Study On Absorption Mechanism Of Deinagkistrodon Acutus Venom Through Digestive Tract And Its Immunomodulatory Effect

Objective:Animal experiment was used to explore whether the Deinagkistrodon acutus venom can be absorbed into the blood in the form of protein peptides and the distribution of the Deinagkistrodon acutus venom protein in the gastrointestinal tract after oral administration.The immunosuppressive mouse model was constructed by intraperitoneal injection of cyclophosphamide.Detected the pharmacological effects of Deinagkistrodon acutus venom on the immune function of mice under normal and immunosuppressive conditions.Detected the mechanism of Deinagkistrodon acutus venom enhancing the immune function of immunosuppressive mice.Provide experimental and theoretical basis for the further development and utilization of Deinagkistrodon acutus venom.Methods:(1)Rats were given Deinagkistrodon acutus venom by gavage.The Shotgun mass spectrometry was used to investigate the presence of the Deinagkistrodon acutus venom proteins in the blood of rats,and the immunohistochemistry was used to detect the distribution of the Deinagkistrodon acutus venom proteins in the gastrointestinal tract of rats.HE staining was used to detect the effect of Deinagkistrodon acutus venom on gastrointestinal tract in rats.(2)Immunosuppressive mice model was established by intraperitoneal injection of cyclophosphamide.Normal mice and immunosuppressive mice were divided into low,medium and high dose of Deinagkistrodon acutus venom groups.Deinagkistrodon acutus venom(30,60,120 mg/kg)was given by gavage for 14 days.Vital signs such as breathing and mental state were observed.Spleen and thymus tissues were weighed,and spleen and thymus index were calculated.Prepared the spleen cell suspension,Con A induced T lymphocyte proliferation and LPS induced B lymphocyte proliferation were detected by CCK-8 method,phagocytic coefficientαwas detected by carbon particle clearance method,phagocytic rate of peritoneal macrophages was detected by neutral red method,delayed type hypersensitivity was induced by dinitrofluorobenzene and detected the degree of auricle swelling.Explore the effect of oral administration of Deinagkistrodon acutus venom on the immune function of normal mice and immunosuppressive mice.(3)Immunosuppressive mice model was established by intraperitoneal injection of cyclophosphamide.Deinagkistrodon acutus venom(30,60,120 mg/kg)was given by gavage for 14 days.Panax quinquefolium was used as positive control group.HE staining was used for histopathological examination of spleen,flow cytometry was used to detect the changes of CD4~+and CD8~+T cell subsets in peripheral blood.ELISA was used to detect the levels of TNF-αand IL-4cytokines in spleen tissue,real-time PCR was used to detect the expression levels of transcription factors T-bet and GATA-3 mRNA in spleen tissue.Western boltting was used to detect the protein expression levels of STING and IRF3 of c GAS-STING signaling pathway in the spleen of mice,and to analyze the immunomodulatory effect of Deinagkistrodon acutus venom on immunosuppressive mice.Results:(1)There were 4 groups and 3 groups of proteins in the plasma of rats after 1 h and 3 h after oral administration of Deinagkistrodon acutus venom which matched the protein database of Deinagkistrodon acutus venom.The main active proteins were snake venom metalloprotease and acid phospholipase A2.After entering the digestive tract,the Deinagkistrodon acutus venom was distributed in the rat stomach and duodenum.It was mainly distributed in the duodenum at 1 h after oral administration and in the stomach and duodenum at 3ours after oral administration.(2)Different doses of Deinagkistrodon acutus venom can significantly increase the spleen index of normal mice(P<0.05),and inhibit the atrophy of the thymus and spleen caused by cyclophosphamide,improve the proliferation activity of B lymphocytes and the reduction of the phagocytic rate of mononuclear macrophages caused by cyclophosphamide(P<0.05).60 mg/kg of Deinagkistrodon acutus venom can promote the proliferation of T and B lymphocytes in normal mice,significantly increase the phagocytic coefficient and phagocytic rate of monocyte macrophages in normal mice,and promote delayed-type hypersensitivity in normal mice(P<0.05),and improve the reduction of T lymphocyte proliferation activity caused by cyclophosphamide(P<0.05).120 mg/kg of Deinagkistrodon acutus venom can promote the proliferation of T and B lymphocytes in normal mice,increase the phagocytic coefficient and phagocytic rate of mononuclear macrophages in normal mice,promote the delayed type hypersensitivity of normal mice(P<0.05),improve the decrease of phagocytic coefficient of mononuclear macrophages induced by cyclophosphamide and restore the delayed type hypersensitivity of immunosuppressive mice(P<0.05).(3)30,60 mg/kg of Deinagkistrodon acutus venom can significantly increase the expression level of T-bet mRNA and T-bet/GATA-3 value of immunosuppressed mice,reduce the expression level of GATA-3 mRNA(P<0.05).60,120 mg/kg of Deinagkistrodon acutus venom can significantly increase the percentage of CD4~+T lymphocytes and reduce the percentage of CD8~+T lymphocytes in the peripheral blood of immunosuppressed mice(P<0.05).60 mg/kg of Deinagkistrodon acutus venom can significantly down regulate the expression of STING and IRF3 protein of immunosuppressive mice(P<0.05).120 mg/kg of Deinagkistrodon acutus venom can increase the serum cytokines TNF-αand IL-4 concentration of immunosuppressed mice(P<0.05).Conclusion:Deinagkistrodon acutus venom can be absorbed into the blood through the digestive tract in the form of peptides.After being absorbed by the digestive tract,Deinagkistrodon acutus venom has varying degrees of influence on the immune function of normal mice and immunosuppressed mice.It can significantly enhance the humoral,cellular and non-specific immune functions of mice.Deinagkistrodon acutus venom can improve the immunosuppressive state of mice caused by cyclophosphamide.Deinagkistrodon acutus venom can increase the concentration of spleen cytokines TNF-αand IL-4 of immunosuppressive mice,regulate the expression levels of transcription factors T-bet and GATA-3 mRNA,regulate the cGAS-STING signal pathway.The results show that Deinagkistrodon acutus venom has low toxicity and can significantly improve the immune function,which has potential drug development prospects.