牛至提取散及其有效成分的抗炎作用研究OA

[Objective]This study aimed to investigate the in vitro and in vivo anti-inflammatory effects of herba origani extract pulvis(HOEP)and its volatile marker components thymol(Thy)and carvacrol(Car),water-soluble marker components rosmarinic acid(RA),chlorogenic acid(CA),and protocatechuic acid(PA).[Method]RAW264.7 cells in the logarithmic growth phase were selected,and treated with different concentrations of HOEP(200,400,800,1 000,1 200,and 1 600 μg/mL)and its active ingredients(20,40,80,100,120,and 160 μg/mL).The cell survival rate was determined using CCK-8 method to screen and determine the maximum non-toxic dose and the maximum test concentration of the tested drugs.An inflammatory model of RAW264.7 cells was constructed using lipopolysaccharide(LPS).The cells were divided into 8 groups:Blank control group(NC),model group(MC),and HOEP,Thy,Car,RA,CA,and PA groups.The in vitro activities of HOEP,Thy,Car,RA,CA,and PA were evaluated through cell survival rate assays,cell morphology observations,determination of nitric oxide(NO)release levels,and detection of cell inflammatory factor contents.A BALB/c mouse toe swelling model was constructed using λ-carrageenan.The mice were divided into 9 groups:NC,MC,HOEP,Thy,Car,RA,CA,PA,and aspirin(ASA)groups.The toe volume was measured to calculate the degree of toe swelling.HE staining were used to observe the histological changes of mouse toe.The content of inflammatory factors in serum was detected by ELISA.The in vivo activities of HOEP,Thy,Car,RA,CA,and PA was evaluated.[Result]Based on the maximum non-toxic dose and considering the practical operation,the final determined maximum test concentration of HOEP was 1 000 μg/mL,and the maximum test concentrations of Thy,Car,RA,CA,and PA were all 100 μg/mL.The in vitro experiments revealed that,compared with MC group,after treatment with HOEP,Thy,Car,RA,CA,and PA,the survival rate of RAW264.7 cells induced by LPS,the amount of NO released,and the contents of IL-1β,IL-6,IL-10,and TNF-α were all significantly decreased(P<0.05).The in vivo experiments revealed that compared with MC group,the color changes and swelling degrees of the mouse toe pads in HOEP,Thy,Car,RA,CA,and PA groups were significantly alleviated.At 2 hours after injection of λ-carrageenan solution,the swelling degree of mouse toe in HOEP,ASA,Thy,Car,RA,and PA groups was significantly reduced(P<0.05).At 4 hours,the swelling degree of mouse toe in HOEP,Thy,Car,RA,and PA groups was significantly reduced(P<0.05).At 6 hours,the toe swelling degrees of mice in ASA,HOEP,Thy,Car,RA,PA,and CA groups were all significantly reduced(P<0.05).Pathological observation and serological analysis revealed that,compared with MC group,after treatment with HOEP,ASA,Thy,Car,RA,CA,and PA,the infiltration of lymphocytes and granulocytes in the mouse toe swelling caused by λ-carrageenan was reduced to a certain extent.The contents of IL-1β,IL-6,IL-10,and TNF-α in mouse serum were all significantly decreased(P<0.05).[Conclusion]HOEP,Thy,Car,RA,CA,and PA could inhibit cell proliferation and differentiation,NO release,and secretion and expression of inflammatory factors induced by LPS.And they could effectively alleviate the toe swelling of mouse,the pathological damage of toe,and reduce the content of inflammatory factors in mouse serum,and achieved anti-inflammatory effects.This results provided reference for the basic study of anti-inflammatory active substances of HOEP and the elucidation of its mechanism of action.