Objective To examine the anti-inflammatory effects and potential mechanisms of polypeptide from Moschus(PPM)in lipopolysaccharide(LPS)-induced THP-1 macrophages and BALB/c mice.Methods The polypeptide was extracted fr...Objective To examine the anti-inflammatory effects and potential mechanisms of polypeptide from Moschus(PPM)in lipopolysaccharide(LPS)-induced THP-1 macrophages and BALB/c mice.Methods The polypeptide was extracted from Moschus and analyzed by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Subsequently,LPS was used to induce inflammation in THP-1 macrophages and BALB/c mice.In LPS-treated or untreated THP-1 macrophages,cell viability was observed by cell counting kit 8 and lactate dehydrogenase release assays;the proinflammatory cytokines and reactive oxygen species(ROS)were measured by enzyme-linked immunosorbent assay and flow cytometry,respectively;and protein and mRNA levels were measured by Western blot and real-time quantitative polymerase chain reaction(qRT-PCR),respectively.In LPS-induced BALB/c mice,the proinflammatory cytokines were measured,and lung histology and cytokines were observed by hematoxylin and eosin(HE)and immunohistochemical(IHC)staining,respectively.Results The SDS-PAGE results suggested that the molecular weight of purified PPM was in the range of 10–26 kD.In vitro,PPM reduced the production of interleukin 1β(IL-1β),IL-18,tumor necrosis factorα(TNF-α),IL-6 and ROS in LPS-induced THP-1 macrophages(P<0.01).Western blot analysis demonstrated that PPM inhibited LPS-induced nuclear factorκB(NF-κB)pathway and thioredoxin interacting protein(TXNIP)/nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domain containing 3(NLRP3)inflammasome pathway by reducing protein expression of phospho-NF-κB p65,phospho-inhibitors of NF-κB(IκBs)kinaseα/β(IKKα/β),TXNIP,NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),and pro-caspase-1(P<0.05 or P<0.01).In addition,qRT-PCR revealed the inhibitory effects of PPM on the mRNA levels of TXNIP,NLRP3,ASC,and caspase-1(P<0.05 or P<0.01).Furthermore,in LPS-induced BALB/c mice,PPM reduced TNF-αand IL-6 levels in serum(P<0.05 or P<0.01),decreased IL-1βand IL-18 levels in the lungs(P<0.01)and alleviated pathological injury to the lungs.Conclusion PPM could attenuate LPS-induced inflammation by inhibiting the NF-κB-ROS/NLRP3 pathway,and may be a novel potential candidate drug for treating inflammation and inflammation-related diseases.展开更多
基金the Establishment of Key Disciplines of Traditional Chinese Medicine in Sichuan Province-Pharmacology of Chinese Materia Medica(No.2020ZDXK01)the Systemic Research and Development of Moschus(No.D-2019-6 and D-2019-8)。
文摘Objective To examine the anti-inflammatory effects and potential mechanisms of polypeptide from Moschus(PPM)in lipopolysaccharide(LPS)-induced THP-1 macrophages and BALB/c mice.Methods The polypeptide was extracted from Moschus and analyzed by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Subsequently,LPS was used to induce inflammation in THP-1 macrophages and BALB/c mice.In LPS-treated or untreated THP-1 macrophages,cell viability was observed by cell counting kit 8 and lactate dehydrogenase release assays;the proinflammatory cytokines and reactive oxygen species(ROS)were measured by enzyme-linked immunosorbent assay and flow cytometry,respectively;and protein and mRNA levels were measured by Western blot and real-time quantitative polymerase chain reaction(qRT-PCR),respectively.In LPS-induced BALB/c mice,the proinflammatory cytokines were measured,and lung histology and cytokines were observed by hematoxylin and eosin(HE)and immunohistochemical(IHC)staining,respectively.Results The SDS-PAGE results suggested that the molecular weight of purified PPM was in the range of 10–26 kD.In vitro,PPM reduced the production of interleukin 1β(IL-1β),IL-18,tumor necrosis factorα(TNF-α),IL-6 and ROS in LPS-induced THP-1 macrophages(P<0.01).Western blot analysis demonstrated that PPM inhibited LPS-induced nuclear factorκB(NF-κB)pathway and thioredoxin interacting protein(TXNIP)/nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domain containing 3(NLRP3)inflammasome pathway by reducing protein expression of phospho-NF-κB p65,phospho-inhibitors of NF-κB(IκBs)kinaseα/β(IKKα/β),TXNIP,NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),and pro-caspase-1(P<0.05 or P<0.01).In addition,qRT-PCR revealed the inhibitory effects of PPM on the mRNA levels of TXNIP,NLRP3,ASC,and caspase-1(P<0.05 or P<0.01).Furthermore,in LPS-induced BALB/c mice,PPM reduced TNF-αand IL-6 levels in serum(P<0.05 or P<0.01),decreased IL-1βand IL-18 levels in the lungs(P<0.01)and alleviated pathological injury to the lungs.Conclusion PPM could attenuate LPS-induced inflammation by inhibiting the NF-κB-ROS/NLRP3 pathway,and may be a novel potential candidate drug for treating inflammation and inflammation-related diseases.
