| Purpose:Damp-heat syndrome is a common clinical witness of traditional Chinese medicine,the course of the disease is repeated,throughout the development of many diseases,often manifested as fever,loose stool thin,sticky,foul odor,etc.,its pathological mechanism is not completely clear.In this study,by establishing a rat model of damp-heat syndrome,the pathological mechanism of damp-heat syndrome was explained from the perspective of intestinal mucosal barrier,and the therapeutic effect of Chinese herbal compound Gegenqinlian Decoction was initially discussed.Material and method:Twenty-four SPF wistar male rats were randomly divided into control group(C),damp-heat syndrome group(M),Gegenqinlian Decoction low-dose group(0.98·kg-1· d-1)(T1)and Gegenqinlian decoction high-dose group(4.9·kg-1·d-1)(T2),with6 rats in each group.The control group was fed ordinary diet and given 2 m L normal saline every day,and placed in a normal environment with temperature(18±3)℃ and humidity(45±15)%.The other groups were fed high-fat diet and 20% honey with unlimited water every day,and were given 2 m L 56° liquor,and placed in an artificial climate chamber with temperature of(32±2)℃ and humidity of(90±5)% from 9:00 to 17:00 to prepare damp-heat syndrome model for 42 days.After the modeling,the treatment group was given the corresponding dose of drugs,and the control group and the damp-heat syndrome group were given the same volume of normal saline for 14 days.During this period,the mental state,body shape,diet and water intake,tongue image,coat,scrotum and defecation of the rats were dynamically monitored every two weeks,and score statistics were performed every two weeks.The autonomous behavior,exploratory behavior and activity of the rats were recorded by open field experiment before and after modeling and after treatment,so as to determine whether the damp-heat syndrome rat model was successfulully established.The pathological changes of colonic tissue were observed by HE staining after the animals were killed.The levels of D-LA and DAO in plasma and IL-6,IL-10,TNF-α and IFN-γ in serum were determined by ELISA.The expression levels of tight junction proteins Occludin,Claudin-1,ZO-1 and ZO-2 in colon tissue of rats were detected by Western Blot.Results:1.Observation of the general situation of rats: the rats in the control group have bright eyes,moderate diet and drinking water,moderate activity,flexible response,soft and smooth fur,moderate shape and size of testicles,hidden under the coat,light yellow urine color,soft and hard stool medium brown granular,tongue color light red;Compared with the control group,the rats in the dampened heat syndrome group gradually appeared lethargy,lethargy,reduced sensitivity,slow reaction,hair yellow dye,slow weight growth,reduced diet and water intake,red swelling and swelling of the scrotum,short urine,yellow,cloudy and thick brown,loose stool thin,even perianal filthy,dark red tongue a little prickly and so on.Compared with the damp-heat group,the spirit of the rats in the Gegenqinlian decoction low-dose and high-dose groups gradually improved,the activity increased,the fur became shiny,the body weight gradually returned to the normal growth rate,the amount of diet and water increased,the scrotum returned to normal,the urine color was light yellow,the stool was soft and hard moderate granular,and the tongue color was light red.2.Symptom scores of rats in each group: The symptom scores of rats in each group changed with time.With the development of the experiment,the symptom scores of rats in the control group maintained at a stable level without obvious fluctuations.Compared with the control group,the symptom scores of the rats in the damp-heat syndrome group increased slightly(P< 0.01),and the subsequent scores fluctuated at a higher level(P < 0.01),indicating that the rats had signs of damp-heat syndrome.Compared with the damp-heat syndrome group,the symptom scores of the low-dose Gegenqinlian decoction group decreased and gradually recovered to a lower level(P < 0.01),indicating that the signs of damp-heat syndrome were reduced.Compared with the damp-heat syndrome group,the symptom scores of the high-dose Gegenqinlian decoction group decreased gradually,indicating that the signs of damp-heat syndrome were relieved(P < 0.05).3.Results of open field behavior experiment: Before modeling,there were no significant differences in the total distance and cumulative duration of the boundary between the four groups of rats(P > 0.05).After modeling,compared with the control group,there were significant differences in the total movement distance and boundary accumulation duration of rats in model group and Gegenqinlian Tang treatment group(p < 0.01),indicating that the motor power of rats was weakened,activity was decreased,inquiry was poor,and anxiety was increased.After treatment,compared with the damp-heat syndrome group,there were significant differences in the total exercise course of rats in the Gegenqinlian decoction low-dose and high-dose groups(P < 0.01),indicating that the motor power of rats was gradually enhanced,and the activity was improved,and the exploration ability was better.Compared with the dampness-heat syndrome group,the cumulative duration of the boundary behavior of rats in the low-dose Gegenqinlian decoction group was significantly different(p <0.01),indicating that the anxiety of rats was relieved and the motor power was enhanced.Although there was no significant difference in the high-dose Gegenqinlian decoction group(P > 0.05),there was a trend.4.Morphological results showed that:(1)Visual observation: in the control group,the intestinal tube was tender and light yellow,with smooth intestinal mucosa,no gas,edema,bleeding,and other phenomena,and good elasticity.Compared with the control group,the intestinal tissue of the damp-heat syndrome group was darker in color,with poor elasticity,adhesion to surrounding tissues,uneven surface,thickening and edema,varying degrees of gas accumul ation in the intestinal cavity,and beaded shape in severe cases.Compared with the damp-heat syndrome group,the intestinal condition of the low and high dose Gegenqinlian decoction groups was alleviated.(2)Pathological morphology under light microscope: after HE staining,the colon tissue structure of the control group was clear and complete,the mucosa,submucosa,muscle layer and serosa layer were arranged neatly,the epithelial cells contained a large number of goblet cells,and glands were developed.Compared with the control group,the colon tissue of the damp-heat syndrome group was necrotic,the serosa layer was broken and separated and disappeared,the glands were irregularly arranged or even disappeared,the epithelial cells were in poor shape,the goblet cells were significantly reduced,and the crypt structure was incomplete.Compared with the damp-heat syndrome group,the structure of each layer in the low and high dose Gegenqinlian decoction groups was complete,regular and clear,the arrangement of glands was basically restored,goblet cells were significantly increased,and the structure of cryps was significantly improved.5.ELISA results showed that:Compared with the control group,the plasma levels of D-LA and DAO in the damp-heat syndrome group were significantly increased(P<0.05).Compared with the damp-heat syndrome group,the plasma levels of D-LA and DAO in the low-dose Gegenqinlian decoction group were significantly decreased(P<0.05).Compared with the damp-heat syndrome group,the plasma levels of D-LA and DAO in the high-dose Gegenqinlian decoction group tended to decrease(P>0.05).Compared with the control group,the content of TNF-α in the serum of the damp-heat syndrome group was significantly increased(P<0.05),the content of IL-6 was significantly increased(P<0.01),the content of IL-10 was significantly decreased(P<0.05),and the content of IFN-γ was significantly increased(P<0.01).Compared with the damp-heat syndrome group,the content of TNF-α in serum of the low-dose Gegenqinlian decoction group was significantly decreased(P<0.05),the content of IL-6 was significantly decreased(P<0.01),the content of IL-10 was significantly increased(P<0.05),the content of IFN-γ was significantly decreased(P<0.01).Compared with the damp-heat syndrome group,the content of TNF-α in the serum of the high-dose Gegenqinlian decoction group was significantly decreased(P<0.05),the content of IL-6 had no significant change,the content of IL-10 was significantly increased(P<0.01),and the content of IFN-γ had a tendency to decrease(P> 0.05).6.Western Blot results showed that:Compared with the control group,the Occludin expression and Claudin-1 expression of intestinal occludin in the damp-heat syndrome group were significantly decreased(P < 0.01),and the ZO-1 and ZO-2 protein expression were significantly decreased(P < 0.01).Compared with dampness-heat syndrome group,the Occludin expression and Claudin-1 protein expression in Gegenqinlian decoction low-dose group were significantly increased(P < 0.05),and the ZO-1 and ZO-2 protein expression levels were significantly increased(P < 0.01).Compared with the damp-heat syndrome group,the Occludin expression in Gegenqinlian decoction high-dose group was significantly increased(P < 0.05),the Claudin-1 protein expression had a downward trend(P > 0.05),the ZO-1 protein expression was significantly increased(P < 0.01),and the ZO-2 expression level had no significant change(P > 0.05).Conclusion:1.The pathogenesis of damp-heat syndrome may be related to the damage of intestinal mucosal epithelial cells,the decrease of intestinal tight junction protein,the increase of intestinal permeability,and body inflammation.2.Gegenqinlian Decoction may reduce intestinal permeability,repair the damaged mechanical barrier of intestinal mucosa,and reduce inflammation by regulating the expression of related intestinal tight junction protein,thus achieving the effect of treating damp-heat syndrome. |
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