Effect Of Oridonin On Infection And Oxidative Stress Mycobacterium Marinum

Mycobacterium tuberculosis(Mtb),which may infect humans for an extended period of time,is the source of the ancient zoonotic illness known as tuberculosis(TB).Mtb can cause long-term infection in humans,and threaten the life and health of humans and livestock.Mtb mainly infects the lungs of patients and called intrapulmonary tuberculosis.In addition,Mtb infects intestinal tract,liver,bone,kidney,and other parts,collectively referred to as extrapulmonary tuberculosis.Mtb is an intracellular parasite that is difficult to treat with short-term drug medications.The appearance of drug-resistant strains of TB has presented new challenges for treatment of TB patients.Therefore,finding new anti-TB drugs remains urgent.Mycobacterium marinum(Mm)is very similar with Mtb,and the model of Zebrafish infected with Mm can form a structure similar to the human granuloma.Therefore,infected zebrafish with Mm is an important animal model for screening anti-tuberculosis drugs.As a natural medicine,oridonin has anti-inflammatory and antimicrobial effects,so it is studied to be the evidence of finding new drugs for the treatment of tuberculosis.In this experiment,Mm、A549 cell、Raw264.7 cell and zebrafish were materials,and the growth curve of Mm was used to determine the effect of the drug.Colony unit counting and fluorescence scanning were performed on cells infected with Mm to detect the effect of drugs on bacterial infection;the effect of the drug on cellular oxidative stress and inflammatory levels with infected Mm was tested by western-blot and q RT-PCR assays.Finally,the zebrafish was used as the animal model for microinjection,and the effects of the drug on Mm infection and oxidative stress in zebrafish were determined through photographing,counting and testing.Results show,Oridonin had no significant inhibition effect on the growth curve of Mm,while the colony unit count and fluorescence scan count tests showed that oridonin significantly inhibited the infection of Mm on lung epithelial cells.The toxicity test showed that oridonin at the concentration of 30 μM had no effects on the activity of zebrafish.60 μL oridonin was added into the water for feeding zebrafish of20 m L at the final concentration of 30 μM,and then Mm injection was performed on the zebrafish.Seven days later,colony unit count and fluorescence scan count tests showed that oridonin significantly inhibited the number of Mm in zebrafish.The above results indicated that oridonin could inhibit the infection of Mm on cells and limit the proliferation of Mm in zebrafish.Mm infection induced inflammation and oxidative stress,while oridonin significantly inhibited the expression of proinflammatory mediators TNF-α,IL-6,IL-1β,and the inflammatory proteases i NOS and COX-2.Further study indicated that the inhibition effects may be caused by regulating the Nrf2/HO-1/NQO-1 and AKT/GSK3β/AMPKα signaling pathways.Next,we tested the effect of oridonin on Mm-induced oxidative stress in zebrafish.The results showed that pretreatment with oridonin significantly reduced Mm-induced malondialdehyde(MDA)in zebrafish.The above results indicated that oridonin could indeed reduce the oxidative stress in Mm infected zebrafish.In this study,we found that oridonin inhibit the Mm infection in vitro and proliferation in vivo,and inhibit the oxidative stress induced by Mm infection.Additionally,oridonin may be responsible for inhibiting the oxidative stress by regulating the Nrf2/HO-1/NQO-1 and AKT/GSK3β/AMPKα signaling pathways.The conclusion of this study not only extended the biological functions of oridonin but also provided a theoretical basis for its application of oridonin in inhibiting Mtb infection.