| Trichinosis is a kind of zoonoses which is caused by Trichinella spiralis.This parasitic food borne disease is also popular around the world.People are infected mostly by eating raw or undercooked meat containing Trichinella spiralis cysts and have the symptoms of fever,abdominal pain,diarrhea,eyelid edema,myocarditis and systemic muscle pain.Due to great harm to human health,severe patients could even die from complications after infection.Nowadays,albendazole and benzimidazole are used to treat the disease.However,their side effects are obvious.To develop novel drugs against Trichinosis is a focus currently.Plants are natural organisms which have the advantages such assafety,low toxicity,less toxicity to non-targeted pathogen and no pollution to the environment.It is a trend to obtain effective substances from natural plants and then apply them to prevent and control diseases.In this study,we tried to co-culture 20 kinds of natural plant extracts and Trichinella spiralis respectively in vitro.We screened out the plant extracts which had killing effects on muscle larvae,adult and newborn larvae of Trichinella spiralis.Then we designed the experiment to explore the killing mechanism in vitro to provide a new method for the prevention of Trichinosis.The main contents and results are as follows:(1)Screening out the natural plant extracts which have killing effects on the various developmental stages of Trichinella spiralis.Adult,newborn larvae and muscle larvae were collected in vitro.Control group,DMSO group and natural plant extracts group(concentrations: 10mg/L,50mg/L,100mg/L)were esablished.Under the condition of 37 and 5%? CO2 for 24 h,mortality was calculated through microscope examination.The results showed sanguinarine(50mg/L)and cynanversicoside G(50mg/L,100mg/L)had the killing effects on muscle larvae.The mortality reached 100±0.0%,87.3±4.9% and 100±0.0% respectively.Sanguinarine,psoralen,amygdalin and hyperin had the killing effects on adult.At the concentration of 10mg/L,sanguinarine,psoralen and amygdalin had a mortality of 71.6±5.3%,97.4±6.5% and 93.8±5.1%.At the concentration of 50mg/L,the mortality was all 100±0.0%.Hyperin(50mg/L,100mg/L)had a mortality of 87.6±5.8% and 100±0.0% respectively.Sanguinarine,sanggenon C could kill newborn larvae,at the concentration of 10mg/L after 24 h treatment.Sanguinarine had a mortality of 100±0.0%.While sanggenon C(50mg/L,100mg/L)had mortality of 87.6±5.8% and 100±0.0%,respectively.(2)Study on the mechanism of killing effects of sanguinarine on various developmental stages of Trichinella spiralis.We utilized sanguinarine to treat muscle larvae,adult and newborn larvae.The results indicated that among the concentration of 14~30mg/L,with the increasing trend,killing effects on muscle larvae rise.The mortality reached 100% at the concentration of 30mg/L.Among the concentration of 3~15mg/L,with the increasing trend,killing effects on adult worm grew,and the mortality was 100% at the concentration of 15mg/L.With the increase of sanguinarine,the newborn larvae decreased gradually.The killing effects on newborn larvae increased with the concentration from 1.4 mg/L to3mg/L and the mortality reached 100% at the concentration of 3mg/L.Sanguinarine damaged the surface of adult Trichinella spiralis in vitro by scanning electron microscopic.The adult body shrank;annular wrinkles deepened,local skin ulcerated;surface longitudinal cristae disappeared,male worm copulatory appendage collapsed and atrophied,which is difficult to identify two of the mastoid and genital cleft.The results showed that sanguinarine might seriously jeopardize the surface structure of adult Trichinella spiralis.The experiment was divided into 3 periods in vivo.Sixty 8-week-old female BALB/c mice in each period were divided into 6 groups randomly: control group,challenge group,DMSO group,albendazole group.The adult stage,transitional stage and the larval cysts were treated by sanguinarine at the concentration of 70 and 80mg/kg,80 and 100mg/kg,100 and 150mg/kg,respectively.At day 1,day 13,day 35 after Trichinella spiralis infection,the mice were treated with sanguinarine for 3 consecutive days.At day7,day 30,day 46,the mice were sacrificed to collect worms to calculate the worm reduction rate.The results showed that sanguinarine had killing effects on the adult stage,transitional stage and the larval cysts.At the concentration of 70 and 80mg/kg,the reduction rates of adult worms were 37.2% and 36.9%.The reduction rates of newborn larvae in migration period were 26.4% and 39.6% at the concentration of 80 and 100mg/kg respectively.At the concentration of 100 and 150 mg/kg,the rate of reduction for the larvae was 31.7% and 42.3%,respectively.Observing the sections of mouse duodenum,tongue muscle and masseter muscle,pathological changed with HE staining.The intestinal villus length and crypt depth were measured and villus crypt ratio was calculated.Sanguinarine could restore intestinal villus length and villus crypt ratio increased significantly(P<0.05).The number of goblet cells in intestinal mucosa of duodenum were counted by PAS staining.The results indicated that sanguinarine could induce the number of intestinal goblet cells to increase significantly(P<0.05),enhance mucosal immunity and promote the shed of Trichinella spiralis.ROS content in sera was detected by ELISA.The results showed that sanguinarine could increase the content of ROS in the transitional period and maintain balance.Sanguinarine,cynanversicoside G,psoralen,amygdalin,Hyperoside and sanggenon C were screened out to exert obvious killing effects on Trichinella spiralis.Sanguinarine had killing effects on adult,newborn larvae and muscle larvae in vivo and in vitro.Sanguinarine could also reduce the pathological changes of the intestine and muscles by increasing the content of ROS,maintaining ROS balance and increasing the number of goblet cells to promote the shed of the Trichinella spiralis. |
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