Research On The Effect Of Obatoclax Against Toxoplasma Gondii

Toxoplasma gondii is a specialized intracellular parasite that is extremely widespread,with approximately one-third of the world’s population infected with T.gondii and a worldwide pandemic trend.When T.gondii infects immunocompromised hosts,it can cause symptoms such as encephalitis,lymph node enlargement,retinitis and myocarditis,and is an important zoonotic pathogen.The prevalence and spread of toxoplasmosis is a serious threat to human health and public health safety.The current "gold standard" drugs for the treatment of toxoplasmosis are pyrimethamine and sulfadiazine.However,the drugs have strong toxic side effects,causing nausea,vomiting and other symptoms in the host,and in severe cases,causing abnormal liver function in the host,and long-term use may lead to drug resistance.Therefore,it is urgent to explore new drugs with low toxicity and high efficiency for the prevention and treatment of toxoplasmosis.Obatoclax(also known as GX15-070)is a novel anticancer drug for the treatment of hematologic malignancies,as well as for the treatment of coronavirus and parasitic infections,especially for the inhibition of the growth and development of Plasmodium falciparum and Cryptosporidium parvum.Because of the similar growth and development cycle and pathogenic mechanism of P.falciparum,Obatoclax may be a potential drug for the treatment of toxoplasmosis.At the same time,Obatoclax has the advantages of less toxic side effects and less likely to develop drug resistance.Therefore,this study investigated the therapeutic effects of obatoclax on toxoplasmosis through in vivo and in vitro tests,and investigated the mechanism of drug action to provide a theoretical basis for obatoclax as a potential drug for the treatment of toxoplasmosis.In this study,we first screened the safe concentration of 0-100 n M for the action of Obatoxin on Vero cells using the CCK-8 Cell Activity Kit.The ability of obatoclax to resist the invasion of cells by T.gondii tachyzoites was assessed by CCK-8 cell activity assay and Giemsa staining method.After infection of cells with T.gondii at a ratio of 5:1,cells were incubated with different concentrations of obatoclax and the results showed that obatoclax significantly inhibited T.gondii invasion of cells in a dose-dependent manner compared to the control group.Three methods,CCK-8 cell activity assay,Giemsa staining and fluorescence quantitative PCR,were used to investigate the ability of the drug to resist T.gondii proliferation in cells.The results showed that compared with the T.gondii-infected control group,the cellular activity of the drug administration group was significantly increased,and the number of intracellular worms and worm load were significantly reduced,demonstrating that obatoclax could significantly inhibit T.gondii proliferation in a dosedependent manner.A mouse model of acute T.gondii infection was established to investigate the therapeutic effect of obatoclax on T.gondii-infected mice.The results showed that obatoclax significantly increased the survival rate of the infected mice.q PCR results showed that the parasite load in the heart,liver,spleen and kidney of the treated mice was significantly reduced in a dosedependent manner compared with that of the control group.The results of in vitro and in vivo experiments together demonstrated that obatoclax can effectively inhibit T.gondii infection.In order to investigate the mechanism of action of obatoclax against T.gondii infection,transmission electron microscopy(TEM)was used to observe the effect of the drug on the ultrastructure of T.gondii tachyzoites,and ROS detection kit was used to measure the level of ROS in tachyzoites after incubation with the drug,as well as the level of mitochondrial membrane potential(ΔΨm)and ATP production in tachyzoites after incubation with the drug.The results showed that obatoclax disrupted the mitochondrial structure of T.gondii,induced an increase in ROS production,and decreased the mitochondrial membrane potential and ATP level of T.gondii,demonstrating that obatoclax may induce the death of the parasite by disrupting the redox balance of T.gondii.Studies have demonstrated that various drugs exert anti-T.gondii effects by regulating host cell autophagy and apoptosis mechanisms.Therefore,in this study,we assessed the regulatory effects of obatoclax on host cell autophagy and apoptosis by detecting key factors of autophagy and apoptosis.The results showed that the autophagy-related protein m RNA level increased significantly,the autophagyrelated protein expression level increased,and the LC3 II/LC3 I ratio increased significantly when T.gondii infected Vero cells for 4 h.The autophagy level decreased significantly when the infection time of T.gondii was prolonged,especially at 24 h of infection compared with that at 4 h of infection.It was demonstrated that T.gondii could significantly induce autophagy in host cells at 4 h of infection,and that T.gondii might regulate intracellular autophagic clearance after adapting to the intracellular environment in the later stages of infection.The results of obatoclax treatment for T.gondii infection showed that obatoclax could significantly upregulate the expression of autophagyrelated proteins and maintain a higher level of autophagy in cells at 24 h compared with the T.gondii-infected control group.This demonstrates that obatoclax can exert anti-T.gondii effects by enhancing cellular autophagy.Apoptosis is one of the important defense mechanisms against intracellular pathogen infection.WB and AO/EB assays were used to detect key apoptosis proteins and the number of apoptotic cells to assess the regulatory effect of obatoclax on apoptosis.The results showed that T.gondii infection could significantly promote CASP3 and PARP protein shearing and increase the number of apoptotic cells,demonstrating that T.gondii infection could cause apoptosis.The results of obatoclax treatment for T.gondii infection experiment showed that compared with the T.gondii infection group,obatoclax significantly reduced CASP3 and PARP shear levels and the number of apoptotic cells,demonstrating that obatoclax can alleviate apoptosis caused by T.gondii infection.This study demonstrates for the first time that obatoclax inhibits the invasion and proliferation of T.gondii into host cells in a dose-dependent manner with a high therapeutic index.Obatoclax alleviated liver and spleen histopathy caused by T.gondii infection in mice,significantly reduced the T.gondii load in all tissues and promoted survival of mice.Obatoclax increased tachyzoite ROS production,decreased ΔΨm and ATP levels,and disrupted mitochondrial structure,thereby disrupting the oxidative-antioxidative homeostasis of tachyzoites,which in turn led to oxidative stress and inhibited the growth of T.gondii tachyzoites.In addition,Obatoclax also has an indirect inhibitory effect against T.gondii infection by enhancing the level of autophagy in host cells and alleviating the level of apoptosis in infected cells.This study reveals that obatoclax can be a potential candidate compound against T.gondii infection and provides a theoretical basis for the development of novel anti-Toxoplasma drugs.