Study On The Preliminary Efficacy And Mechanism Of Action Of The Immunity-Regulating Active Site Of Oviductus Rana

Oviductus Rana is a valuable animal medicine in China,with medicinal and edible values and a long history.Oviductus Rana is a dried product from the oviduct of female forest frogs and is rich in nutrients,rich in protein,essential amino acids,unsaturated fatty acids,sterols,phospholipids and other active ingredients.Based on previous studies,it was found that oviductus rana has the effect of regulating immunity,but the active site and mechanism of action by which toadstool oil regulates immunity to exert its medicinal effect have rarely been reported.In clinical practice,cyclophosphamide,as a commonly used antitumor drug,has the side effect of immune depression.Therefore,in this paper,we firstly screened the active site of toad oil for immune modulation by establishing an immune depression model through cyclophosphamide(CTX),studied the immune modulating effect of the active site of toad oil through in vivo and in vitro experiments,and used ultra performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry(UPLC-Q/TOFMS)technique to identify and analyze the chemical components of the active site of oviductus rana.The chemical composition of the active parts of oviductus rana was identified and analyzed using ultra performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry(UPLC-Q/TOF-MS)to clarify its chemical composition and lay the material foundation for the exploration of immunity regulation by oviductus rana.Then we combined network pharmacology and molecular docking research strategy to conduct preliminary investigation on the mechanism of action of toad oil in regulating immunity,and further investigated the mechanism of action of oviductus rana in regulating immunity by RAW264.7 cells.The main research aspects of this topic are as follows:(1)Experimental study on the screening of the active site of oviductus rana to regulate immunityIn this study,different extraction methods including CO2 supercritical fluid extraction(CO2-SFE)technology,ethanol extraction and water immersion method were used to classify and extract different compounds in toad oil to obtain three different extraction sites of toad oil,including toad oil supercritical extract(ORO),toad oil alcohol extract(ORE),and toad oil water immersion(ORW).Cyclophosphamide(CTX)established an immunocompromised mouse model,collected serum after gavage administration,spleen and thymus and other immune organs,and determined the active sites of toad oil to regulate immunity by monitoring mouse weight change,immune organ index,immune factor immunoglobulin M(IGM),interferon γ(IFN-γ)and other indicators.The expression of immune-related factors such as IFN-γ indicates that the active site of immunity-modulating of toad oil is supercritical extract(ORO).(2)Study on the immunomodulatory effects of the active parts of toad oilThe supercritical extract(ORO)of toad oil was studied through animal experiments and RAW264.7 cell experiments on the modulation of immunity of Toad Oil(ORO).Firstly,RAW264.7 cells were used to study the immunomodulatory activity of ORO,mainly using CCK-8 kit to determine the effect of ORO on cell proliferation,neutral red experiment to investigate the effect of ORO on the phagocytic activity of RAW264.7,and Griess method to detect the effect of ORO on nitric oxide(NO)secretion in RAW264.7 cells with or without LPS.The ELISA kit detected the effect of ORO on LPS-induced secretion of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in RAW264.7 cells.The q-RT-PCR experiment investigated the expression of i NOS,IL-6and AKT1 m RNA in LPS-induced RAW264.7 cells by ORO.The results showed that ORO had no inhibitory effect on RAW264.7 cell activity,on the contrary,it had a significant proliferative effect.ORO can reduce the secretion of NO,TNF-α and IL-6factors of RAW264.7 by enhancing phagocytic activity and reducing lipopolysaccharide(LPS).The above results show that ORO shows a significant function of regulating the body’s immunity.Secondly,an immunocompromised mouse model was established by cyclophosphamide(CTX)induction,ORO was given by gavage at different doses,and serum and immune organs were collected from the mice.The pharmacodynamics of immune modulation by ORO was investigated by analyzing the body weight change,thymus index,spleen index,expression of immune factors immunoglobulin G(IGG),tumor necrosis factor α(TNF-α)and interleukin 6(IL-6)in mice.The results showed that ORO had a significant restoring effect on body weight and a repairing effect on CTX-injured immune organs in mice;meanwhile,ORO promoted the proliferation conversion rate of splenic lymphocytes and IGG expression of humoral immunity compared with the CTX-induced immune depression model group,indicating that ORO has a modulating effect on immune depression.(3)Exploring the mechanism of immunomodulatory effect of supercritical extract of oviductus rana based on network pharmacologyFor the first time,UPLC-Q-TOF-MS technology was used to analyze the chemical composition of ORO,and a total of 40 active ingredients were identified through UNIFI software.For the first time,the network pharmacology research method was applied to the study of the mechanism of immunity regulation of oviductus rana,and the "component-target-disease" network of immunity regulation of oviductus rana was constructed.(C20:3)and γ-linolenic acid(GLA),and predicted the possible mechanisms of ORO immune modulation.A molecular docking method was used for the preliminary validation of the screened key potent components.(4)Effects of docosahexaenoic acid(DHA),eicosatrienoic acid(C20:3)and gammalinolenic acid(GLA),the key pharmacological components of supercritical extract of oviductus rana,on the immune activity of RAW264.7 cellsIn this paper,based on the preliminary validation of the pharmacodynamic components by molecular docking,the effect of key pharmacodynamic components of ORO on the immune activity of RAW264.7 cells was investigated to verify the immunomodulatory effect of key pharmacodynamic components of ORO.In summary,this study screened the active sites of toad oil for modulating immunity by establishing an immune depression model,and made further studies on the active sites of oviductus rana for modulating immunity.For the first time,UPLC-Q/TOF-MS technique was used to analyze the chemical composition of the active site of oviductus rana in regulating immunity;network pharmacology was applied to predict the mechanism of action of ORO in regulating immunity,to screen the potential active medicinal components of ORO,and to perform preliminary validation of the potential active medicinal components using molecular docking;RAW264.7 cells were used to perform immune The immune activity of ORO was verified by RAW264.7 cells,and the immune modulating effect and mechanism of ORO were investigated in a more systematic way.