| BackgroundAs a global pandemic disease, malaria is a major threat to humans all over the world, causing destructive damage on the human health and heavily burdening economic development. Drug-resistance of plasmodium, especially Plasmodium falciparum, is an increasingly challenging problem, besides, habitats of mosquitos are expanding with global warming. Subsequently, malaria is becoming the most fatal and destructive vector-borne diseases.Asian and African natural environment of high temperature and humid environment as malaria endemic areas have influences on the pathogenesis, development and prognosis of malaria. In Chinese medicine, the incidence of its infection is also closely related to Damp-Heat exopathogen. Treatment on Syndrome Differentiation is the core of Chinese medicine.Setting up animal models with combination of disease and syndrome can provide a laboratory basis for the development of Chinese medicine preventing and treating malaria. Rational use of drugs for delaying falciparum drug resistance has the important significance. Reserches on effects of "Dampness-Heat" factors on development of drug-resistance of Plasmodium berghei against artemisinin and piperaquine in artemisinin-piperaquine compound tablets can be the fundamental evidences for further investigation on the mechanisms for drug resistance and the applications of Artequick in damp and warm regions of Asia and Africa.ObjectiveIn this research, a disease-pattern conjugated rodent malaria model of the damp-heat syndrome was established based on the basic theories of Traditional Chinese Medicine, while factors including environments, physique differences and plasmodium infections were taken into consideration. Strains resistant to artemisinin and piperaquine were cultivated on the damp-heat syndrome animal models, and an effect of "Dampness-Heat" factors on development of drug-resistance of Plasmodium berghei against artemisinin and piperaquine in Artequick was analyzed by comparative. Observed whether damp-heat syndrome animal models would affect the drug resistance to Plasmodium berghei K173 strain of artemisinin and piperaquine or not.Methods1 Kunming mice were given high carbonhydrate and high fat diet and kept in damp and heat environment to optimize the temperature and duration required for establishment of damp-heat syndrome model. Then Plasmodium berghei K173 (P. b. K173) was intraperitoneally injected to infect the mice to establish mice malaria model of damp-heat syndrome. Regular physical status such as body temperature (rectal), urine and faeces were recorded along with expression levels of biochemical markers including aquaporin-4 (AQP-4) and tumor necrosis factor α (TNF-α) for the primary evaluation on the animal model.2 Comparison was made among the malaria-normal environment group, damp-heat environment group and damp-heat syndrome group concerning plasmodium infection rates, INF-γ、IL-4 levels and the ratios.3 By administrating stepwisely increasing doses of artemisinin and piperaquine, resistant strain was cultivated in both malaria moderns in damp and heat environment and normal environment. ED90 and I90 were determined and calculated based Peters four days tests at interval of five generations. Trends of I90 in different groups were recorded and analyzed to elucidate the effect of damp and heat environment on drug resistance of P. b. K173 strain against artemisinin and piperaquine.Results1 In the present research, treatment of high carbonhydrate and high fat diet and damp-heat environment caused the uninfected mice to excrete mucous stool, yellow urine and the rectal temperature to rise. After manual infection with P. b. K173, the rectal temperature kept rising until the experiment was completed, when the mice showed symptoms including less weight, high rectal temperature and decreased food. We can saw symptoms including water intake, spirit burnout, less activity, dull fur, mucous stool, yellow urine and swollen anus. As these symptoms were consistent to identification standards and clinical manifestations of damp-heat syndrome, the model establishment was successfully accomplished. Average levels of AQP-4 and TNF-α were increased to 3.15±0.33ng/mL,706.66±84.59pg/mL, respectively, in the malaria group, comparing to the normal group (2.46±0.12ng/mL,313.51±43.08pg/mL). The damp-heat syndrome malaria group showed higher TNF-α level than damp-heat syndrome colibacillosis mice (537.23±34.78pg/mL). All differences were statistically significant (P<0.05), it indicated that immunological responses were highly activated in damp-heat syndrome malaria mice.2 Observed protozoa infection rate under the damp and heat environment in early stages of infection in mice was lower than the normal. Contrast three groups of mice under the malaria-normal environment, damp-heat environment and damp-heat syndrome concerning IFN-γ, IL-4, the swallow percentage of peritoneal macrophages and NO changes. These indexes early increased in early and decreased late. The serum IFN-y levels in mice on day 4 reached the peaks (1091.51±73.60pg/mL,1186.02±41.14 pg/mL,1112.77±43.23 pg /mL). IL-4 levels on day 6 reached the peaks as 337.31±16.88 pg/mL,333.32 ±18.63 pg/mL,347.51±26.60 pg/mL. The levels of IFN-γ and IFN-γ /IL-4 ratio under the damp and heat environment was higher than the normal in early stages of infection(P<0.05). IFN-y activated and enhanced the the swallow function of peritoneal macrophages in early, but later IFN-γ, IL-4 decreased. Protozoal infections had the explosive growthes, eventually leading to death in mice.3 In the cultivation of resistant strain, for 20 generations the resistant indexes of the malaria group and the damp-heat syndrome malaria group were 5.8 and 2.7 respectively. Dose of artemisinin increased from 185.88 mg/kg to 418.67 mg/kg,293.10mg/kg in 3 days, the strain showed slight resistance to artemisinin, the resistant indexes of the malaria group and the damp-heat syndrome malaria group were 10.9 and 5.2 respectively. Piperaquine-resistant strain in the malaria group gained medium resistance, whereas the strain in the damp-heat syndrome malaria group was slightly resistant. The results indicated that damp-heat syndrome had influences on the development of drug-resistance of Plasmodium berghei against artemisinin and piperaquine.Conclusions1 Usig the way of high carbonhydrate and high fat diet and kept in damp and heat environment could establish the damp-heat syndrome rodent model.2 Kunming mice showed Th1 immunological reaction featured by INF-γ elevation during early-stage of P. b. K173 infection. High plasma INF-γ level was responsible for the initial suppression on the growth of plasmodium in the damp-heat syndrome malaria mice during early-stage infection.3 The resistant indexes for 20 generations indicated that damp-heat syndrome had influences on the development of drug-resistance of Plasmodium berghei against artemisinin and piperaquine. |
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