and an enhancement in crypt integrity,while the average histology score showed a decrease.Western blot analysis re-vealed substantial increase in ZO-1 and occludin expression after 4-HC treatment.Flow cytometry results indicated a dra-matic decrease in the differentiation rate of Th17 cells in spleen lymphocytes and mesenteric lymph nodes,while the dif-ferentiation rate of Treg cells was significantly elevated.Immunohistochemical and Western blot analyses demonstrated that 4-HC markedly reduced the phosphorylation of STAT1 and STAT3,while up-regulating the phosphorylation of STAT6,suggesting that 4-HC modulates CD4+T cell activity through the JAK-STAT pathway.CONCLUSION:The 4-HC may enhance the course of DSS-induced colitis in mice,alleviate colonic tissue damage,and modulate the balance be-tween Th17 and Treg cells,potentially involving the JAK/STAT signaling pathway.

Objective:To investigate the role of Platycoside E(PE)in a mouse model of bleomycin(BLM)-induced pulmonary fibrosis by targeting the JAK/STAT signaling pathway to suppress macrophage M2 polarization.Methods:Forty BALB/c mice were randomly assigned to five experimental groups:blank control group,model group,pirfenidone(PDF)experimental group,PE high-dose group and PE low-dose experimental group,each with eight mice.After adaptive feeding,except for blank control group,all mice were used in the model of BLM nasal drip-induced pulmonary fibrosis.HE and Masson staining were employed to examine pathological alterations of lung tissue in mice;ELISA to detect concentrations of IL-10,IL-4,IL-17A and TNF-α in mice serum;expressions of CD206 and CD11b in lung tissue were detected by immunofluorescence.Western blot to detect protein expressions of JAK1,p-JAK1,STAT6 and p-STAT6 in lung tissues.Results:Compared with blank control group,tissues in model group showed distorted structure and thickened alveolar walls,fibrotic foci were formed,and alveolar inflammatory fraction and collagen volume fraction were significantly increased(P<0.01).ELISA showed that concentrations of IL-4,IL-10,IL-6 and TNF-α in serum were significantly reduced compared to those of model group.Immunofluorescence showed that fluorescence intensity of CD11b and CD206 treated by PE were decreased significantly.Western blot showed that expressions of JAK1,p-JAK,STAT6 and p-STAT6 proteins were significantly elevated in lung tissues of model mice.Following PE treatment,levels of the above proteins were diminished.Conclusion:PE can effectively improve lung fibrosis induced by BLM in mice,the mechanism may be related to the inhibition of JAK/STAT pathway to block macrophage M2 polarization.

Objective To construct a recombinant poxvirus vector vaccine, rVTTδTK-RBD, and to evaluate its safety and immunogenicity. Methods The receptor-binding domain (RBD) gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE, to construct the recombinant poxvirus shuttle vector pSTKE-RBD. This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain (VTT). The recombinant poxvirus rVTTδTK-RBD was successfully obtained after several rounds of fluorescence phage screening. The effect of rVTTδTK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination. The levels of specific and neutralizing antibodies produced by rVTTδTK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly. The effect of rVTTδTK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry. Results Through homologous recombination, enhanced green fluorescent protein (EGFP) screening marker, and multiple rounds of fluorescent phosphorescence phage screening, a recombinant poxvirus rVTTδTK-RBD, expressing RBD with deletions in the thymidine kinase (TK) gene, was successfully obtained, which was validated by PCR. The in vivo experiments on BALB/c mice showed that rVTTδTK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT. Conclusion The recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 is successfully constructed and obtained, with its safety and immunogenicity confirmed through various experiments.
Animals ; Mice ; SARS-CoV-2/genetics* ; COVID-19 ; Vaccines, Synthetic/genetics* ; Genes, Reporter ; Bacteriophages ; Mice, Inbred BALB C

As a potential vectored vaccine,Newcastle disease virus(NDV)has been subject to various studies for vaccine development,while relatively little research has outlined the immunomodulatory effect of the virus in antigen presentation.To elucidate the key inhibitory factor in regulating the interaction of infected dendritic cells(DCs)and T cells,DCs were pretreated with the NDV vaccine strain LaSota as an inhibitor and stimulated with lipopolysaccharide(LPS)for further detection by enzyme-linked immunosorbent assay(ELISA),flow cytometry,immunoblotting,and quantitative real-time polymerase chain reaction(qRT-PCR).The results revealed that NDV infection resulted in the inhibition of interleukin(IL)-12p40 in DCs through a p38 mitogen-activated protein kinase(MAPK)-dependent manner,thus inhibiting the synthesis of IL-12p70,leading to the reduction in T cell proliferation and the secretion of interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),and IL-6 induced by DCs.Consequently,downregulated cytokines accelerated the infection and viral transmission from DCs to T cells.Furthermore,several other strains of NDV also exhibited inhibitory activity.The current study reveals that NDV can modulate the intensity of the innate?adaptive immune cell crosstalk critically toward viral invasion improvement,highlighting a novel mechanism of virus-induced immunosuppression and providing new perspectives on the improvement of NDV-vectored vaccine.

AIM:To investigate the potential of atractylenolide Ⅲ(AⅢ)in mitigating dextran sulfate sodium(DSS)-induced injury in a mouse model of chronic inflammatory bowel disease(IBD),and to explore the mechanisms in-volved,particularly the modulation of signal transducer and activator of transcription 3(STAT3)signaling,which plays a crucial role in the homeostasis of T helper 17(Th17)and regulatory T(Treg)cells.METHODS:A mouse model of DSS-induced chronic IBD was established,and the mice were divided into 4 groups:control,model(DSS),high-dose(50 mg/kg)AⅢ,and low-dose(30 mg/kg)AⅢ.The disease activity index(DAI)was utilized to assess disease severity.Histo-pathological damage in the colons of IBD mice was evaluated by hematoxylin-eosin(HE)staining.The protein levels of phosphorylated STAT3,occludin and zonula occludens-1(ZO-1)were analyzed using immunohistochemical staining and Western blot.Flow cytometry was employed to examine the differentiation of splenic lymphocytes into Th17/Treg cells.RESULTS:Both DAI assessments and HE staining indicated that AⅢ significantly alleviated inflammatory injury in mice with DSS-induced chronic IBD.Immunohistochemical analysis demonstrated that AⅢ enhanced the expression of ZO-1 and occludin in colonic tissues.Flow cytometry results revealed that AⅢ helped maintain the balance between splenic Th17 and Treg cells.Furthermore,immunohistochemical staining and Western blot showed that AⅢ inhibited the phos-phorylation of STAT3.CONCLUSION:Treatment with AⅢ effectively reduced inflammatory injury in a mouse model of chronic IBD by preserving Th17/Treg homeostasis through the inhibition of STAT3 phosphorylation.As a natural com-pound,AⅢ exhibits significant therapeutic potential for the treatment of chronic IBD.

AIM:This study examined the inhibitory effect of peiminine on the human colonic adenocarcino-ma cell line SW480 and explored the underlying mechanisms.METHODS:SW480 and human normal colonic epithelial CCD-841CoN cells were treated with different concentrations of peiminine and subjected to the CCK-8 assay to select the optimal treatment time and concentration of the compound.SW480 cell migration and invasion were evaluated by the wound-healing and Transwell assays.Cell cycle progression was analyzed by flow cytometry.The expression levels of cell cycle-related proteins were examined by Western blot.SW480 xenograft tumor model was established in nude mice to ex-amine the effect of peiminine on tumor growth and the expression of cell cycle-related proteins in vivo.RESULTS:Peimi-nine(110 mg/L)significantly inhibited the proliferation of SW480 cells compared with the control group(P<0.01),caused cell cycle arrest at G1 phase,and significantly downregulated the expression of cyclin dependent kinase 2(CDK2),CDK4,CDK6,cyclin D1,p-Rb/Rb,E2F1,E2F3,and E2F4(P<0.05).Peiminine inhibited SW480 xenograft tumor growth,prolonged the survival of model mice,and affected the expression of CDK2,CDK4,CDK6,and cyclin D1 in tu-mor tissues.CONCLUSION:Peiminine promotes G1 phase arrest by down-regulating the expression of CDK2,CDK4,CDK6,and cyclin D1,thereby inhibiting the proliferation of SW480 cells.

AIM:To explore the effect and mechanism of action of the aqueous extract of Fritillaria ussuriensis(FU-AE)against nonalcoholic fatty liver disease(NAFLD).METHODS:The association between Fritillaria ussuriensis Maxir.(FU)and NAFLD was analyzed by network pharmacology.A mouse model of NAFLD was induced in mice by high fat diet(HFD)+10%fructose drinking water,and three doses of Fritillaria ussuriensis aqueous extract were given to the mice for intervention.Colorimetric assay was used for detection of aspartate aminotransferase(AST),alanine aminotrans-ferase(ALT),triglyceride(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C)levels in the serum of experimental mice.Hematoxylin and eosin staining was used to as-sess the pathological and histological changes in the liver of mice and to clarify the anti-NAFLD effect of aqueous extracts of Fritillaria ussuriensis.Liver tissue proteins were extracted,and expression of proteins related to the AMP-activated pro-tein kinase(AMPK)/acetyl-CoA carboxylase(ACC)pathway was detected by Western blot to clarify the mechanism of an-ti-NAFLD action of Fritillaria ussuriensis.The microbial composition of cecum contents was explored using 16S rRNA se-quencing to reveal the modulatory effect of the aqueous extract of Fritillaria ussuriensis on the structure of intestinal flora in mice with nonalcoholic fatty liver disease.RESULTS:Aqueous extract of Fritillaria ussuriensis(high dose)ameliorated exogenous adipocyte infiltration in the liver of mice with NAFLD(P<0.05).AST,ALT,TG,TC and LDL-C levels were significantly decreased(P<0.05)and HDL-C levels were significantly increased(P<0.05)in the high-dose group.Aque-ous extract of Fritillaria ussuriensis(high dose)significantly increased expression of phosphorylated AMPKα,AMPKα,and phosphorylated ACC in the livers of the model mice(P<0.05),significantly reduced expression of ACC(P<0.05),and significantly increased the relative abundance of the potentially beneficial bacteria Faecalibaculum rodentium,Lacto-bacillus johnsonii,Akkermansia muciniphila(P<0.05).CONCLUSION:Aqueous extract of Fritillaria ussuriensis may ameliorate NAFLD in mice by activating the AMPK/ACC pathway and modulating the structure of intestinal flora.