Effection And Mechanism Of Pulsatilla Chinensis (Bunge) Regel Saponins On Schistosoma Japonicum And Its Host

Objective:To study the toxicity of Pulsatilla chinensis (Bunge) Regel saponins (PRS) againstOncomelania hupensis (O. hupensis) of the Schistosoma japonicum (S. japonicum)intermediate, the effect on the metabolism of key enzymes of O. hupensis snails, thetoxicity on zebra fish and mammalian, the effects against in vitro or vivo S. japonicumin different developmental stages, and its immunomodulatory effect on mice infectedwith S. japonicum, provide theoretical and experimental basis for discovering newantischistosomal drug.Methods:1. The immersion test to kill snails, snails climbing test and zebra fish toxicity testwere detected by immersion method. O. hupensis snails were exposed to40%and80%of24h LC50of PRS for24h, and then choline esterase (CHE), alanine aminotransferase(ALT), alkaline phosphatase (AKP), lactate dehydrogenase (LDH) activities incephalopodium and liver of snail were determined by enzyme kinetic assay. Theultrastructure in cephalopodium and liver of snail after immersing24h in24h LC50PRSwas observed by Transmission Electron Microscopy (TEM). Mammalian toxicologytest, acute oral toxicity, and skin or eye irritation test were practised according to thestate standard of the People’s Republic of China (GB15670-1995).2. ICR mice were infected by patching abdominal infection using cercariae. Micelivers after infected42d were grinded, screened, and then eggs were obtained. Miracidiawere hatched using the eggs. Cercariae were obtained using infected snails with positivelight. The eggs, miracidia and cercariae of S. japonicum were incubated with0,1,2,3,4,5,6,7and8μg/ml PRS for different time then to observe percent hatching rates ofthem. Three-hour-old schistosomula were prepared by penetrating the mouse skin with schistosome cercariae, while7-,14-and42-day-old schistosomes were collected frommice infected with S. japonicum cercariae for7,14and42days by perfusion.3h,7,14dschistosomula and42d adult schistosome were incubated with0,1,5,10,20and30μg/ml PRS for4,24,48and72h then to observe the state of them. Mice infected with50S. japonicum cercariae were administrated orally using different doses of PRS indifferent developmental stages of S. japonicum in vivo. Mice were killed by bleeding42d post infection, and adult schistosomes were collected by perfusion and computed.Mice infected with80S. japonicum cercariae were injected by tail vein using25mg/kgdose of PRS in different developmental stages of S. japonicum in vivo. Mice were killedby bleeding42d post infection, and adult schistosomes were collected by perfusion andcomputed. Mice infected with80S. japonicum cercariae for42d were injected by tailvein using25mg/kg dose of PRS only one time in vivo. Mice were killed by bleeding24h post injected, and adult schistosomes were collected by perfusion and grinded.Glycogen, protein and enzymes of adult schistosomes were detected.3. C57BL/6mice infected with50S. japonicum cercariae were administratedorally using150mg/kg/d dose of PRS in vivo for49d. Mice were killed by bleeding49dpost oral administration, the detection of mouse body weight, liver and spleen weightchanges, worm reducing rates were estimated therapeutic efficacy of PRS. The changesof total white blood cells counts (WBCs), the rates of differential leukocyte count,immunohistochemistry of mouse liver, serum IFN-γ, IL-4, TGF-β and IL-17levelchanges, soluble egg antigen (SEA) specific IgG, IgG1, IgG2a between the infecteduntreated group and the infected treated group were estimated immunomodulatoryeffect of PRS on mice infected S. japonicum.Results:1. PRS showed the highest toxicity on snails with24h,48h,72h LC50vales of0.48,0.23,0.17mg/l and LC90of2.64,0.81,0.34mg/l. No snails climbed up in4and8mg/lPRS and the climbing up rate of0.5mg/l PRS was26.67%after24h,0.5mg/l NIC was47.78%after24h. Sublethal dose in vivo24h exposure to40%and80%24h LC50ofPRS could decrease the activities of different enzymes such as CHE, AKP, ALT indifferent body tissues of snails and PRS could not significantly inhibit LDH. Theultrastructure in cephalopodium and liver of snail was injured after immersing24h in24h LC50of PRS by TEM. Mammalian toxicology test, acute oral toxicity, and skin or eye irritation tests showed that PRS was safe to mammalian.2. PRS could supress the hatching rates of eggs for24h slightly superior to controldrug PZQ at different concentrations, especially in the4μg/ml concentration. PRS andcontrol drug PZQ showed time-and dose-dependent mortality effects on the miracidiaand cercariae of S. japonicum. PRS also exhibited in vitro impaired effect against3h,7,14d schistosomula and42d adult worms. There was a certain degree of damage to thetemporal apophysis and cortex of the surface of adult male and female S. japonicumaffected by30μg/ml PRS in vitro for4h. Oral administration of high, medium and lowdoses of PRS showed schistosomicidal effects on the every life stages of S. japonicum,but the high dose of PRS was the best. Schistosomicidal effects and killing the femaleworm results on mice infected1and21d for administration were superior to the samedose of ART and PZQ. The results about25mg/kg dose PRS of tail vein injectionshowed a better schistosomicidal effect on the every life stages of S. japonicum.Glycogen, protein, AKP, ACP, SOD, GR of S. japonicum were decreased at25mg/kginjected dose of PRS for24h.3. After C57BL/6mice infected with50S. japonicum cercariae were at150mg/kg/d oral administrated dose of PRS for49d, infected treated group miceshowed a highly significant increase in body weight as compared with infecteduntreated group (P＜0.01). On the other hand, infected treated group mice showed ahighly significant decrease in both liver and spleen weight as compared with infecteduntreated group (P＜0.01). WBCs of infected treated group mice showed highlysignificant decrease compared with infected untreated group (P＜0.01). Percentage ofneutrophil, monocyte, eosinophil of infected treated group mice showed highlysignificant decrease (P＜0.01), compared to the infected untreated group mice.Percentage of lymphocyte and basophilic of the infected treated group mice showedhighly significant increase (P＜0.01), compared to the infected untreated group mice.PRS treatment significantly reduced total worms (P＜0.01) and worm reduction rateswere55.27%compared to the infected untreated group mice. There was a significantdifference (P＜0.01) of reduction eggs in liver between the infected treatment groupmice and infected untreatment group mice. Mouse liver after49d infected treatment ofPRS showed fewer inflammatory cells surrounding the eggs than the infecteduntreatment group mice through hematoxylin and eosin staining. α-SMA, TIMP-1, TGF-β1, collagen typeⅠ, collagen type Ⅳ expression of mouse liver after49d infectedtreatment of PRS showed highly significant decrease (P＜0.01) than the infecteduntreatment group mice through immunohistochemistry. Mouse serum IFN-γ, IL-4,TGF-β and IL-17levels were significantly different (P＜0.01) between the infectedtreatment group mice and the infected untreatment group. Both IgG and IgG1levels ofthe infected treatment group mice significantly decreased in response to SEA, comparedto the infected untreatment group. Response of IgG2a against SEA did not significantly(P＞0.05) alter between the infected treatment group mice and the infected untreatmentgroup. The IgG1/IgG2a ratio was decreased with PRS treatment.Conclusions:1. PRS which could be effective at lower concentrations to kill O. hupensis of the S.japonicum intermediate host is a very promising molluscicide and almost no toxicity tomammal, lower toxicity to the zebra fish than NIC.2. The effects of PRS against in vitro or vivo S. japonicum in differentdevelopmental stages showed that PRS may eventually has therapeutic potential in thetreatment or prevention of disorders involving S. japonicum infection and is expected tobecome a new anti-schistosome drugs.3. During the treatment process of PRS against mice infected with S. japonicum,the changes of serum cytokines and related antibodies of infected mice showed thatthere was a certain relationship between the schistosomicidal role of PRS and immuneregulation.