| BackgroundPrimary liver cancer(PLC)is insidious onset and progresses rapidly.The mortality rate is second only to lung cancer,seriously endangering human health.Traditional Chinese medicine is an important adjuvant treatment for patients with primary liver cancer.It can control the progression of cancer,repress tumor metastasis and prolapse,and prolong the survival time of patients.This study is the main pharmacodynamics and safety evaluation of Qinghe Huayu Particles in the treatment of primary liver cancer(Z171100001717008).Qinggan Huayu Particle is an effective recepie based on years of experience in the treatment of liver cancer by Professor Yao Shukun.The previous animal experiments and cell experiments of this group have clarified the main effective components of Qinggan Huayu Recipe,such as oxymatrine,which has clear anti-tumor and cell proliferation inhibition effects in various tumors.PurposeThis study will provide a stable,experimentally relevant liver cancer model for acute phlegm and blood stasis research and conduct acute toxicity tests to provide a reference for clinical drug safety.Method:Experiment 1:80 ICR mice,weighing 18-21g,were randomly di-vided into control group and 7 Qinggan Huayu Particlesdose groups of 26.8,35.8,48.3,64.4,85.9,116.3,153.8 g crude drug/kg,10 in each group.After fasting for 15h,the drug was administered once,the volume of administration was 40 ml/kg body weight,and the control group was given the same volume of water.The toxicity was observed after administration,and adverse reactions were recorded.At the end of the observation period on day 14,the surviving animals were dissected and observed for each organ change.Experiment 2:72 healthy male SD rats of SPF grade,weighing(220±20g),about 6 weeks old,were divided into blank group(n=12),modeling group 1(n=30),modeling group2(n=30)after one week of adaptive feeding,The drug-administered group(modeling group 1 and modeling group 2)were intraperitoneally injected with DEN,50 mg/kg;group 1 was administered once a week,and group 2 were administered twice a week.Observe the general condition,coat color,activity,gait,demeanor,stool,urine,etc.of the rats;record changes in body weight and food intake;The survival of each group of rats was recorded,and the natural mortality of each group of rats was calculated.Three rats were randomly selected for observation in each group in W3,W5,W6,and W9;The rats were dissected,observed on the color of the liver tissue,with or without congestion,edema,sclerosis,and cancer nodules;Pathological light microscopy was used to observe whether there were lesions and their severity in each group,and whether the liver tissue lesions in rats were related to the frequency of administration.Results1.Experimental study on acute toxicity of QingganHuayuParticles1.1 Observation of mortality and adverse reactions in miceQinggan Huayu Particleswere administered orally to mice at a dose of 26.8,35.8,48.3,64.4,85.9,116.3,153.8 g of crude drug/kg,administration volume was 40 ml/kg,and the man toxicity observed was acute death.The death of the animals occurred within 1-24 hours after the administration.The animal mortality on the same day of the above dose administration was 0%,0%,0%,20%,40%,100%and 60%,respectively;The mortality of the animals within 24 hours after the administration in each dose group was 0%,0%,0%,20%,100%,100%and 100%.The maximum dose for clinical use of this product is 0.67g crude drug/kg.There were 2 deaths in the 64.4g crude drug/kg dose group after the Qinggan Huayu Particles was given to the mice by oral administration;10 of the 85.9,116.3,153.8g crude drug/kg dose groups all died acutely.The Bliss method was used to calculate the median lethal dose(LD50)as 69.94 g crude drug/kg(equivalent to 104 times the clinical dose),the maximum non-lethal dose is 48.3 g crude drug/kg,and the maximum non-toxic reaction dose is 26.8 g crude drug/kg.In addition to acute death,the main adverse reactions were diarrhea,lying still,gait instability,disappearance of righting reflex and limb stiffness.1.2 Effects on body weight and food intake in miceCompared with the control group,the D1-14body weight of the 153.8 g crude drug/kg dose group of Qinggan Huayu Granule male rats was lower than that of the control group within 14 days after administration,and the D1 body weight was significantly lower than that of the control group(P<0.01),no significant statistical differences were observed at other time points.The other dose groups’ male rats and the female rats were basically equal to the weight of the control group at each time point,and there was no statistically significant difference compared with the control group.Compared with the control group,the D1 intake of the 153.8g crude drug/kg dose group of Qinggan Huayu Granule male group was significantly lower than that of the control group,which was about 52%of the control group.The food intake after D2 was basically the same as that of the control group.The other dose groups of male rat and the female rats were substantially equivalent to the food intake of the control group at each time point.1.3 Gross anatomy observationAt the end of the 14-day observation period,after anatomical observation,no exudate,hemorrhage and adhesion were observed in the chest and abdomen of the animal.No congestion,bloody,bleeding,exudate,adhesions,erosion,ulcers and other visible lesions was observed in the heart,liver,spleen,lung,kidney,adrenal gland,gastrointestinal tract,bladder,testis or epididymis.2.The improvement of rat primary liver cancer model2.1 Observation on natural mortality and adverse reactions in ratsThe normal control group(1 to 4 cages)had a natural mortality rate of 0%from the beginning to the end of the observation,and the survival rate was 100%;In group A,the death from W17 to the end of W22 observation,except for the dissected rats,the natural mortality rate was 22.22%,38.89%,44.44%,44.44%,50.00%,100.00%;The toxicity of group B was obviously from W5.There were 4 natural deaths at W5,6 natural deaths at W6,4 natural deaths at W7,1natural death at W8,and 3 natural deaths at W9.All animals died in Group B by the 9th weekend.The natural mortality rates were 22.22%,55.56%,77.78%,83.33%,and 100.00%,respectively.Kaplan-Meier survival analysis showed that there was a significant difference in survival between group A and group B.After Log-Rank test,the difference in overall survival between the two groups was statistically significant(P<0.01).The general state of each group except for deathwas observed,and there were no obvious abnormalities in the urine color,feeding,drinking water and body hair of the control group;The adverse reactions seen in group A and group B were mainly characterized by erect hair,yellow urine,and nasal bleeding and no recovery after symptoms appeared.Some rats have combined symptoms;The incidence of erect hair,yellow urine,and nasal bleeding in group A was 22.22%,11.11%,and 11.11%;The incidence of erect hair,yellow urine,and nasal bleeding in group B was 27.78%,88.33%,and 66.67%.2.2 Effects on body weight and food intake in ratsCompared with the control group,the body weight of group B showed a slow growth trend from the first to third weeks after administration,and decreased from the fourth to the ninth week.At the end of 22 observing weeks,the growth trend of the control group and the A group was basically close,and there was no continuous decline.Compared with the control group,the food intake of group B decreased significantly after administration,and the growth trend of food intake in the control group and group A was basically close.2.3Toxic effects on ratsIn the same administration reagent and the same amount of each administration,the degree of the disease gradually increases as the frequency of administration increases.Rats in group B began to die at the 5th week,and the formation of cancer nodules;liver tissue changed from bright red to deep red to dark purple at the 8th week;rats produced massive ascites at the9th week,and the natural mortality rate reached 100%.Until the death of group B rats,pathological sections(HE staining)of liver tissues of each group were observed.Microscopic observation showed that the structure of liver lobules in the control group was clear,and the hepatocytes were radially distributed around the central vein,which were arranged in a strip shape.The cell size was consistent,the cytoplasm was abundant,the nuclear membrane and nucleolus were clear and distinct,no degeneration and necrosis were observed,and no inflammatory cell infiltration was observed.At the 5th week,the formation pseudolobules in group B rat liver tissue were observed under microscope.The arrangement of hepatocytes was focal,nested and beam-shaped,with large nuclei and partial hepatocytes with binuclear or multinuclear.Hepatic cell vacuolar degeneration can be seen in the internal division of hepatic lobules.At the 6th week,in group B,it is observed the structure of the hepatic lobule was destroyed,and it became a false leaflet of different sizes and irregular shapes.Hepatocyte degeneration,necrosis,disordered arrangement,cells are binuclear or multinuclear,see intratumoral hemorrhage,necrosis,inflammatory cell infiltration and nuclear division.In the 9th week,the B group observed under the microscope:visible cirrhosis,destruction of the structure of the hepatic lobule,and the formation of false leaflets of different sizes and irregular shapes.Hepatocyte degeneration,necrosis,disordered arrangement,cells are binuclear or multinuclear,cell atypicality is obvious,mitotic figures are more common,and pathological mitotic figures can be seen.B group was observed at week 6,and group A was observed atweek 17 under microscope:there is cirrhotic hepatic lobular structure destruction,which becomes unequal,sized,and irregular pseudolobules.The arrangement of hepatocytes is disordered,there is degeneration and necrosis,and the cells are binuclear or multinucleated.See intratumoral hemorrhage,necrosis,inflammatory cell infiltration and nuclear division.Conclusion:1.Qinggan Huayu Particles were administered to mice with 26.8 g of crude drug/kg(equivalent to 40 times the clinical dose)without toxicity;When the dose reached 104 times the clinical dose,that is,69.94 g of crude drug/kg,the half lethal dose of the mice was reached.It is suggested that the clinical dose of Qinggan Huayu Granule(0.67g crude drug/kg)is safe.2.With the increase of frequency of administration,the degree of liver tissue lesions in group B was aggravated,the mortality of rats increased,and the survival period was significantly reduced.The model of DEN was given once a week,and death occurred after 17 weeks.Due to its long survival period,it is similar to the natural pathogenesis of human liver cancer,and is more suitable for the development of new drugs for antigenic liver cancer. |
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