Killing Muscular Larvae Of Trichinella Spiralis And Anti-fibrotic Effect Of Combination Of Wortmannilatone F And Recombinant G31P In A Murine Model Of Trichinellosis

Objective: trichinosisis a serious global food-borne zoonosis which may jeopardize human health.Research topics on trichinosis concentrate mainly on etiology and immunology.However,prevention and treatment drugs on trichinosis have been little investigated.Broad-spectrum type medicines such as albendazole and mebendazole not only are patented from overseas,but also have many side effects.Therefore,developing potential drugs with independent intellectual property rights in killing Trichinella spiralis is looming ahead.This is of important significance in the development of social economy.Wortmannilactone F is a newtype compound separated from Talaromyces Wortmannii.It inhibits respiratory electron-transport chain and blocks cellular respiration.G31 P is a competitive inhibition of chemokines by high affinity binding chemokine receptors-CXCR1/2.It blocks biological effect of IL8 and other chemokines,such as preventing the inflammatory cell infiltration and reducing the inflammatory responses.So that applying G31 P clinically can alleviate or repair serious injury of infectious diseases.Ideal insecticides for Trichinella should not only kill Trichinella larvae,but also prevent collagen fibers accumulation and angiogenesis.The aims of this research were to evaluate the therapeutic efficacy of Wortmannilactone F&G31P in killing Trichinella larvae and affecting the formation of encapsulated Trichinella.Method: 1.To establish the trichinosis mouse model:6 weeks old male BALB/c mice(n=32).Random 8 mice served as negative control.Each mouse was orally fed with 150±5 infective Trichinella larvae except the ones in negative control group.Process of infection was carried out as previously described [1].24 Trichinella-infected mice were randomly divided into Wortmannilactone F treatment group,Wortmannilactone F&G31P treatment group and model group.Each mouse in four groups was fed in the same condition.2.Experimental method:10 days after infection,mice in Wortmannilactone F&G31P treatment group were subcutaneously injected with 500?g/kg G31 P in every other day for consecutive 14 days,meanwhile prepared with 200mg/kg Wortmannilactone F by gavage in every 24 h for consecutive 3 days.Mice in Wortmannilactone F treatment group were prepared with 200mg/kg Wortmannilactone F by gavage in every 24 h for consecutive 3 days,meanwhile subcutaneously injected with saline in every other day for consecutive 14 days.Mice in model group and negative control group were subcutaneously treated with the same amount of saline in every other day for consecutive 14 days.Mice were sacrificed under ether narcotization 24 h after last treatment and aseptically dissected.Diaphragm tissues were collected for experimental observation of drug effects.3.Observation of experimental parameters(1)Observation of the effect of killing Trichinella larvae:Diaphragm tissues of four groups were collected for subsequent HE staining.To observe drug effects of killing Trichinella larvae,we count the number of encapsulated Trichinella and compared the size in different groups.(2)Morphological observation of encapsulated TrichinellaDiaphragm tissues of four groups were collected for subsequent HE staining.We observed the drug effects of Wortmannilactone F&G31P on morphological changes of encapsulation and immunopathological changes under light microscope.Integrity of encapsulation and development status of Trichinella larvae were observed under electron microscope.(3)Immunohistochemistry staining was used to detect the expression of FSP1,in order to observe the effects of the number of gathering fibroblasts around diaphragm tissues and integrity of encapsulation impacted by G31 P.(4)Masson staining was used to detect level of fibrosis and collagen fiber accumulation by G31 P,so that we realized the G31P's effect to integrity of encapsulation.(5)Partial diaphragm tissues were used for immunofluorescence staining to detect expression of vWF in order to know the effect of angiogenesis by G31 P during the forming process of encapsulation.Result: 1.Observation of the effect of killing Trichinella larvae by Wortmannilactone F&G31P:(1)In Wortmannilactone F treatment group and Wortmannilactone F&G31P treatment group,the numbers of diaphragm muscle Trichinella larvae under light microscope were 11.5±2.1 and 6.4±1.2.Worm reduction rate achieved to 58.0% and 76.6%.There was significant difference in the comparison(P<0.01).Compared with the one in model group which was 27.4±2.5,there was also significant difference in the comparison(P<0.01).(2)We observed the structure of larvae inside encapsulation in model group under light microscope.Corneum of larvae were smooth and intact.The linellae of larvae were downy.Worms were tortuous and folding in nurse cells.Thick encapsulation was around the larvae.Under electron microscope,larvae possessed uniform thickness,and horizontal grains appeared at regular distances.Larvae surface structures between Wortmannilactone F treatment group and Wortmannilactone F&G31P treatment group had no significantly difference.However,compared with larvae in model group,they had changed remarkably.Worms were tiny and kept rigid under light microscope.Corneum of muscle larvae were uneven and out of order under electron microscope.2.The effect of Trichinella larvae encapsulation by Wortmannilactone F&G31P through immunohistochemistry staining and Masson staining(1)In negative control group,FSP1 positive cells were rarely observed in mouse diaphragm tissues.However,great quantities of FSP1 were located around larvae in model group.After treated with Wortmannilactone F or Wortmannilactone F&G31P,FSP1 were statistically reduced(P<0.01).There were also significant difference between latter two groups(P<0.01).(2)Moreover,Masson staining was performed to reflect the content of collagen fibers.In negative control group,no Trichinella larvae were found,so that no collagen fibers accumulated.However,in model group and Wortmannilactone F treatment group,there were great quantities of collagen fibers observed in mouse diaphragm tissues.In Wortmannilactone F & G31 P treatment group,collagen proliferation in mouse diaphragm tissues was forbidden.(3)In addition,Smart V3500 software was applied to measure the areas of larvae encapsulation.We found in model group and in Wortmannilactone F treatment group,the areas of larvae encapsulation are 4445.1±402.2(?m2)and 4332.2±493.7(?m2).There were no significant difference in comparison(P>0.05).The areas in Wortmannilactone F&G31P group were 1949.6±346.6(?m2),which were reduced in size compared with the areas in former two groups(P<0.01).3.The effect of Wortmannilactone F&G31P on angiogenesis around larvae encapsulation(1)We found that vWF positive cells were scarcely observed in normal diaphragm tissues in negative control group.Stimulated by Trichinella larvae,vWF positive cells had mushroomed in model group and in Wortmannilactone F treatment group.After treated with Wortmannilactone F&G31P,v WF's expression was inhibited.(2)We applied Image Process plus 6.0 software to detect the percentage of vWF positive cells.We found in model group and in Wortmannilactone F treatment group,the percentages of vWF positive cells are 6.7±1.1%.,6.2±1.2%.There were no significant difference in comparison(P>0.05).The percentage of vWF positive cells in Wortmannilactone F&G31P group was 2.2±0.4%,which were reduced observably compared with the areas in former two groups(P<0.01)Conclusion: 1.Wortmannilactone F has the effect on killing Trichinella larvae.Compared with which using any drug alone,the number of encapsulation in diaphragm tissues decreases significantly by using Wortmannilactone F& G31 P.2.The destructive effect of Wortmannilactone F&G31P on muscle larvae surface structure is remarkable.3.After treated with Wortmannilactone F& G31P(mainly G31P),angiogenesis around encapsulation was forbidden,the scanty blood vessels couldn't provide larvae for enough nutrients and metabolic pathways of waste,so that encapsulation couldn't inform intactly.4.After treated with Wortmannilactone F& G31 P,collagen fibers accumulation was inhibited,and integrity of encapsulation and development status of Trichinella larvae were affected,in order that Trichinella larvae presented with growth retardation.Meanwhile through destructing integrity of encapsulation,secondary damage of fibrosis to host was alleviated.5.Given integrity of encapsulation destroyed,humoral immunity and cellular immunity were used to its fullest potential.Trichinella larvae inside encapsulation could be killed and inflammatory injury site caused by Trichinella larvae was repaired.6.Worm reduction rate by Wortmannilactone F& G31 P was 76.1%,and insecticidal efficiency could achieve to ivermectin's effect performed by Basyoni.Drug combination could come to the same effect of foreign clinical medicine.Fortunately,they have little side effect and great application value with independent intellectual property rights.Using Wortmannilactone F& G31 P for killing Trichinella larvae has good prospects for economic benefit and market development.