【摘要】：目的： 通過研究白頭翁總皂苷（Pulsatilla chinensis (Bunge) Regel saponins，PRS）對日本血吸蟲（Schistosoma japonicum）中間宿主釘螺（Oncomelania hupensis）的殺滅作用、對釘螺新陳代謝關(guān)鍵酶的影響、對斑馬魚和哺乳動物的毒性、對日本血吸蟲體內(nèi)外不同發(fā)育階段的作用及對感染日本血吸蟲小鼠的免疫調(diào)節(jié)作用，為白頭翁總皂苷成為新的抗血吸蟲新藥提供理論和實驗依據(jù)。 方法： 1.采用浸泡法，進行浸殺釘螺試驗、釘螺上爬試驗及斑馬魚毒性試驗；采用酶促動力學(xué)方法檢測釘螺暴露在PRS濃度為浸泡24h LC50的40%和80%藥液中24h后的頭足及肝臟膽堿酯酶（choline esterase，CHE）、丙氨酸氨基轉(zhuǎn)移酶（alanineaminotransferase，ALT）、堿性磷酸酶（alkaline phosphatase，AKP）、乳酸脫氫酶（lactate dehydrogenase，LDH）的酶活值；采用透射電鏡觀察濃度為24h LC50的PRS作用釘螺后其頭足及肝臟的超微結(jié)構(gòu)；采用中華人民共和國國家標準GB15670-1995進行哺乳動物急性經(jīng)口毒性試驗和皮膚、眼睛刺激試驗。 2.采用尾蚴腹部貼片法感染ICR小鼠，感染后42d的小鼠肝臟經(jīng)過研磨、過篩獲得蟲卵，毛蚴由蟲卵孵化，尾蚴由陽性感染釘螺光照逸出，用含有0、1、2、3、4、5、6、7、8μg/ml的PRS藥液分別在不同的時間作用于蟲卵、毛蚴、尾蚴；PRS對體外培養(yǎng)的日本血吸蟲不同發(fā)育階段的作用采用經(jīng)皮膚或灌注沖蟲法在3h、7、14、42d對感染不同數(shù)量尾蚴的小鼠進行剖殺取蟲，用含有0、1、5、10、20、30μg/ml的PRS培養(yǎng)液對童蟲及成蟲進行培養(yǎng)，觀察藥物在不同時間段的殺蟲作用；將感染50條日本血吸蟲尾蚴的小鼠在血吸蟲不同發(fā)育階段用不同劑量的PRS藥液灌服，42d后灌注沖蟲計算荷蟲量及減蟲率；將感染80條尾蚴的小鼠在感染后的不同時間經(jīng)過尾靜脈注射25mg/kg PRS藥液，42d后灌注沖蟲計算荷蟲量及減蟲率；PRS對日本血吸蟲糖原、蛋白及酶的影響采用對感染42d的小鼠一次性尾靜脈注射25mg/kg的PRS藥液，24h后剖殺取蟲研磨，檢測PRS對蟲新陳代謝的影響。 3.采用感染50條尾蚴的C57BL/6小鼠連續(xù)49d口服150mg/kg/d劑量的PRS，通過檢測小鼠體重、肝脾重量的變化及減蟲效果估計長期用藥的治療效果；通過檢測感染治療組與感染不治療組之間小鼠白細胞及其組成細胞的比率變化、肝臟的免疫組化、血清IFN-γ、IL-4、TGF-β和IL-17水平的變化、蟲卵可溶性抗原（solubleegg antigen，SEA）特異性抗體IgG、IgG1、IgG2a值的改變探究PRS對日本血吸蟲感染小鼠的免疫調(diào)節(jié)作用。 結(jié)果： 1. PRS經(jīng)過24h、48h、72h浸殺釘螺后的LC50分別為0.48、0.23、0.17mg/l，LC90分別為2.64、0.81、0.34mg/l；8和4mg/l PRS作用后無釘螺上爬，0.5mg/l PRS作用24h后總上爬率為26.67%，0.5mg/l NIC作用24h后總上爬率為47.78%。釘螺用PRS濃度為40%及80%的24h LC50浸泡24h后，其頭足及肝臟的CHE、AKP、ALT的活力明顯降低（P＜0.05），LDH變化不大；24h LC50PRS作用24h后，釘螺頭足及肝臟細胞的透射電鏡顯示PRS對釘螺的超微結(jié)構(gòu)有一定程度的損傷；斑馬魚試驗顯示相對于氯硝硫胺（niclosamide，NIC），PRS對魚類的致死濃度與殺螺濃度差別較大；哺乳動物急性經(jīng)口毒性試驗、眼刺激實驗、急性皮膚刺激試驗結(jié)果顯示PRS對哺乳動物無毒性。 2.不同濃度的PRS及對照藥物吡喹酮（praziquantel，PZQ）對日本血吸蟲蟲卵作用24h后的孵化結(jié)果顯示PRS對蟲卵孵化的抑制效果略優(yōu)于PZQ的作用，尤其是在4μg/ml濃度時，日本血吸蟲蟲卵對PRS的作用更敏感；PRS及對照藥物PZQ在不同時間對毛蚴、尾蚴的作用結(jié)果顯示其死亡率對PRS及PZQ的濃度及作用時間有一定的依賴性；在不同濃度PRS作用下日本血吸蟲各個發(fā)育時期體外培養(yǎng)的結(jié)果顯示藥物對日本血吸蟲的3h、7、14d童蟲及42d成蟲均有殺傷作用；日本血吸蟲雄蟲及雌蟲經(jīng)過30μg/ml PRS體外作用4h后蟲體體表的感覺乳突及皮層均有一定程度的損壞；不同劑量PRS對不同感染時期的小鼠口服用藥結(jié)果表明高、中、低劑量的PRS在體內(nèi)均有一定的殺蟲作用，但300mg/kg劑量的PRS有較好的療效，感染1d和21d開始用藥的殺蟲及殺雌蟲效果均優(yōu)于相同劑量的青蒿琥酯（artesunate，ART）和PZQ；尾靜脈注射25mg/kg劑量PRS的實驗結(jié)果表明在日本血吸蟲感染小鼠的不同階段用藥均有比較好的殺蟲作用；25mg/kg劑量PRS作用24h后日本血吸蟲雄蟲、雌蟲的糖原、蛋白、AKP、酸性磷酸酶（acid phosphatase，ACP）、超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽還原酶（glutathionereductase，GR）均有所降低。 3.用150mg/kg/d劑量的PRS連續(xù)49d灌服感染50條尾蚴的C57BL/6小鼠發(fā)現(xiàn)，PRS治療組小鼠體重的增加與感染不治療組比較存在明顯差異（P＜0.01），肝脾重量的降低與感染不治療組比較存在明顯差異（P＜0.01）；PRS治療后小鼠白細胞的降低與感染不治療組比較存在明顯差異（P＜0.01），中性粒細胞、單核細胞及嗜酸性細胞比率的降低與感染不治療組比較存在明顯差異（P＜0.01），淋巴細胞和嗜堿性細胞比率的增多與感染不治療組比較存在明顯差異（P＜0.01）；PRS長期用藥對感染50條尾蚴小鼠的荷蟲量結(jié)果顯示PRS用藥組與感染不治療組比較減蟲效果存在明顯差異（P＜0.01），減蟲率為55.27%。肝臟蟲卵的減少與不治療組比較存在明顯差異（P＜0.01）；用藥49d后小鼠肝臟的蘇木素伊紅染色（hematoxylin andeosin staining，HE染色）顯示PRS用藥組肝臟的蟲卵及卵周圍炎性細胞相對于感染不治療組較少；免疫組化顯示PRS藥物治療后肝臟的α-平滑肌動蛋白（alpha-smooth muscle actin，α-SMA）、金屬蛋白酶抑制因子-1（tissue inhibitor ofmetalloproteinase1，TIMP-1）、轉(zhuǎn)化生長因子-β1（transforming growth factor beta1，TGF-β1）、Ⅰ型膠原（collagen typeⅠ）及Ⅳ型膠原（collagen type Ⅳ）的表達與感染不治療組比較明顯減弱；PRS治療組與感染不治療組比較小鼠血清中的IFN-γ、IL-4、TGF-β和IL-17水平均有明顯差異（P＜0.01）；PRS治療組SEA特異性抗體IgG、IgG1水平與感染不治療組相比明顯降低（P＜0.01）；PRS治療組與感染不治療組相比SEA特異性抗體IgG2a水平變化不大（P＞0.05），PRS治療組與感染不治療組相比較IgG1/IgG2a比率降低。 結(jié)論： 1.實驗數(shù)據(jù)顯示PRS對哺乳動物幾乎沒有毒性，對斑馬魚毒性低于NIC，能夠在較低濃度下有效殺死日本血吸蟲中間宿主湖北釘螺，是一種非常有潛力的滅螺劑，， 2.體內(nèi)外實驗結(jié)果顯示PRS對日本血吸蟲各個發(fā)育階段均有殺傷作用，有望成為新的抗血吸蟲藥物。 3. PRS在治療日本血吸蟲感染宿主小鼠的過程中，小鼠血清中與免疫應(yīng)答有關(guān)的細胞因子及特異性抗體發(fā)生了一定的改變，說明PRS的殺蟲作用與皂苷的免疫調(diào)節(jié)有一定的關(guān)系。
[Abstract]:Purpose :

In this paper , the effects of total saponins of tilla chinensis ( Bunge ) Regel ( PRS ) on Onlyhupensis ( Schistosoma japonicum ) in the middle host of Schistosoma japonicum were studied . The effects of the key enzymes on the metabolism of Ononhupensis were studied . The effects of different developmental stages and the immunoregulation on the mice infected with Schistosoma japonicum were studied .

Method :

1 . Immersion method is used to carry out the test of snail test , snail climbing test and the toxicity test of zebrafish ;
The enzyme activity values of head and foot and liver cholinesterase ( CHE ) , alanine aminotransferase ( ALT ) , alkaline phosphatase ( ALP ) , lactate dehydrogenase ( LDH ) were measured in 40 % and 80 % of patients exposed to the concentration of PRS by enzymatic kinetics .
The ultrastructure of the head and liver was observed by transmission electron microscope ( TEM ) .
The acute oral toxicity test and skin and eye irritation test in mammals were conducted using the national standard GB15670 - 1995 of the People ' s Republic of China .

2 . The ICR mice were infected with the abdominal patch method of cercariae . After the infection , the livers of mice were ground and screened to obtain the eggs . The cercariae were hatched by the eggs . The cercariae were hatched from the positive infected snails .
The effect of PRS on different developmental stages of Schistosoma japonicum cultured in vitro was performed on 3 h , 7 , 14 , and 42 days with different numbers of cercariae by skin or perfusate method . The mice were cultured with PRS culture solution containing 0 , 1 , 5 , 10 , 20 and 30 渭g / ml to observe the insecticidal effect of the drugs in different time periods .
The mice infected with 50 Japanese cercariae were administered with different doses of PRS solution at different developmental stages of Schistosoma japonicum .
The mice infected with 80 cercariae were injected intravenously with 25 mg / kg PRS at different times after infection , and the amount of charged worm and the rate of insect reduction were calculated after 42 days .
The effects of PRS on glycogen , protein and enzyme of Schistosoma japonicum were studied by intravenous injection of 25 mg / kg of PRS solution at one time tail of infected mice . After 24 h , the effects of PRS on the metabolism of Schistosoma japonicum were detected .

3 . In C57BL / 6 mice infected with 50 cercariae , a dose of 150 mg / kg / day PRS was orally taken for 49d , and the effect of long - term administration was estimated by detecting the weight of mice , the changes of liver and spleen weight , and the effect of long - term administration .
The changes of serum IFN - 緯 , IL - 4 , TGF - 尾 and IL - 17 levels , the changes of serum IFN - 緯 , IL - 4 , TGF - 尾 and IL - 17 levels , the change of specific antibody IgG , IgG1 , and IgG1 , and the change of the values of soluble egg antigen ( SEA ) were investigated by detecting the changes of serum IFN - 緯 , IL - 4 , TGF - 尾 and IL - 17 levels between the infected treatment group and the untreated group .

Results :

1 . After 24 h , 48 h and 72 h of PRS , the LC _ ( 50 _ ( 50 ) were 0.48 , 0.23 , 0.17 mg / l and 2.64 , 0.81 , 0.34 mg / l respectively .
After 24 hours , the total climbing rate was 26.67 % , the total climbing rate was 47.78 % after 24h after 24h after the action of 8 and 4mg / l PRS .
After 24 hours of 24h LC50PRS , the transmission electron microscope of the head and liver cells showed that PRS had some degree of damage to the ultrastructure of the nail .
Zebrafish test showed that the lethal concentration of niclosamide ( NIC ) and PRS was significantly different from that of niclosamide ( NIC ) .
Acute oral toxicity test in mammals , eye irritation test , acute skin irritation test results show that PRS is non - toxic to mammals .

2 . The incubation results of different concentrations of PRS and control drug pramide ( PZQ ) for 24 h after the action of Schistosoma japonicum showed that the inhibition effect of PRS on egg hatching was slightly better than that of PZQ , especially in the concentration of 4 渭g / ml , the eggs of Schistosoma japonicum were more sensitive to PRS .
The effect of PRS and control drug PZQ on cercariae and cercariae in different time showed that the mortality of PRS and PZQ had a certain dependence on the concentration of PRS and PZQ .
In vitro culture of Schistosoma japonicum at different concentrations of PRS showed that the drug had a killing effect on 3 hours , 7 , 14 days , 14 days and 42 days of Schistosoma japonicum .
After 30 渭g / ml PRS in vitro , the sensory mastoid and cortex of Schistosoma japonicum were damaged in some degree after 30 渭g / ml PRS in vitro .
The results of oral administration of different doses of PRS in mice with different infection periods showed that the high , middle and low doses of PRS had some insecticidal effect in vivo , but the dosage of 300 mg / kg of PRS had better effect , and the killing and female killing effects of the drug at the beginning of 1d and 21d were better than those of the same dose of arteannuum ( ART ) and PZQ .
The experimental results of the dosage of 25 mg / kg / kg tail vein showed that there was a better insecticidal effect in different stages of the mice infected with Schistosoma japonicum .
After 24 h of 25 mg / kg dose of PRS , the glycogen , protein , alkaline phosphatase , acid phosphatase ( ACP ) , superoxide dismutase ( SOD ) and glutathione reductase ( GR ) of Schistosoma japonicum decreased .

3 . C57BL / 6 mice infected with 50 cercariae were administered with a dose of 150 mg / kg / d . The results showed that the weight of mice in the PRS group was significantly different from that in the untreated group ( P < 0.01 ) , and the decrease of liver and spleen weight was significantly different from that in the untreated group ( P < 0.01 ) .
Compared with the untreated group , there was a significant difference in the ratio of neutrophils , monocytes and eosinophils ( P & lt ; 0.01 ) , and the ratio of lymphocytes and basophilic cells was significantly different from that in the untreated group ( P & lt ; 0.01 ) .
The results of long - term use of PRS in mice infected with 50 cercariae were significantly different from those in untreated group ( P < 0.01 ) , and the rate of reduction was 55.27 % . There was a significant difference between the reduction of the eggs of liver and the untreated group ( P < 0.01 ) .
After 49d administration , eosin staining ( HE staining ) of the liver of mice showed that the eggs of the liver and the inflammatory cells around the eggs were less than those in the untreated group .
The expression of 偽 - smooth muscle actin ( 偽 - SMA ) , metalloproteinase - 1 ( TIMP - 1 ) , transforming growth factor - 尾1 ( transforming growth factor beta1 , TGF - 尾1 ) , type 鈪

本文編號：2136847