| As an important zoonotic parasite,Toxoplasma gondii severely affects human health,public sanitary and husbandry production.For the prevention and control of this parasite,the ideal vaccines and drugs are still on their roads.As Chinese herbal medicines with abundant species contain extremely complex compositions and have been used in our country for a long time,they can be as an imperative breach for developing anti-T.gondii effective drugs.Thus,twelve kinds of Chinese herbal medicines were selected according to the normal symptoms of toxoplasmosis for decoction preparation,which were subsequently used to assess the efficacy against T.gondii infections.A general research method of Chinese herbal medicine for anti-T.gondii was established,and the foundation will be laid for developing the specific therapeutic drugs of toxoplasmosis and resolving the molecular mechanism of Chinese herbal monomers inhibiting proliferation of this parasite in the future.Firstly,twelve kinds of Chinese herbal decoctions were prepared based on the processing methods of traditional medicines in this study.The data collected from their optical densities and dry weights,between which there was no significant correlation,indicated that all the obtained decoctions contain pigments with different degrees.Afterwards,five pairs of specific primers designed according to the reference sequence of T.gondii ACT1 gene were synthesized to optimize reaction condition.According to the results of PCR amplification and agarose gel electrophoresis assays,a Real-time PCR quantitative analysis system with stronger applicability,wider application scope and no influence by the decoctions was finally created.Cytotoxicity tests showed that all the decoctions possessed significantly different toxicity that can be ignored after 64-time dilution for host cells,suggested that two more works should be done before anti-Toxoplasma investigation:(I)confirm the selected herbs are useful for this parasite in vitro,and(II)unify concentrations of all the obtained decoctions.As evaluation for the decoction anti-T.gondii infection in vitro in this study was mainly performed in 24-well plate,nine curves were thus subsequently matched according to the normal regression models based on the number of tachyzoites in plates;and the infectious dose,that is 1542.21 parasites per well,was confirmed by solving their extremums.Preliminary tests showed that Semen arecae catechu(Sa),Radix cynanchi atrati(Rc),Herba artemisiae apiaceae(Ha),Hr teraxaci mongolici cum radice(Ht)and Radix bupleuri(Rb)decoctions had a significant effect on anti-T.gondii infection in vitro(P < 0.05),indicating that the selected herbs are able to inhibit the proliferation of this parasite.After unify the concentrations of decoctions,we found that the cytotoxicity of Sa,Rc,Ha,Herba eupatorii fortunei(He)and Rb at 64.0 mg/m L are lethal for host cells;another seven kinds of decoctions and their combinations with equal proportions significantly decreased the intracellular proliferation of T.gondii in comparison with blank control(P < 0.001).Moreover,Ht can uni-axially enhance the efficacy of Rhizoma atractylodis(Rh),and the synergism exists between Rh and Flos lonicerae japonicae(Fl).Data from influence of different combinations of decoctions on T.gondii infection showed that the anti-infection effect of Rh-Ht with 1:2 was lower than that of the equal proportion combination in vitro(P < 0.05),that is,excessive Ht weakened the combined anti-infection efficacy between Rh and Ht.Furthermore,the effect of Rh-Fl with 2:1 was also significantly lower than their equal proportion combination in vitro(P < 0.05),that is,excessive Rh was not benefit for Fl anti-T.gondii.The results of median effective concentration measurement showed that several genotypes of T.gondii including RH,PRU,VEG and C7 strains are sensitive to Radix lithospermi seu arnebiae(Rl),Radix aconiti(Ra)and Ht,among which the stroppiest was atypical strain C7.When prevention,all the decoctions had no statistical effects on the body weights of mice and their CD19+ B lymphocytes in spleens(P > 0.05).Interestingly,several kinds of decoctions are useful for T lymphocytes,e.g.He for CD3e~+CD4~+ T,Radix albus paeoniae lactiflorae(Rap)for CD3e~+CD8a+ T cells,and Rh,Radix angelicae pubescentis(Rp)and Fl for both of them(P < 0.05).In addition,He,Rap and Fl can also promote the proliferation of lymphocytes in spleen tissues of mice,among which the most sensitive was Rap(P < 0.01).The results for challenge showed that all the twelve kinds of decoctions with intragastric administration were not suitable to prevent the acute infection of T.gondii RH strain,whereas several decoctions were useful for PRU isolate,e.g.Rh,Sa,He,Ht for PRU acute,and Rl,Rb for PRU chronic infections.When treatment using the obtained decoctions,the data revealed that Sa and Ht can reduce the body weights of T.gondii RH-or PRU-infected mice respectively,and all decoctions have no effects on the RH and PRU acute infections(P > 0.05).Notably,Sa,Rl and Rap were suitably used for treatment of chronic infection caused by brain cysts of this parasite;Rh and Ha were benefit for the transformation from acute to chronic infection of PRU isolate.In summary,the twelve kinds of Chinese herbal medicines have different effects on mice against T.gondii infections in vitro and in vivo,even through the same decoction in the acute and chronic infections of this parasite.The above conclusions can provide important references for T.gondii prevention and control using Chinese herbal medicines,toxoplasmosis treatment,further exploration of monomer anti-Toxoplasma molecular mechanism,and the development of ideal drugs against T.gondii infections in the future. |
PDF Full Text Request