| Gastrointestinal heat retention refers to the syndrome of gastrointestinal heat caused by a high-calorie diet that swells in the gastrointestinal tract.Consuming high-calorie food is likely to cause gastrointestinal heat retention in children since their viscera and gastrointestinal tract are delicate and fragile.Retention of heat in the gastrointestinal system can encourage the emergence and progression of respiratory tract infections,leading to co-disease of the lungs,stomach,and intestine.In the treatment of gastrointestinal heat retention attached to pneumonia,Yinlai Decoction,a valid prescription of the simultaneous treatment of the lung,stomach,and intestines,has a major impact.However,the precise process is yet unclear.And 5-HT which is an important immune medium in the body can regulate the immune response in a variety of ways.Previous studies have found that rats of the gastrointestinal heat retention combined with pneumonia exhibited lower levels of 5-HT and an imbalance of Th17/Treg cells.This study was designed to explore the mechanism of the gastrointestinal heat aggravating pneumonia and the therapeutic mechanism of Yinlai Decoction based on 5-HT level of serum and the balance of Th17/Treg cells.Experiment 1:Effects of Yinlai Decoction on 5-HT Levels of Serum and Th17/Treg Cell Balance in Rats of the Gastrointestinal Heat Retention Combined with PneumoniaPurpose:To explore 5-HT level of serum and Th17/Treg cell balance in rats of the gastrointestinal heat retention combined with pneumonia,and the intervention effect of Yinlai Decoction.Method:A total of 30 SPF rats(4-week-old)were randomly divided into a normal group(Group N),a pneumonia group(Group P),a gastrointestinal heat retention group(Group G),a gastrointestinal heat retention combined with pneumonia group(Group GP),and a Yinlai Decoction treatment group(Group T).While the rats with gastrointestinal heat retention were fed a high-calorie diet and milk gavaged,the pneumonia rats were generated by inhaling LPS aerosol.The gastrointestinal heat retention combined with pneumonia rats were prepared by the combination of the two,and were intervened by Yinlai Decoction.The general conditions of the rats were observed,and the lung and colon tissues were detected by HE staining.The levels of serum IL-1β,TNF-α,IL-6,IL-17A,IL-10 as well as serum 5-HT level were detected by ELISA.The expression levels of Tphl mRNA and protein in colonic tissues were detected by RT-PCR and WB.The expression levels of Roryt and Foxp3 proteins in mesenteric lymph nodes were detected by WB.Result:1.General(1)Sign of rats:After modeling of gastrointestinal heat retention combined with pneumonia,the rats showed malaise,decreased activity,dark and lusterless fur,soft yellow stool texture,and greasy odor.Yinlai Decoction could improve the mental state of rats and reduce the greasy and unpleasant odor of feces.(2)Body weight:After modeling of gastrointestinal heat retention combined with pneumonia,the body weight of rats increased slowly;Yinlai Decoction had no significant effect on the body weight growth rate of rats.2.Lung and colon histopathology(1)Pulmonary histopathology:Rats’ alveolar structures became disorganized after pneumonia modeling,along with edema and inflammatory cell infiltration.The inflammatory injury in the rat lung was further exacerbated,with a significant amount of inflammatory exudation,once the model of gastrointestinal heat retention combined with pneumonia was developed.Rats’ alveolar structures might be altered by Yinlai Decoction,which could also lessen inflammatory exudation and inflammatory cell infiltration.(2)Colonic histopathology:The amount of goblet cells in rat colon tissue marginally increased after pneumonia modeling.The tissue structure of the colon in rats was unclear,the number of goblet cells was reduced,and the recess space was widened after modeling of gastrointestinal heat retention combined with pneumonia.3.Serum inflammatory factor levelAfter pneumonia modeling,the levels of serum pro-inflammatory factors IL-6,TNF-α,and IL-1β in rats increased(P<0.01).After modeling of gastrointestinal heat retention combined with pneumonia,the levels of IL-6 and TNF-α were further increased(P<0.01),and the level of IL-1β was further increased(P<0.05).Yinlai Decoction could significantly reduce the levels of serum pro-inflammatory factors IL-6,TNF-α,and IL-1βin rats(P<0.01).4.Th17/Treg cell balance1)Roryt and Foxp3 protein levels in mesenteric lymph nodesAfter pneumonia modeling,the protein expression level of Roryt in rat mesenteric lymph node was increased(P<0.01),and the protein expression level of Foxp3 was decreased(P<0.01).After modeling of gastrointestinal heat retention combined with pneumonia,the protein expression level of Roryt in rat mesenteric lymph nodes was further increased(P<0.01),and the protein expression level of Foxp3 was further decreased(P<0.05).Yinlai Decoction could significantly reduce the expression level of Roryt protein in mesenteric lymph nodes of rats(P<0.01),and increase the expression level of Foxp3 protein(P<0.01).2)Levels of IL-17a and IL-10 cytokinesAfter pneumonia modeling,the serum IL-17A level of rats increased(P<0.01),and IL-10 level decreased(P<0.01).After modeling of gastrointestinal heat retention combined with pneumonia,the serum IL-17A level of rats further increased(P<0.01),and the IL-10 level further decreased(P<0.01).Yinlai Decoction could reduce the level of serum IL-17A in rats(P<0.01)and increase the level of IL-10(P<0.01).5.Tphl mRNA transcription level of colon tissueAfter pneumonia modeling,the expression levels of colonic Tphl mRNA and protein were decreased in rats(P<0.01).After modeling of gastrointestinal heat retention combined with pneumonia,Tphl mRNA and protein expression levels of colonic TPhl in rats were further decreased(P<0.05,P<0.01).Yinlai Decoction could significantly increase the expression levels of Tphl mRNA and protein in rat colon(P<0.01).6.Expression level of Tph1 protein in colon tissueCompared with the group N,the levels of colonic Tphl protein in the groups P and G were significantly reduced(P<0.01).Compared with the group P,the level of colonic Tphl protein in the group GP was further reduced(P<0.01).Compared with the GP group,the Tphl protein level in the colon of the T group was significantly increased(P<0.01).7.Serum 5-HT levelsAfter pneumonia modeling,the serum 5-HT level of rats decreased(P<0.05).After modeling of gastrointestinal heat retention combined with pneumonia,the serum 5-HT level of rats further decreased.Yinlai Decoction could significantly increase the level of serum 5-HT in rats(P<0.01).Conclusion:The mechanism of aggravation of pneumonia by gastrointestinal heat retention is related to the decrease of serum 5-HT level and imbalance of Th17/Treg cells.Yinlai Decoction can play a role in the treatment of heat accumulation in gastrointestinal tract combined with pneumonia by regulating 5-HT level and Th17/Treg cell balance.Experiment 2:Exploration of the Mechanism of Yinlai Decoction in the Treatment of the Gastrointestinal Heat Combined with Pneumonia Based on the Effect of 5-HT on the Balance of Th17/Treg CellsPurpose:To explore the effect of 5-HT on the balance of Th17/Treg cells in the gastrointestinal heat combined with pneumonia rats and the intervention effect of Yinlai Decoction.Method:Forty SPF rats(4 weeks old)were randomly divided into normal group(N group),the gastrointestinal heatcombined with pneumonia group(GP group),Yinlai Decoction treatment group(T group),Tphl inhibitor group(Ⅰ group),and Yinlai Decoction combined with Tphl inhibitor group(IT group).The pathological changes of lung and colon tissues were detected by HE staining.Serum IL-6,TNF-α,IL-1β,IFN-γ,IL-17A,IL-10 and other inflammatory factors were detected by ELISA,and the levels of 5-HT and 5-HIAA in colon and serum were detected by ELISA.The expression level of colonic Tphl protein was detected by WB.The levels of Ror yt and Foxp3 proteins in the mesenteric lymph nodes and lung tissues as well as the 5-HT receptor proteins 5-HT7,5-HT2A,and 5-HT1B in the mesenteric lymph nodes were detected by WB.Result:1.General(1)Sign of rats:After the establishment of the model of gastrointestinal heat retention combined with pneumonia and the administration of the inhibitor,the rats displayed depression,decreased activity,dull fur,and soft,yellow,sticky,and odorous feces.Rats’mental health can be enhanced,and feces tend to be normal,via Yinlai decoction.(2)Body weight:Rats gained weight slowly after gastrointestinal heat retention combined with pneumonia modeling.2.Lung and colon histopathology(1)Pulmonary histopathology:After the inhibitor was administered and the model of gastrointestinal heat retention combined with pneumonia was set up,the rats’ alveolar structure became disordered,along with edema and a significant infiltration of inflammatory cells.Rats’ alveolar structure can be altered by yinlai decoction,which can also lessen inflammatory cell infiltration and lessen lung tissue inflammation in rats.(2)Colonic histopathology:The colon tissue structure of rats was unclear after the model of gastrointestinal heat retention combined with pneumonia was made up,and the recess space was wider than that of the normal group.The colon tissue structure of the rats was unclear,the mucosa decreased,and the recess area clearly widened after the inhibitor was administered.Rat colon tissue structure can be altered by yinlai decoction,and colon tissue inflammation can be lessened.3.Serum inflammatory factor levelThe levels of proinflammatory factors IL-6,TNF-,IL-1β,IFN-γ,and IL-17A in the serum of rats increased significantly(P<0.01)after the model of gastrointestinal heat retention combined with pneumonia was established,whereas the level of anti-inflammatory factor IL-10 decreased significantly(P<0.01).Yinlai decoction can significantly reduce the levels of IL-6,TNF-α,IL-1β,IFN-γ and IL-17A in rats(P<0.01)and significantly increase the level of IL-10(P<0.01).Yinlai decoction also significantly decreased the levels of IL-6,TNF-α,IL-1β,IFN-γ and IL-17A in the inhibitor group(P<0.01),and increased the level of IL-10(P<0.01).4.Expression level of colonic Tphl proteinThe expression level of Tphl protein in colon of rats decreased after the model of gastrointestinal heat retention combined with pneumonia was established(P<0.01).Yinlai decoction can significantly increase the expression level of Tphl protein in rat colon(P<0.01).Yinlai decoction also significantly increased the expression level of Tphl protein in colon of rats in inhibitor group(P<0.01).5.Serum and colon tissue 5-HT levelsThe level of 5-HT in serum and colon of rats decreased(P<0.01).Yinlai decoction can significantly increase the level of 5-HT in serum and colon of rats with gastrointestinal heat retention combined with pneumonia(P<0.01).Yinlai decoction also increased the level of 5-HT in serum and colon of rats in the inhibitor group(P>0.05,P<0.01).6.Serum and colon tissue 5-HIAA levelsThe level of 5-HIAA,a metabolite of 5-HT in serum and colon of rats,decreased after the model of gastrointestinal heat retention combined with pneumonia was established(P<0.01).Yinlai decoction can significantly increase the level of 5-HIAA in serum and colon of rats(P<0.01).Yinlai decoction also increased the level of 5-HIAA in serum and colon of rats in the inhibitor group(P<0.01).7.Expression levels of Foxp3 and Roryt proteins in mesenteric lymph nodes and lung tissue The expression level of Roryt protein in mesenteric lymph nodes and lung tissue of rats increased(P<0.01)and the expression level of Foxp3 protein decreased(P<0.01)after the establishment of the model of gastrointestinal heat retention combined with pneumonia.Yinlai decoction can significantly reduce the expression level of Roryt protein in mesenteric lymph nodes and lung tissues of rats(P<0.01)and significantly increase the expression level of Foxp3 protein(P<0.01).Yinlai decoction also decreased the expression level of Roryt protein in mesenteric lymph nodes and lung tissues of rats in inhibitor group(P<0.01),and increased the expression level of Foxp3 protein in mesenteric lymph nodes and lung tissues of rats in inhibitor group(P<0.01).8.Mesenteric lymph node 5-HTRS expression levelsThe expression levels of 5-HT receptor proteins 5HT7,5HT2A and 5HT1B in mesenteric lymph nodes of rats decreased after the establishment of the model of gastrointestinal heat retention combined with pneumonia(P<0.01).Yinlai decoction can significantly increase the expression levels of 5-HT receptor proteins 5HT7,5HT2A and 5HT1B in mesenteric lymph nodes of rats(P<0.01).Yinlai decoction also increased the expression levels of 5-HT receptor proteins 5HT7,5HT2A and 5HT1B in mesenteric lymph nodes of rats in inhibitor group(P<0.01).Conclusion:The mechanism of Yinlai decoction regulating Th17/Treg cell balance in rats with gastrointestinal heat retention combined with pneumonia is related to the intervention of 5-HT production and metabolism. |
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