Experimental Study On The Pathogenesis Of Clonorchiasis Induced By Clonorchis Sinensis Infection And Drug Therapy In Mice

OBJECTIVE:Clonorchis sinensis,also known as Chinese liver fluke,is a food-borne parasite.The patient is due to ingesting raw or insufficiently cooked food containing the larvae-metacercariae in the host.In vivo development of adult parasites in the liver and bile duct,causing the pathological changes of the liver and bile duct inflammation around,can induce jaundice,cholangitis,cholelithiasis,fibrosis and other bile duct disorders.The epidemic areas of the disease in the world are mainly in China,South Korea,North Korea,Japan,Vietnam and Russia's Far East and other countries and regions.In our country,there are 27 provinces with varying degrees of prevalence of clonorchiasis,Guangdong province is the most prevalent area,with a population infection rate of up to 30%,and its incidence has shown an upward trend.In 2012,the International Agency for Research on Cancer?IARC?officially identified Clonorchis sinensis as a type I carcinogen of cholangiocarcinoma.Cholangiocarcinoma?CCA?ranks second in the incidence of hepatobiliary malignancies,drug and surgical treatment is difficult,it does great harm to human health with poor prognosis.Due to mechanical injury and immunopathological damage of the liver flukes,chronic hepatocyte oxidative damage,liver fibrosis and bile duct epithelial cell proliferation are the pathological basis of cholangiocarcinoma.Although WHO has clarified carcinogenic effects of the liver fluke,the molecular mechanism of its pathogenesis is not yet fully understood and still under intensive study.Praziquantel?PZQ?is currently the first-line anti-trematode drug with good insecticidal ability and is the drug of choice for the treatment of clonorchiasis.Its medicinal mechanism is mainly acting on the Ca²⁺channel of trematodes,making its muscles tonic contraction,decreased mobility,inhibition of secretion and metabolism;at the same time,exposure of the surface antigen make it susceptible to an immune attack by the host.However,there is an increasing trend in clinical resistance to this drug.Therefore,the development of new anti-parasitic drugs?including anti-tremor drugs?has become a hot issue for scholars both at home and abroad.Wortmannilatone F?WF?is a novel anthelmintic that inhibits the activity of NADH-reductase in worms,which blocks the anaerobic oxidative phosphorylation and destroys the worms by inhibiting the transfer of electrons through the mitochondrial inner membrane of the respiratory chain,affecting the worm ATP synthesis,resulting in obstruction and death of parasites.Wortmannilatone F belongs to a class of biological agents,the side effects on mammals is extremely small,our previous experiments have proved that the drug has obvious inhibitory and killing effect on adult and capsule of Trichinella spiralis..Interleukin-8?IL-8?plays an important role not only in immune regulation,anti-pathogen infection,inhibition of tumorigenesis and development,but also in the inhibition of tissue fibrosis formation.G31P is an IL-8 analogue that competitively antagonizes the secretion of IL-8 and its biological activity and has the effect of antagonizing the secondary damage to the structure and function of the host organism caused by severe fibrosis of the tissue due to parasitic infection.Our earlier experiments also proved these facts.In this study,we investigated the imbalance of oxidative stress response in host liver cells induced by the hepatic infection and the long-term fibrosis of liver tissue,and expression of tumor markers such as PCNA and CK19 in order to reveal the pathological mechanism of the onset of Clonorchis sinensis infection in mice.At the same time,we also analyzed the changes of the above-mentioned pathogenic factors of cholangiocarcinoma from the inhibition of the liver flukes by drugs,so as to provide a certain experimental basis for the prevention and treatment of clonorchiasis.METHODS:1.Establishment of animal model1.1 Clonorsinensis rabbit model:Through oral infection,a male rabbits infected liver fluke metacercariae about number 1000.After 30 days,the eggs of liver fluke were positive on the inspection stool.The rabbit was anesthetized to sacrifice,collected Liver flukes adult in the bile duct,for the drug anti-clonorchis experimental observation in vitro.1.2 Clonorsinensis mouse model:There are 102 male BALB/C mice,weight15-20g,oral infected of liver fluke metacercariae.Each mouse were inoculated with 70±5.After 30 days,the eggs found were confirmed to be modeled successfully.The mouse hepatobiliary tissue and serum were isolated?stored at-80??for the next experiments.1.3 Therapeutic drugs to use:Wortmannilatone F:administered after the infection in mice to 30 days,200mg/kg,gavage once daily for 3 days;praziquantel:administered after infection in mice to 30d,5mg/kg,gavage method,once daily for 3days;G31P:administered after infection in mice to 30d,500?g/kg,subcutaneously,once/2 days,until the end of the experiment.Combination therapy groups:The administration mode is that the two administration methods in the above methods are performed simultaneously.2.Experimental Groups2.1 In vitro inhibition of hepatic inhalation groups:Praziquantel groups,Wortmannilatone F groups and control groups were set.Praziquantel groups:administrationconcentrationswere0.1,0.5,1,5,10,50,100,500?mol/L;WortmannilatoneFgroups:administrationconcentrationswere0.1,0.5,1,5,10,50,100,500?mol/L.The number of adults in each experiment was 20?n=20?.2.2 Animal model groups:positive mice infected with infection were randomly divided into two groups.The infection groups were divided into 30d groups?30d,n=8?,40d groups?40d,n=8??50d,n=8?,60d groups?60d,n=8?,90d groups?90d,n=8?and 180d groups?180d,n=8?.The administration groups were subdivided into PZQ?n=18?,praziquantel combined with G31P?PZQ+G31P,n=18?and Wortmannilatone F combined with G31P?n=18?.Three groups were taken at the 40th day,the 50th day and the 60th day after infection and 6 mice were randomly selected from each group.Normal negative groups were 8 mice.3.Experimental Method3.1 Inhibition of worms in vitro experimental observation:After administration to1,4,6,12,24,48,72h,the number of adult death count by observing under microscope or staining adult morphology.After drug treatment,liver flukes were observed by transmission electron microscopy and mitochondrial activity.Morphological observation:Take normal control groups,infection groups,administration groups mice liver tissue on the same part,the same size of the tissue mass to making paraffin sections,with HE and Masson staining.Comparing of mice infected with liver fluke between before and after treatment,the result on the dynamic pathology of liver tissue and liver tissue/bile duct tissue fibrosis development process were suspected.3.3.Oxidative stress in liver and expression of fibrotic markers:in the experimental groups,the liver tissue and paraffin sections were respectively analyzed by Immunohistochemistry to detect the levels of NOX,LOX,8-OHdG and FSP-1.For the amount of ROS,some liver tissues of mice were detected by immunofluorescence staining.3.4 Liver tumor markers PCNA,CK19 expression:the liver tissue were made to paraffin sections,then detected the liver PCNA,CK19 expression by immunohistochemistry,respectively.3.5 Serological test-IL-6:using the serum reserved at-80?serum,ELISA kit,measured IL-6 levels.3.6 The above groups of data were statistically analyzed.RESULTS:1.Study on the pathogenesis of mice infected by liver flukes1.1 Pathological changes of liver in mice with clonorchiasisGross and microscopic pathological changes of liver:compared with the control group,the mice in the infected groups had enlarged liver,multiple inflammatory nodules and gray-white cysts in the margins of the liver,and extrahepatic bile ducts were significantly enlarged.Microscopic examination showed that the liver cell edema,portal area broadening,partial necrosis of hepatocytes around bile ducts,hyperplasia of acinar area and deposition of collagen fibers;a large number of inflammatory cell infiltration,disorganization of hepatic lobules and formation of pseudolobules.1.2 Peribiliary fibrosis level in mouse liver:Masson staining of the liver and FSP-1immunohistochemical results showed that compared with the control group,more collagen fibers were found in the liver tissue of the infected groups,in which a large amount of collagenous fiber deposition occurred in the portal area of the 180 d group after infection.1.3 Serum IL-6 level in mice:The level of IL-6 in the infected mice reached its peak at 30⁶0 days,then returned to the normal level at 180 days.The median maximum level of IL-6 ranged from 40.3 to55.3 pg/ml,about twice as high as the serum level in uninfected control mice.1.4 Changes of Tumor Markers?PCNA,CK19?in mice with clonorchiasisThe expression of PCNA and CK19 in infected mice began to increase 30d after infection,and reached the highest level at 60d to 90d.With the prolonged survival of adult liver parasites in the host,the expression of PCNA gradually increases in the area of acinar hyperplasia.1.5 Oxidative Stress Response in mice with clonorchiasisCompared with the negative mice,in the infected group COX-2 and LOX immunoreactivity were observed in the inflammatory area on the 30th day.At the 60th day,COX-2 immunoreactivity decreased,while the LOX immunoreactivity in the 90d acinar area gradually decreased,but in inflammatory area is still obvious.8-OHdG first appeared in the inflammatory cell area and extended to the bile duct epithelial cells on the 90th day.At the 180th,the number of positive cells decreased.At the 30th day after infection,ROS accumulation occurred,and the highest amount was found on the 60th day.The expression of ROS in the liver after liver injury was consistent with the above oxidative stress products.2.Results of in vitro administration to inhibit the liver flukes2.1 Worm Activity and Morphology:2.1.1 Praziquantel groups:adult worms contracted intensely immediately after the drug action,the body size reduced gradually,and the activity decreased;1 h after the administration,the head and body surface of the parasites began to appear vacuoles.All of the parasites in these groups started to die at 4 h,and the number of dead worms in500?mol/L group was 18;at 8 h,9 died in 1?mol/L praziquantel group,and 12 died in 0.5 and 1?mol/L praziquantel groups;at 24 h all the worms died in 100 and 500?mol/L praziquantel groups.At 48 h,all the parasites in 5,10 and 50?mol/L praziquantel groups died.At 72 h,all parasites died.2.1.2 Wortmannilatone F groups:at the beginning,the parasites in these groups showed more active than the control group,but after 12 h,the worms appear shrinkage,curling,activity attenuated.In 500?mol/L Wortmannilatone F group,there were 5 dead bodies when cultured 4 h and 18 died when 12 h;at 48 h,the worms all died.In100?mol/L Wortmannilatone F group,when cultured 8 h,the worm death occurred,when cultured 72 h,the worms all died.In0..5,1,5,10,50?mol/L Wortmannilatone F groups,the worms died at 48 h,when cultured 72h,about half of them died.2.2 Transmission electron microscopy observation:in praziquantel groups,the liver flukes showed cortical matrix and mitochondria body swelling,some of the epidermal cells completely dissolved,muscle fiber swelling.In the epithelial layer of the intestine wall,the filamentous protrusions appeared disordered,and the endoplasmic reticulum,ribosome,mitochondria and lysosomes in the intestinal epithelial cell layer swelled.In Wortmannilatone F groups,the mitochondria swelled in the cortical tissue of the flukes,the epidermal cell nucleus appears swelling,too,and filamentous protuberances in the epithelial layer of the intestinal wall showed disorder and contraction.2.3 Mitochondrial activity test:compared with praziquantel groups and the control group,low rate of substrates consumed by mitochondrial NADH-fumarate reductase in Wortmannilatone F groups.3.Effect of Drug Combination Therapy on Mouse Infection Model3.1 Pathological changes of liver in mice with clonorchiasisLiver abscess come into being in mice in praziquantel groups,and a large amount of fibrosis appeared on the edge of the liver.Inflammatory cells were found in a large number in the focus area.In Wortmannilatone F and praziquantel combined with G31P groups,there was a small amount of fibrosis on the edge of liver tissue in mice.Microscopically,the pathological sections of the mice in the infected groups showed hyperplasia of bile ducts and acinar regions.Collagen fibers deposited around the bile duct and infiltration of inflammatory cells were observed.In some tissue sections,the liver flukes were catched in the bile ducts,and inflammatory cell infiltration was obvious in the lesion area.However,the number of inflammatory cells and collagen deposition were significantly less than those in infected mice.3.2 Peribiliary fibrosis level in mouse liver:Compared with the infected groups,the G31P-treated groups?PZQ+G31P/WF+G31P group?had less expression of collagen fibers and FSP-1 in the hepatobiliary duct;while in praziquantel groups,a great deal of obvious collagen fibers appeared,of which60 days after infection,showed the heaviest degree.3.3 Serum IL-6 level in mice:The level of serum IL-6 in infected mice was significantly higher than that of all those treated with G31P groups?PZQ+G31P/WF+G31P group?.3.4 Changes of Tumor Markers?PCNA,CK19?in mice with clonorchiasisThe levels of PCNA and CK19 in the liver of infected mice were significantly higher than those of the other groups treated with G31P?PZQ+G31P/WF+G31P?.However,the PCNA level of praziquantel group was significantly higher than that of the untreated group at 40d after infection,and the level of CK19 was also significantly higher than that of the untreated group at 60d after infection.3.5 Oxidative Stress Response in mice with clonorchiasisThe levels of COX-2 and LOX immune responses in the PZQ+G31P/WF+G31P group were significantly lower than those in the infection group.At 40 days after infection,the levels of COX-2 and LOX in PZQ group were significantly higher than those in Control group.There was no significant difference between PZQ group and the other groups at 60 days after infection.There was no significant difference in COX-2and LOX levels between WF+G31P group and PZQ+G31P group at 60 days after infection.3.6 Mouse Liver Index ComparisonLiver index is the ratio of liver weight to body weight of mice?%?.Compared with the negative control group,the liver index of the infected group and each group of drug-treated group were significantly increased?P<0.05?;Compared with the infected groups,the liver index of Wortmannilatone F and praziquantel combined with G31P groups and praziquantel groups decreased significantly?P<0.05?.Liver index is proportional to the degree of liver damage.CONCLUSION:1.During the infection of Clonorchis sinensis,the hepatobiliary tissue in diseased mice suffered long-term mechanical and chemical stimulation of the parasites. Inflammatory reaction and oxidative stress continued to occur,leading to high expression of COX-2,LOX and IL-6.The inflammatory response index increased first and then decreased,while the degree of fibrosis and the expression of 8-OHdG continued to increase,and ROS persisted,which promoted long-term inflammatory lesions in the hepatobiliary tissue and led to the proliferation of hepatobiliary epithelial cells.2.Chronic liver fluke infection,making tumor markers for cholangiocarcinoma,such as PCNA,CK19 expression increased,might promote the bile duct epithelial cell gene mutations,leading to the occurrence of hepatobiliary cancer.3.Wortmannilatone F has a potent direct inhibitory effect on adults of the liver flukes.4.Combining Wortmannilatone F with G31P not only inhibits insecticides,but also reduces the degree of hepatobiliary cell proliferation and fibrosis and reduces the possibility of cholangiocarcinoma.5.Combination of praziquantel with G31P did not affect the worm killing effect,while the combination might reduce the possibility of cholangiocarcinoma.6.G31P can antagonize the formation of fibrosis around the bile duct and repair the damage to the host tissue caused by the parasites,at the same time,it may also reduce the possibility of cholangiocarcinoma.