| Damp-heat diarrhea in livestock and poultry is a common disease in the southwest of China in summer and autumn,which brings huge economic cost to the breeding industry.According to the traditional Chinese medicine,the pathologic location of diarrhea is mainly in the spleen,stomach,and intestine;Yet mainly in the spleen.The pathogenic factors include exogenous pathogen,Dietary disorders,emotional disturbance,spleen-stomach weakness and spleen deficiency syndrome,etc..However,the main pathogenic factor is dampness pathogen that recorded in the book of Nanjing,which leads to the disorder of spleen transport and then causes diarrhea.That is to say,dampness leads to five evils.Traditional Chinese medicine has good effects on diarrhea.According to clinical syndrome differentiation and treatment,Zhukuqin compound has the functions of heat-clearing,detoxifying,eliminating-dampness and anti-diarrhea.The previous research showed that Zhukuqin granule(ZKQG)can effectively treat the diarrhea of piglets with damp-heat.Whereas,the mechanisms of promoting the spleen transport in ZKQG was still unclear.The purpose of this study was to establish the model of damp-heat diarrhea in rats and then they would be treated by ZKQG,to explore the clinical therapeutic effect of ZKQG on rats with damp-heat diarrhea and its mechanisms of promoting the spleen transport,and to provide a theoretical basis for the clinical application of ZKQG.1.Studied on the therapeutic effect of ZKQG on rats with damp-heat diarrhea.Established the model of damp-heat diarrhea in rats(administration by high fat and high sugar,high temperature and high humidity,senna leaf)and they were treated by ZKQG,the clinical symptoms were observed,and the related indexes in the blood were measured.The results showed that the rats in blank group were in clear fur,had good mental state,and normal eating and drinking,and they had healthy posture,and the body-temperature,feces,urine,and anus were normal;The rats in model group were in fluffy hair fur,they curled up and didn’t want to move,they were short of appetite,and suggested red or umid anus,and defecate were thin and soft,or loose stools,and urine were dark and yellow.After administration of 5 d,the effective rate in Baitouweng group,ZKQG high/middle/low dose groups were 100%,and the cure rate was respectively 100%,100%,62.5% and 50%,respectively.Compared with the blank group,the diarrhea index and the contents of RBC,HGB,PCV,WBC,PMN in model group were increased,the indexs of spleen and thymus was decreased,and the level of IL-2 in serum was decreased on the 3 d and 5 d after administration.Compared with model group,the diarrhea index and the contents of RBC,HGB,PCV,WBC,PMN in Baitouweng group and ZKQG high/middle/low dose groups were decreased,the indexs of spleen and thymus and level of IL-2 in serum were increased.It was suggested that ZKQG could effectively treat the rats with damp-heat diarrhea and improve the immune ability.2.Studied the effect of ZKQG on caspase-3 and caspase-9 in spleen of rats with damp-heat diarrhea.The spleen pathological sections were made and observed,The levels of caspase-3 and caspase-9 in spleen were detected by ELISA.The results showed that the spleen tissue were damaged with a large number of constricted lymphoid nucleis,splenic sinus dilatation,red blood cell leach,and the levels of caspase-3 and caspase-9 were significantly increased(P<0.05 or P<0.01)in model group in comparsion to blank group.Compared with model group,the injured spleen tissue recovered,and the levels of caspase-3 and caspase-9 were decreased in Baitouweng group and ZKQG high/middle/low dose groups,in which the effect of ZKQG high/middle/low doses were dose-dependent.It was suggested that ZKQG could reduce the injury of spleen tissue and pathological apoptosis of spleen cells in rats with damp-heat diarrhea.3.Studied the effect of ZKQG on MDA and SOD in spleen of rats with damp-heat diarrhea.The levels of MDA and SOD of spleen in rats were detected by ELISA.The results showed that the levels of MDA and SOD in model group were significantly increased and decreased,respectively(P<0.01)in comparison with blank group.Compared with model group,The levels of MDA and SOD in Baitouweng group and ZKQG high/middle/low dose groups were decreased and increased,respectively.It was suggested that ZKQG could alleviate oxidative stress injury of spleen.4.Studied the effect of ZKQG on autophagy related proteins in spleen of rats with damp-heat diarrhea.The levels of LC3-Ⅱ,LC3-Ⅰ,Beclin1,mTORCI and p-mTORCI of spleen in rats after administration of 5 d,10 d and 15 d were detected by ELISA.The results showed: After administration of 5 d,compared with blank group,the levels of LC3-Ⅱ,LC3-Ⅰ,Beclin1,LC3-Ⅱ/LC3-Ⅰ and mTORCI/p-mTORCI in the spleen of model group were increased,the levels of mTORCI and p-m TORCI were decreased(P>0.05);Compared with model group,the levels of LC3-Ⅱ,LC3-Ⅰ,Beclin1,LC3-Ⅱ/LC3-Ⅰ and mTORCI/p-mTORCI of the spleen in Baitouweng group and ZKQG high/middle/low dose groups were increased,the levels of mTORCI and p-m TORCI were decreased(P<0.01).After administration of 10 d and 15 d,compared with blank group,the levels of LC3-Ⅱ,Beclin1,LC3-Ⅱ/LC3-Ⅰ were significantly increased(P<0.01),the levels of LC3-Ⅰ,mTORCI/p-mTORCI were increased,the levels of mTORCI and p-mTORCI were significantly decreased(P<0.01)in the spleen of model group;Compared with model group,the levels of LC3-Ⅱ,LC3-Ⅰ,Beclin1,LC3-Ⅱ/LC3-Ⅰ and m TORCI/p-mTORCI were decreased,the levels of mTORCI and p-m TORCI were significantly increased in the spleen of Baitouweng group and ZKQG high/middle/low dose groups.It was suggested that ZKQG could promote the autophagy of spleen cells within a certain limit,also could inhibit the excessive autophagy of the spleen cells.5.Studied the effect of ZKQG on the mRNA expressions of mTOR,Akt and PI3 K in spleen of rats with damp-heat diarrhea.After administration of 5 d,10 d and 15 d,the spleen of rats wasaseptically collected and RNA was extracted,and then the mRNA expressions of mTOR,Akt and PI3 K were detected by qRT-PCR.The results showed: After administration of 5 d,there was no significant difference in the mRNA expressions of mTOR,Akt and PI3 K between model group and blank group(P>0.05);Compared with model group,the mRNA expressions of mTOR,Akt and PI3 K of spleen in Baitouweng group and ZKQG high/middle/low dose groups were significantly decreased(P<0.01).After administration of 10 d and 15 d,compared with blank group,the mRNA expressions of mTOR,Akt and PI3 K in the spleen of the model group were significantly decreased(P<0.01);Compared with model group,the mRNA expressions of mTOR,Akt and PI3 K were increased in the spleen of Baitouweng group and ZKQG high/middle/low dose groups.It was suggested ZKQG could regulate the autophagy of spleen cells in rats with damp-heat diarrhea by regulating PI3K/Akt/mTOR signal pathway,so as to achieve the effect of regulating spleen transport.In conclusion,ZKQG has good therapeutic effect on rats with damp-heat diarrhea.ZKQG could regulate the autophagy of spleen cells in rats with damp-heat diarrhea by regulating PI3K/Akt/mTOR signal pathway,so as to eliminate the damaged organelles or proteins,reduce the pathological apoptosis,improve the immunity of body,and achieve the effect of regulating spleen transport. |
