The Study On Treatment Of Damp-heat Syndrome Of Mice Malaria By Artemisinin Combined With Shuang Huanglian Injection

Objective:This study explored the establishment and evaluation of damp-heat syndrome of mouse malaria in Traditional Chinese Medicine（TCM）,and preliminarily explored the syndrome essence.According to the “combination of disease and chinese medicine syndrome” theory,artemisinin combined with Shuang Huanglian injection or not was used to treat damp-heat syndrome of mouse malaria,to compare the efficacy of the two and initially explored the mechanisms.Author hope this study would enrich research of damp-heat syndrome in Traditional Chinese Medicine and provide a basis for the prevention and treatment of malaria by the integrated medicine of Traditional Chinese medicine and the West.Methods:1.Establishment and evaluation of mouse malaria model with TCM damp-heat syndrome: SPF female ICR mice were randomly divided into 7 groups,with 6 mice per group: Control group,damp-heat group（DH）,damp-heat drug group（DH-D）,mouse malaria group（P.bK173）,mouse malaria drug group（P.bK173-D）,mouse malaria dampheat syndrome group（DHP.bK173）,mouse malaria damp-heat syndrome drug group（DHP.bK173-D）.Groups DH,DH-D,DHP.bK173 and DHP.bK173-D were placed in an artificial climate chamber with high temperature and humidity（temperature: 37±1℃,humidity 91±0.5%）for 21 days.After 21 days,groups P.bK173,P.bK173-D,DHP.bK173 and DHP.bK173-D were intraperitoneally infected with Plasmodium berghei strain K173（5*10⁶ pRBC per mouse）.The day of infection was recorded as D0.Groups DH-D,P.bK173-D and DHP.bK173-D were given Haoqin Qingdan decoction by oral administration at D5,and the other groups were given physiological saline.The weight, temperature,food intake,water intake,parasitemia and the overall state of mice were observed.Besides,performed open field test to evaluate the voluntary activity ability,weighed liver,spleen and kidney organs,and calculated organ indexes.ELISA kits were used to detect cytokines such as TNF-α,IL-8,ET-1,NF-κB,IL-1,VEGF in serum and liver tissue,AQP-4 in kidney and colon.Western blot was used to detect PI3 K Class Ⅲ,Akt,p-Akt,mTOR,p-mTOR,p-IKK,Bcl-xL,IL-6,and CD81 in liver,and qPCR was used to detect PI3Kr1/Akt1 mRNA expression in liver.Pathological sections of liver,spleen,kidney and tongue were made to observe pathological changes after hematoxylineosin staining.2.Artemisinin combined with Shuang Huanglian injection for the treatment of damp-heat syndrome of mouse malaria: SPF female ICR mice were randomly divided into 8 groups,with 6 mice per group: Control,the malaria damp-heat syndrome mouse model group（DHP.bK173）,artemisinin low-dose group（ART-L,12.5mg/kg）,artemisinin medium-dose group（ART-M,25mg/kg）,and artemisinin high-dose group（ART-H50mg/kg）,artemisinin low-dose combined with Shuang Huanglian injection group（ARTL+SHL,ART-12.5mg/kg+SHL-10.72ml/kg）,artemisinin medium-dose combined with Shuang Huanglian injection group（ART-M+SHL,ART-25mg/kg+SHL-10.72ml/kg）,artemisinin high dose combined with Shuang Huanglian injection group（ART-H+SHL,ART-50mg/kg+SHL-10.72ml/kg）.Groups DHP.bK173,ART-L,ART-M,ART-H,ARTL+SHL,ART-M+SHL,and ART-H+SHL were intraperitoneally infected with Plasmodium berghei strain K173（5*10⁶ pRBC/mouse）after 21 days in artificial climate chamber with high temperature and humidity（temperature: 37±1℃,humidity 91±0.5%）.The day of infection was recorded as D0.At D5-D8,ART-L,ART-M,ART-H,ARTL+SHL,ART-M+SHL and ART-H+SHL groups were continuously treated for 4 days.The weight and the overall state of mice were observed for determining formation of mouse malaria model with TCM damp-heat syndrome.Besides,observed parasitemia rate on D5 and D9 after inoculation,calculated ED₅₀ and ED₉₀ by probit regression,weighed the liver,spleen and kidney organs and analyzed their organ index,and did the blood biochemistry.The cytokines ICAM-1,IL-8,ET-1,TNF-α,PECAM-1 and VCAM-1in serum and liver were detected by ELISA kit.Western blot was used to detect mTOR,p-mTOR,PI3Kp110,PI3 K Class Ⅲ,p-IKK,Akt,p-Akt,PTEN,Bcl-xL,VEGF,IL-6 and CD81,and qPCR was used to detect PI3Kr1/Akt1 mRNA expression in liver. Pathological sections of liver,spleen,and kidney were made to observe pathological changes after hematoxylin-eosin staining.Results:1.Establishment and evaluation of mouse malaria model with TCM damp-heat syndrome: After 21 days living in high temperature and high humidity environment,the mice had developed into damp-heat syndrome of TCM with certain but mild pathological changes,including wet yellow fur,head hair lose,limb fatigue,increased mucus in feces,dry stool,increased food intake,increased water intake,decreased weight and decreased body temperature（P<0.05）.Pathological sections showed organ tissue had pathological changes,including varying degrees of hyperaemia,loose swelling of cells,increased lymphocytes,increased thickness of lamina propria of tongue tissue,etc.And decreased serum ET-1 and NF-κB（P<0.05）was observed.The mice infected with plasmodium in normal environment showed increased food intake,reduced stool volume and water content,and decreased voluntary activity（P<0.05）.Parasitemia rate increased rapidly at D7 and the organ index of liver and kidney were increased（P<0.05）.Pathological sections showed destruction of liver,spleen and kidney tissue structure,a large of malaria pigment deposition and red blood cell obstruction in liver,spleen and kidney,and immune cell infiltration.The expression of IL-8,NF-κB,VEGF and ET-1 in liver were increased（P<0.05）,while the expression of AQP-4 in kidney,Akt,p-mtor and CD81 in liver were decreased（P<0.05）.The physical signs of mouse malaria with damp-heat syndrome were similar to those of mice with damp-heat syndrome but aggravated.The fur of DHP.bK173 was moist,yellow and disorderly,some of the mice were depilated on the head,lethargy,aggregation,and the body weight and temperature were significantly decreased（P<0.01）.The intake of food and water was decreased,the stool was soft and partially unformed,and the mucus in stool increased significantly.The voluntary activity decreased（P<0.05）,and the liver organ index increased（P<0.05）.Pathological sections showed malarial pigment was deposited in the liver and spleen,red blood cells were aggregated and obstructed,the structure of the liver,kidney and spleen were damaged sincerely,the keratinization of the tongue was obvious,and the density of papillae decreased and the thickness of the lamina propria was increased.Besides,the expression of ET-1 in serum,PI3 K Class Ⅲ,p-mTOR and CD81 in liver decreased,and the expression of PI3Kr1 and Akt1 mRNA were higher than group DH（P<0.05）.Compared with P.bK173,the parasitemia rate of DHP.bK173 during D0-D5 increased slowly（P<0.05）,the trend of growth was earlier and shifted to the left,the weight of liver,spleen and kidney increased less（P<0.05）,the structural damage of liver,spleen and kidney was aggravated,the infiltration of immune cells was reduced,and the thickness of lamina propria of tongue tissue increased.IL-8 and VEGF in liver decreased（P<0.05）,and AQP-4 in kidney increased（P<0.05）.After the administration of Haoqin Qingdan decoction,for DH-D,the food intake decreased,the water intake increased,the stool mucus decreased,the stool was harder.Besides,the expression of TNF-α,ET-1,NF-κB,VEGF in serum and Bcl-xL,IL-6,Akt1 mRNA in liver were increased（P<0.05）,while AQP-4 in kidney was decreased（P<0.05）.For P.bK173-D,food intake further reduced,fecal volume increased,parasitemia rate grow faster than P.bK173 within 36 hours,tongue tissue lamina propria thickness decreased,and the expression of ET-1 in serum,CD81 and PI3Kr1 mRNA in liver were increased（P<0.05）.For DHP.bK173,water intake and voluntary activity increased（P<0.05）,fecal water and mucus reduced,the growth of parasitemia rate slowed down（P<0.05）,and lamina propria thickness reduced.The expression of TNF-α,ET-1 in serum,IL-8,PI3 K Class Ⅲ,CD81,Akt1 mRNA in liver and AQP-4 in colon were increased,while the levels of renal AQP-4 decreased（P<0.05）.2.Artemisinin combined with Shuang Huanglian injection for the treatment of dampheat syndrome of mouse malaria: The mouse malaria model with damp-heat syndrome had damp,yellow and disorderly fur,the head was epilated,tend to aggregation and lethargy,stool reduced but mucus increased,and weight loss（P<0.05）.The liver and kidney function was seriously damaged,the liver,spleen and kidney organ indices were increased（P<0.05）,and the tissue structure was seriously damaged.the expressions of IL-8 in serum and PI3 K Class Ⅲ,CD81 in liver were decreased（P<0.05）,while Akt1 mRNA expression was increased（P<0.05）.After medication intervention,the characteristics of damp-heat syndrome were improved.Among them,fecal mucus of ART-M+SHL and ART-H+SHL were significantly reduced,and ART-H+SHL vigour was significant improved.With the increase of artemisinin dose,parasitemia reduced,liver and kidney function improved and the tissue damage reduced.Compared with the artemisinin group,the ED₅₀,ED₉₀ and the structural damage of liver,spleen and kidney of the combination medication groups were reduced.The inflammatory cell infiltration,malaria pigment deposition and red blood cell obstruction were also reduced.In terms of cytokine and protein expression,ART-L could reduce the expression of PTEN and Bcl-xL of liver.ART-M increased the expression of IL-8 and PI3 K Class Ⅲ,and reduce the expression of PTEN and Bcl-xL in liver.ART-H significantly increased expression of IL-8 and PECAM-1 in serum as well as PI3 K Class Ⅲ,VEGF,Bcl-xL,CD81,PI3Kr1 mRNA in liver,and decreased the expression of the IL-8 in liver（P<0.05）.ART-L+SHL significantly reduced expression of VCAM-1 in serum,and p-mTOR and IL-6 in liver（P<0.05）.ART-M+SHL increased expression of ICAM-1 and IL-8 in serum and VCAM-1,TNF-α,PECAM-1 and ET-1 in liver,decreased expression of TNF-α in serum and PI3Kp110,PI3 K Class Ⅲ,Akt and mTOR in liver（P<0.05）.ART-H+SHL decreased expression of ICAM-1 and TNF-α in serum and PI3 K Class Ⅲ,mTOR and Bcl-xL expression in liver,increased expression of IL-8 and ICAM-1 in liver（P<0.05）.ART-M and ART-H could increase the expression of PI3Kr1 mRNA while ART-L,ART-M,ARTH and ART-M+SHL inhibited the expression of Akt1 mRNA（P<0.05）.Conclusion1.A stable mouse malaria model with damp-heat syndrome can be established by the method of "high temperature and humidity environment + infected with plasmodium".It has specific physical symptom,pathological changes and the disease site are liver and spleen in TCM.2.The animal model of TCM damp-heat syndrome can be assessed and diagnosed by“intervention factors + limb fatigue/decreased activity + other signs”.3.Compared with artemisinin monotherapy,artemisinin combined with Shuanghuanglian injection has certain advantages in the treatment of damp-heat syndrome of mouse malaria.It can significantly improved the symptoms of damp-heat syndrome,reduce the dosage application of artemisinin and organ tissue damage,which may be related to multi-target intervention of Akt pathway and bidirectional regulation of inflammatory factors,adhesion factors and endothelial cytokines.4.The essence and the therapeutic mechanism of damp-heat syndrome of mice malaria may be related to the complex regulation mechanism of Akt pathway.