Innate immunity,particularly macrophages,is critical for intestinal homeostasis.Sulforaphane,a dietary isothiocyanate from cruciferous vegetables,has been reported to protect against intestinal inflammation.However,th...Innate immunity,particularly macrophages,is critical for intestinal homeostasis.Sulforaphane,a dietary isothiocyanate from cruciferous vegetables,has been reported to protect against intestinal inflammation.However,the role of macrophages in sulforaphane mediated intestinal inflammation and the underlying molecular mechanisms have not been studied yet.In this study,sulforaphane effectively attenuated dextran sodium sulphate(DSS)induced intestinal inflammation in murine model.Of note,sulforaphane skewed the switching from classically(M1)to alternatively(M2)activated phenotype both in intestinal and bone marrow-derived macrophages(BMDMs).The expression levels of M1 associated maker genes induced by DSS or lipopolysaccharide(LPS)plus interferon gamma-γ(IFN-γ)were suppressed by sulforaphane while M2 marker gene expression levels were improved.This resulted in alteration of inflammatory mediators,particularly interleukin-10(IL-10),both in colon tissues and culture medium of BMDMs.Subsequently,IL-10 was found to mediate the sulforaphane induced M2 phenotype switching of BMDMs through the activation of STAT3 signaling.This was confirmed by immunofluorescence analysis with increased number of p-STAT3-positive cells in the colon sections.Moreover,anti-IL-10 neutralizing antibody significantly interfered M2 phenotyping of BMDMs induced by sulforaphane with reduced STAT3 phosphorylation.Findings here introduced a potential utilization of sulforaphane for intestinal inflammation treatment with macrophages as the therapeutic targets.展开更多
Objective:Acute lung injury(ALI)is characterized by inflammation and currently lacks an efficacious pharmacological intervention.The medicine combination of Lonicerae Japonicae Flos(LJF)and Forsythiae Fructus(FF)demon...Objective:Acute lung injury(ALI)is characterized by inflammation and currently lacks an efficacious pharmacological intervention.The medicine combination of Lonicerae Japonicae Flos(LJF)and Forsythiae Fructus(FF)demonstrates combined properties in its anti-infective,anti-inflammatory,and therapeutic effects,particularly in alleviating respiratory symptoms.In previous studies,Chinese medicine has shown promising efficacy in lipopolysaccharides(LPS)-induced ALI.However,there have been no reports of LJF and FF pairing for lung injury.The aim of this study is to compare the efficacy of herb pair Lonicerae Japonicae Flos-Forsythiae Fructus(LF)with LJF or FF alone in the treatment of ALI,and to explore whether LJF and FF have a combined effect in the treatment of lung injury,along with the underlying mechanism involved.Methods:A total of 36 mice were divided into six groups(control,model,LJF,FF,LF,dexamethasone)based on the treatments they received after undergoing sham-operation/LPS tracheal instillation.H&E staining and pulmonary edema indexes were used to evaluate lung injury severity.Alveolar exudate cells(AECs)were counted based on cell count in bronchoalveolar lavage fluid(BALF),and neutrophil percentage in BALF was measured using flow cytometry.Myeloperoxidase(MPO)activity in BALF was measured using enzyme-linked immunosorbent assay(ELISA),while the production of IL-1β,TNF-α,and IL-6 in the lung and secretion level of them in BALF were detected by quantitative polymerase chain reaction(qPCR)and ELISA.The effect of LJF,FF,and LF on the expression of Caspase-1 and IL-1βproteins in bone marrow derived macrophages(BMDMs)supernatant was assessed using Western blot method under various inflammasome activation conditions.In addition,the concentration of IL-1βand changes in lactatedehydrogenase(LDH)release levels in BMDMs supernatant after LJF,FF,and LF administration,respectively,were measured using ELISA.Furthermore,the effects of LJF,FF and LF on STING and IRF3 phosphorylation in BMDMs were detected by Western blot,and the mRNA changes of IFN-β,TNF-α,IL-6 and CXCL10 in BMDMs were detected by qPCR.Results:LF significantly attenuated the damage to alveolar structures,pulmonary hemorrhage,and infiltration of inflammatory cells induced by LPS.This was evidenced by a decrease in lung index score and wet/dry weight ratio.Treatment with LF significantly reduced the total number of neutrophil infiltration by 75%as well as MPO activity by 88%.The efficacy of LF in reducing inflammatory factors IL-1β,TNF-α,and IL-6 in the lungs surpasses that of LJF or FF,approaching the effectiveness of dexamethasone.In BMDMs,the co-administration of 0.2 mg/mL of LJF and FF demonstrated superior inhibitory effects on the expression of nigericin-stimulated Caspase-1 and IL-1β,as well as the release levels of LDH,compared to individual treatments.Similarly,the combination of 0.5 mg/mL LJF and FF could better inhibit the phosphorylation levels of STING and IRF3 and the production of IFN-β,TNF-α,IL-6,and CXCL10 in response to ISD stimulation.Conclusion:The combination of LJF and FF increases the therapeutic effect on LPS-induced ALI,which may be mechanistically related to the combined effect inhibition of cyclic-GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)and NOD-like receptor family protein 3(NLRP3)inflammasomes pathways by LJF and FF.Our study provides new medicine candidates for the clinical treatment of ALI.展开更多
基金the financial support provided by“Modern Food Processing,Food Storage,Transportation Technology,and Equipment”State Key Research and Development Plan(2017YFD0400204)the National Science Foundation of China(31972091).
文摘Innate immunity,particularly macrophages,is critical for intestinal homeostasis.Sulforaphane,a dietary isothiocyanate from cruciferous vegetables,has been reported to protect against intestinal inflammation.However,the role of macrophages in sulforaphane mediated intestinal inflammation and the underlying molecular mechanisms have not been studied yet.In this study,sulforaphane effectively attenuated dextran sodium sulphate(DSS)induced intestinal inflammation in murine model.Of note,sulforaphane skewed the switching from classically(M1)to alternatively(M2)activated phenotype both in intestinal and bone marrow-derived macrophages(BMDMs).The expression levels of M1 associated maker genes induced by DSS or lipopolysaccharide(LPS)plus interferon gamma-γ(IFN-γ)were suppressed by sulforaphane while M2 marker gene expression levels were improved.This resulted in alteration of inflammatory mediators,particularly interleukin-10(IL-10),both in colon tissues and culture medium of BMDMs.Subsequently,IL-10 was found to mediate the sulforaphane induced M2 phenotype switching of BMDMs through the activation of STAT3 signaling.This was confirmed by immunofluorescence analysis with increased number of p-STAT3-positive cells in the colon sections.Moreover,anti-IL-10 neutralizing antibody significantly interfered M2 phenotyping of BMDMs induced by sulforaphane with reduced STAT3 phosphorylation.Findings here introduced a potential utilization of sulforaphane for intestinal inflammation treatment with macrophages as the therapeutic targets.
基金granted by the Natural Science Foundation of Beijing,China(Grant No.7232321)Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(No:ZYYCXTD-C-202005)National Natural Science Foundation of China(No.81721002).
文摘Objective:Acute lung injury(ALI)is characterized by inflammation and currently lacks an efficacious pharmacological intervention.The medicine combination of Lonicerae Japonicae Flos(LJF)and Forsythiae Fructus(FF)demonstrates combined properties in its anti-infective,anti-inflammatory,and therapeutic effects,particularly in alleviating respiratory symptoms.In previous studies,Chinese medicine has shown promising efficacy in lipopolysaccharides(LPS)-induced ALI.However,there have been no reports of LJF and FF pairing for lung injury.The aim of this study is to compare the efficacy of herb pair Lonicerae Japonicae Flos-Forsythiae Fructus(LF)with LJF or FF alone in the treatment of ALI,and to explore whether LJF and FF have a combined effect in the treatment of lung injury,along with the underlying mechanism involved.Methods:A total of 36 mice were divided into six groups(control,model,LJF,FF,LF,dexamethasone)based on the treatments they received after undergoing sham-operation/LPS tracheal instillation.H&E staining and pulmonary edema indexes were used to evaluate lung injury severity.Alveolar exudate cells(AECs)were counted based on cell count in bronchoalveolar lavage fluid(BALF),and neutrophil percentage in BALF was measured using flow cytometry.Myeloperoxidase(MPO)activity in BALF was measured using enzyme-linked immunosorbent assay(ELISA),while the production of IL-1β,TNF-α,and IL-6 in the lung and secretion level of them in BALF were detected by quantitative polymerase chain reaction(qPCR)and ELISA.The effect of LJF,FF,and LF on the expression of Caspase-1 and IL-1βproteins in bone marrow derived macrophages(BMDMs)supernatant was assessed using Western blot method under various inflammasome activation conditions.In addition,the concentration of IL-1βand changes in lactatedehydrogenase(LDH)release levels in BMDMs supernatant after LJF,FF,and LF administration,respectively,were measured using ELISA.Furthermore,the effects of LJF,FF and LF on STING and IRF3 phosphorylation in BMDMs were detected by Western blot,and the mRNA changes of IFN-β,TNF-α,IL-6 and CXCL10 in BMDMs were detected by qPCR.Results:LF significantly attenuated the damage to alveolar structures,pulmonary hemorrhage,and infiltration of inflammatory cells induced by LPS.This was evidenced by a decrease in lung index score and wet/dry weight ratio.Treatment with LF significantly reduced the total number of neutrophil infiltration by 75%as well as MPO activity by 88%.The efficacy of LF in reducing inflammatory factors IL-1β,TNF-α,and IL-6 in the lungs surpasses that of LJF or FF,approaching the effectiveness of dexamethasone.In BMDMs,the co-administration of 0.2 mg/mL of LJF and FF demonstrated superior inhibitory effects on the expression of nigericin-stimulated Caspase-1 and IL-1β,as well as the release levels of LDH,compared to individual treatments.Similarly,the combination of 0.5 mg/mL LJF and FF could better inhibit the phosphorylation levels of STING and IRF3 and the production of IFN-β,TNF-α,IL-6,and CXCL10 in response to ISD stimulation.Conclusion:The combination of LJF and FF increases the therapeutic effect on LPS-induced ALI,which may be mechanistically related to the combined effect inhibition of cyclic-GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)and NOD-like receptor family protein 3(NLRP3)inflammasomes pathways by LJF and FF.Our study provides new medicine candidates for the clinical treatment of ALI.
