The Traditional Chinese Medicine Ejiao Is An Immunoprotective Agent Against Apoptosis

Ejiao(Latin:Colla corii asini)is a traditional Chinese medicine that has a history of more than 2,000 years in China.It was written by Li Shizhen in compendium of materia meclica as a top-class promotion of hematopoietic drugs.Ejiao is refined from the skin of black donkey(Equus asinus L.),the main bioactive component in ejiao is thought to be collagen.It has been widely accepted that ejiao promotes hematopoiesis and immune system function.However,the pharmacological efficacy of ejiao has not been clearly demonstrated in cellular,animal models and the human body.We all know that apoptosis and cell regeneration are in equilibrium under normal physiological conditions.However,apoptosis occurs abnormally,it is accompanied by the occurrence of disease.At present,cell apoptosis or oxidative stress plays an extremely important role in the development of many clinical diseases.For example,sepsis is a multi-organ dysfunction disease caused by bacterial and other pathogenic microorganisms infected.Until now,the treatment of sepsis mainly utilizes antibiotics and other auxiliary therapeutic measures in clinical practice.However,these methods are often unable to exert a good therapeutic effect due to drug resistance,antibiotic toxicity,and disease complexity.The immune system of patients with sepsis is prone to oxidative stress and apoptosis.Therefore,the study attempts to investigate whether ejiao can promote oxidative stress resistance in cell cultures,mouse sepsis model and human peripheral blood mononuclear cells,and whether ejiao can be used for the treatment of common clinical diseases?We studied the pharmacological mechanism of ejiao by the cultured cells.Ejiao is not toxic to the cultured cells;and it promoted cell division and proliferation under a serum free medium.In addition,ejiao had an anti-apoptotic effect on the RAW264.7 cells under oxidative stress.To prove the universality of ejiao protection through other immune cells and non-immune cells,the data indicated that the anti-apoptotic effect is immune cell specific.We quantified apoptosis by two commonly used protein markers,immunoblotting the cleaved caspase-3 and cleavage of PARP1,a substrate of caspase-3.And ejiao treatment reduced the cleavage of these proteins.Under oxidative stress conditions,we found that ejiao significantly reduced intracellular ROS levels and improved mitochondrial function.In Paraquat-induced Drosophila oxidative stress model,we proved that ejiao has directly anti-oxidative stress.To explore the anti-apoptotic effect in vivo,we tested lipopolysaccharides(LPS)model of sepsis.By DNA ladder assay and TUNEL staining,we found that ejiao can significantly reduce the number of apoptosis in the immune organs(thymus and spleen)of sepsis mice.Furthermore,we extracted the mouse serum,and determined inflammatory cytokines including IL-6,IL-10,IL-1β,HMGB1 and TNF-a by ELISA(enzyme-linked immunosorbent assay).Ejiao could significantly increased the level of anti-inflammatory cytokines IL-10 in the early stage of sepsis,made the immune system of mice positively to resist the inflammatory response;and reduced the pro-inflammatory cytokines IL-1β,IL-6,TNF-α,HMGB1 levels in the late stages of sepsis.Finally,improved the survival rate of sepsis mice.We observed that these cells(THP1 cell and PBMC cell)were very sensitive to H2O2 treatment and caused a rapid cell death.Therefore,we applied a mild inducer of apoptosis,Etoposide,Staurosporine,and LPS plus Nigericin.Determined by PARP1 cleavage,Caspase 3/7 activity assay and ATP assay,ejiao rescued the apoptosis in those cells.Apoptosis induced by sepsis affects the host’s immune function in different ways.Moreover,apoptosis in immune cells induced by oxidants plays key pathological roles in aging and cancer radiation therapy.As an immunoprotective agent,ejiao is expected to become an anti-apoptotic drug that be used clinically to treat a variety of diseases caused by apoptosis and oxidative stress.