In Vitro And In Vivo Effect Of MAPK Signal Transduction Pathway Inhibitors On Echinococcus Multilocularis

Objective: To evaluate the effect of mitogen-activated protein kinase(MAPK)signal transduction pathway inhibitors against alveolar echinococcosis in vitro and in vivo.Methods: Echinococcus multilocularis metacestode cysts and protoscolices were obtained from infected mice.Protein chip technology was utilized to screen for key highly expressed target proteins in the MAPK pathway in this parasite and their corresponding inhibitors.Four-week-old BALB/ c female mice used for the in vivo experiment underwent inoculation of E.multilocularis by intraperitoneal injection,as well as intragastric administration of MAPK inhibitors for 6 weeks.We included 6 groups of mice: a phosphate-buffered saline(PBS)group(negative control);an albendazole-treated group(positive group);and 4 experimental groups treated with TRx0237 mesylate,GDC-0994,Pifithrin-? hydrobromide,or Selonsertib.Echinococcus multilocularis protoscolices were collected and cultured in 1066 medium with penicillin/ streptomycin and 10% fetal bovine serum.The in vitro experiment included a PBS group(negative control),a dimethyl sulfoxide–treated group(solvent group),and 4 inhibitor-treated groups as in the in vivo experiment(experimental groups).Each inhibitor group received 4 drug concentrations(5,30,55,and 80 ?M),and the experiment was performed in triplicate per sample.Fluorescence microscopy was used to evaluate the survival rate of the protoscolices every 48 hour beginning from the first 24 hour.The same grouping was used to evaluate cytotoxicity on E.multilocularis germinal cells and L02 cells.Results: The target of MAPK signal pathway that screened from the protein chip experiment were Tau,ERK,p53 and ASK1.Corresponding inhibitors were TRx0237 mesylate,GDC-0994,Pifithrin-? hydrobromide and Selonsertib.The average weights of E.multilocularis metacestode cyst tissue from each group of the in vivo experiment were 873 mg(PBS),335 mg(albendazole),323 mg(TRx0237 mesylate),370 mg(GDC-0994),340 mg(pifithrin-? hydrobromide),and 642 mg(Selonsertib).Weight loss rate of Echinococcus multilocularis cysts from each drug groups were 61.63 %,63 %,57.61 %,61.05 % and 26.46 %.Results showed albendazole,TRx0237 mesylate,and pifithrin-? hydrobromide had significant inhibitory effects on inhibition of E.multilocularis(P < 0.05).We found a positive correlation between drug concentrations and the inhibitory effects seen in the in vitro experiment,with the differences in contrast with the control group becoming statistically significant after 72 hour of treatment(P < 0.05).The inhibition rates of TRx0237 mesylate to germinal cells by drug concentration were 23.73 %,46.59 %,74.71 %,and 77.44 %.Other drugs had no effect on germinal cells.All the inhibitors had low toxicity on L02 cells.Conclusions: MAPK signal pathway targets Tau,ERK,and p53 have important regulation effects on the physiological activities and biological potency in Echinococcus multilocularis.The corresponding MAPK inhibitors have different inhibition against Echinococcus multilocularis in vitro and in vivo.The targets have the potential to become a potential drug target for the treatment of Alveolar Echinococcosis.