Animal Model Validation Of Gouty Arthritis With Damp-Heat Syndrome Based On Literature Review

Objective:Part Ⅰ:The analysis of the modeling method of gouty arthritis with damp-heat syndrome by literature mining.To summarize the construction methods of animal model of gouty arthritis with damp heat syndrome,and provides data support for establishing a feasible model of gouty arthritis with damp-heat syndrome.Part Ⅱ:To compare the modeling effects of two methods with honey water + lard+ White Spirit + High temperature and humidity environment + Inject MSU crystal suspension into the joint and honey water + lard + White Spirit+ Salmonella typhimurium liquid + Inject MSU crystal suspension into the joint.To explore a stable and reliable disease-syndrome combination model of damp-heat syndrome gouty arthritis,in order to study the relationship between GA and syndrome,the disease-syndrome combination animal model for drug research.Methods:Part Ⅰ:By formulating the literature screening criteria,and select Web of Science,Clinical Key,Pub Med,the Chinese Academic Journal Full-text Database(CNKI),Wanfang Science and Chinese VIP Science and Technology Journal Database to retrieve relevant literature.A database was established through EXCEL to summarize the distribution of research institutions,animal species,gender distribution,intervention factors,evaluation indicators,evaluation standards of "disease" and "syndrome" models,etc.Then analyze and organize valuable information.Part Ⅱ:1.Animal grouping: 44 SPF male SD rats were randomly divided into 4groups according to the random number table method: the control group,the gout group,the model group A,and the model group B.Except for the control group and the gout group with 10 animals,other groups 12 in each group.The control group was fed with normal diet and free drinking water under normal temperature and humidity environment.The Gout group was fed under the same conditions as the control group,and MSU crystal suspension were injected into the articular cavity.The Model group A was fed with high-glucose-fat food and added 200 g/L honey water to drink freely,and the rats were fed with oil and liquor alternately every other day for 14 days,and then continued for 14 days at high temperature and high humidity.and finally inject the MSU crystal suspension into the joint cavity to make a model.The Model group A was fed with high-glucose-fat food and added 200 g/L honey water to drink freely,and the rats were fed with oil and liquor alternately every other day for 14 days.fed with Salmonella typhimurium solution on the 10 th day,and boosted it once on the 15 th day.and finally inject the MSU crystal suspension into the joint cavity to make a model.2.According to the scoring rules of the syndrome scoring scale,the rats were characterized and scored from seven aspects,including mental state,diet and drinking water,coat color,tongue appearance,stool character,urination,and scrotum,etc.The body weight,food intake,ankle gait grading,joint swelling,ankle infrared calorific value,were measured.and then Serum levels of AQP2,TC,TG,HDL-C,LDL-C,PG,MTL,GAS,ET-1,CGRP,HSP70,IL-1β,IL-6,TNF-α and NF-κB were measured by Elisa.HE staining was used to observe the pathological changes of ankle join colon under microscope,and Comprehensively evaluate two different animal models of gouty arthritis with damp heat syndrome.3.SPSS software(version number: 26.0)was used to process the data,the data of each group was expressed as(x±s),and the statistical data between each group was compared by one-way ANOVA.For multiple comparisons between groups,LSD test was used when the variance was homogeneous.Otherwise,Dunnett’s T3 test was used.α=0.05 was used as the test level,P<0.05 indicated statistical difference,and P<0.01 indicated extremely significant statistical difference.Graphing was performed using Graphpad Prism software(version number: 8.0.2).Results:Part Ⅰ:1.Two different animal models of gouty arthritis with damp-heat syndrome were found in 4 selected experimental literatures,(1)honey water+ lard + White Spirit + High temperature and humidity environment + Inject MSU crystal suspension into the join.(2)honey water + lard + White Spirit+ Salmonella typhimurium liquid + Inject MSU crystal suspension into the joint.2.Its research institutions include not only the Comprehensive University of traditional Chinese medicine but also clinical hospital institutions.3.The model animals involved were mainly 200±20g male SD rats.4.The intervention factors included high-glucose-fat,White Spirit,damp heat environment,biological factors,etc.5.The evaluation of the model is mainly based on the multi-dimensional comprehensive judgment of animal macroscopic signs,symptoms,behavioral observation,serum biochemical detection,ankle pathology and so on.Part Ⅱ:1.Rat mortality: No death occurred in the control group and the gout group;One animal died in the model group A,with a mortality rate of 8.3%;Three rats in model group B died,and the mortality rate was 25%;Autopsy revealed pulmonary congestion,extensive gastrointestinal flatulence,mucosal congestion and edema.The liver is bluish-gray,and the abdominal cavity is moderately ascites.2.Rats in model group A and model group B both appeared "Damp-heat" performance.for example: mental burnout and drowsiness,withered wet hair loss,The scrotum is flabby and Saggy,the weight and diet are reduced,the amount of defecate is little even shapeless.After injection of MSU crystal suspension,the gout group,model group A and model group B rats all showed irritability,and licked their right foot with their tongue.The right hind limb was bent,lightly touched and even lifted off the ground in a three-legged gait.The right ankle joint has the features of redness,swelling,heat and pain similar to clinical GA patients during inflammatory attacks.The TCM syndrome scores of rats in model group B were higher than those in model group A,but there was no significant significance(P<0.05).It may be that the damp heat syndrome is the result of long-term factor intervention,and the modeling time is too short.3.Changes in body weight and food intake in rats: The body weight of each group increased steadily in the first 7 days.The body weight of rats in model group A and model group B was slightly lower than that in normal control group and gout control group,but there was no statistical significance(P>0.05).On the 21 st day,the body weight and food intake of rats in model group A and model group B were significantly lower than those of control group and gout group(P<0.01),The results showed that both modeling methods could inhibit the growth of body weight and appetite of rats,which led to similar clinical signs of damp-heat syndrome.The body weight and food intake of the rats in the model group B were lower than those in the model group A(P<0.05),which suggested that the effect of oral administration of Salmonella typhimurium on the rats was more obvious,the results were consistent with that of the above-mentioned Group B rats.4.Changes of infrared calorific value,joint swelling,gait grade and inflammatory index of ankle joint in rats: Compared with the control group,the infrared calorific value,gait,arthritis index and swelling degree of ankle joint in Gouty group,model group A and model group B were significantly increased(P<0.01),and the swelling degree of ankle joint reached its peak after 12 hours.After that,even without intervention,the symptoms were gradually relieved,indicating the success of this experiment,This also suggests that the golden time of treatment is within12 h of clinical GA attack,which can greatly alleviate the symptoms of GA attack.In addition,the degree of joint swelling in model group A and model group B was higher than that in gout group at 72h(P<0.05),which may indicate that damp-heat syndrome has an inhibitory effect on the self-limiting of GA attacks.5.Compared with the control group,the expression of HSP70,NF-κB,IL-1β,IL-6 and TNF-α in gout group,model group A and model group B were significantly increased(P<0.01),which indicated that the model was successfully replicated.Compared with the control group and the gout group,the expressions of AQP2,TG,LDL-C,MTL,GAS and ET-1 in model group A rats were significantly increased(P<0.01),while the expressions of CGRP and HDL-C were decreased(P<0.05);the expressions of TG,TC,LDL-C,MTL,GAS,ET-1 and HSP70 in model group B rats were increased(P<0.05),while the expressions of CGRP and HDL-C were decreased(P<0.05).Compared with the gout group,the levels of TG,TC,MTL,GAS,ET-1,NF-κB and TNF-αin model group A and model group B were increased(P<0.05),while CGRP and HDL-The expression of C was significantly decreased(P<0.01),which indicated that the two modeling methods both caused changes in the microscopic indicators of "humidity" and "heat" in rats,and the model of "damp-heat" may stimulate the body to produce chronic inflammatory reaction.Compared with model group A,the expressions of ET-1,HSP70,IL-1β,IL-6 and TNF-α in model group B were higher(P<0.05),which may indicate that the inflammatory reaction of model group B is more obvious than that of model group A.6.Pathology of ankle joint in rats: Compared with the control group,the synovial tissues of the gout group,the model group A and the model group B were significantly congested and swollen,and the synoviocyte proliferation disorder was arranged irregularly in layers with a lot of fibrous hyperplasia,cartilage is severely damaged,and pannus is formed.7.Pathology of rat colon: Compared with the control group and the gout group,the glands of model group A and model group B were disorderly,the crypts were deformed,the mucosa was eroded in a small area,and there was a small amount of neutrophil infiltration.Conclusions:Part Ⅰ:In the animal model of gouty arthritis of damp-heat syndrome,200±20g male SD rats were mainly selected.At present,there are two main methods to combine the symptoms and signs of gouty arthritis of damp-heat syndrome with animal model,(1)one is honey water + lard + White Spirit + High temperature and humidity environment + Inject MSU crystal suspension into the joint.(2)honey water+ lard + White Spirit + Salmonella typhimurium liquid + Inject MSU crystal suspension into the joint.Part Ⅱ:1.The animal model of gouty arthritis with damp-heat syndrome induced by two different methods had the symptoms and signs of "Damp heat syndrome" and the typical symptoms of GA acute attack,and the model group A is safer and more stable.2.The symptomatic manifestations,joint swelling,inflammatory index and serum biochemical indexes of model group A and model group B have both similarities and their own characteristics.The main manifestation is that the model group A is more inclined to "wet",while the model group B has more obvious inflammatory reaction,so group B was more prone to "Hot".