Study On Material Basis And Mechanism Of Antipyretic Effect Of Gypsum And Its Compatibility

Objective: The antipyretic mechanism and substance basis of Gypsum and its compatibility were analyzed and evaluated from in vivo,in vitro and protein levels through the research methods of replication of fever model animals,in vitro culture of mouse macrophages,changes in intestinal flora and serum metabolomics,etc.The specific ways and action nodes of gypsum and its compatibility antipyretic mechanism were explored in depth,and the traditional efficacy of Gypsum in clearing heat,purging fire,relieving restlessness and quenching thirst was preliminarily explained,providing basis for the rational application of Gypsum.Methods: 1.Analysis of metal elements in different processed gypsum products.Through ICP-MS detection technology,qualitative and quantitative analysis of metal elements in gypsum and calcined gypsum produced in Yingcheng,Hubei Province was carried out to analyze the difference in types and contents of metal elements contained in gypsum and calcined gypsum.2.Pharmacodynamic evaluation of antipyretic effect of gypsum and its compatibility: 2.1 Two fever rat models were established by intraperitoneal injection of lipopolysaccharide and subcutaneous injection of yeast suspension back respectively,and the effects of gypsum and calcined gypsum on body temperature of fever model rats at various time points within 8 hours were observed.2.2 The fever rat model was established by subcutaneous injection of yeast suspension on the back,and the proportion change of animal body composition was detected by the composition analyzer of experimental animal body.Blood gas analyzer was used to detect the changes of blood gas in animal blood,and the effects of gypsum and its compatibility on the changes of body fluid composition and blood gas composition in febrile rats were investigated from the perspective of physiological function;2.3 The levels of interleukin-6（IL-6）,interleukin-1（IL-1β）and tumor necrosis factor（TNF-α）in serum of model animals were detected by enzyme-linked immunosorbent assay.The contents of prostaglandin E₂（PGE₂）,arginine vasopressin（AVP）,adrenocorticotropic hormone releasing hormone（CRH）,cyclic adenosine monophosphate（cAMP）and calcium ion(Ca²⁺)in serum and cerebrospinal fluid are comprehensively compared and analyzed,and the specific intervention ways of gypsum and its compatibility on fever model rats are comprehensively analyzed;2.4 The anti-inflammatory and analgesic effects of gypsum and its compatibility were observed through mice hot plate test,xylene induced ear swelling test and acetic acid writhing test,decreased ear swelling.3.Study on the mechanism of antipyretic effect of gypsum and its compatibility: 3.1 21 samples were selected and divided into 7 groups for metabolic study.In view of the antipyretic mechanism of gypsum and its compatibility,find its fever-related characteristic biomarkers and screen its possible antipyretic pathways.3.2 Western blot analysis was used to analyze the expression level of gypsum and its compatibility on NF-κB pathway related proteins in hypothalamus and liver tissues of fever model rats.To further determine the specific ways and targets of gypsum and its compatibility for antipyresis;3.3 By culturing mouse macrophages in vitro and using lipopolysaccharide to induce cell inflammatory model,the effects of gypsum and its compatibility on inflammatory cell viability,nitric oxide（NO）and nitric oxide synthase（iNOS）levels were investigated;3.4 Western blot analysis was used to analyze the protein expression level of NF-κB,IκBα and IκKβ in lipopolysaccharide-induced cellular inflammatory model induced by gypsum and its compatibility,and the specific mechanism and approach of antipyretic effect of gypsum and its compatibility were explained from the cellular level;3.5 Using 16 Sr RNA flora biodiversity analysis technology,gypsum and its compatibility were used to analyze intestinal flora biodiversity of fever model rats,and the intervention effect of gypsum and its compatibility on intestinal flora of fever model rats was investigated;Results: 1.ICP-MS test results show that the trace elements of gypsum and calcined gypsum are quite different（the specific changes are obvious）;2.1 Compared with the model group,gypsum can reduce the body temperature of the two fever model animals,which is to restore the basic body temperature to achieve the antipyretic effect,while calcined gypsum and calcium sulfate cannot reduce the body temperature of the fever model animals and have no antipyretic effect;2.2 Compared with the model group,gypsum and its compatibility can increase the ratio of total water content（P<0.05）and the ratio of extracellular fluid（P<0.05）in the model animals,but there is no obvious change in the percentage of fat content.Compared with the model group,gypsum and its compatibility can reduce pCO₂ in blood gas（P<0.05）,increase oxygen saturation（sO₂%）and carbon oxygen hemoglobin fraction（FO₂Hb%）in blood oxygen（P<0.05）,and decrease Ca²⁺ in electrolyte（P<0.05）;2.3 Compared with model group,gypsum and its compatibility can significantly reduce the expression of IL-6,IL-1β and TNF-α in serum（P<0.05）.Compared with the model group,the expression of PGE₂,CRH and cAMP in cerebrospinal fluid decreased significantly（P<0.05）,the expression of AVP and Ca²⁺ increased significantly（P<0.05）,the expression of PGE₂,CRH,cAMP and Ca²⁺ in serum decreased significantly（P<0.05）,and the expression of AVP increased significantly（P<0.05）;2.4 Compared with the blank group,gypsum and its compatibility can prolong the licking time of mice（P<0.05）and reduce the twisting times of mice caused by acetic acid（P<0.05）;3.1 A total of 482 metabolites were detected based on metabolic analysis of extensive targeted metabolome technology.Serum metabolic analysis showed 17 biomarkers related to fever,and 21 biomarkers related to gypsum and its antipyretic effect and recalled.Among the recalled metabolites,7 different metabolites,L-O-phosphoserine,L-arginine,D-glucuronolactone,glycocholic acid,prostaglandin E2,L-glutamic acid,and 18-hydroxycorticosterone,are highly correlated with fever.The main metabolic pathways and pathways involved by these different metabolites are all related to the regulation of TRP channel by inflammatory media,NF-κB signal pathway,HIF-1 signal pathway,and mTOR signal pathway.3.2.Compared with the model group,gypsum and its compatibility can significantly reduce the expression of NF-B protein in hypothalamus tissue（P<0.05）,and the protein expression is significantly increased（P<0.05）.Compared with the model group,gypsum and its compatibility can significantly down-regulate the expression of NF-κB,IκBα and IκKβ proteins in liver tissue（P<0.05）;3.3 Gypsum and its compatibility have NO effect on the activity of mouse macrophages.Compared with model group,gypsum and its compatibility can significantly reduce the NO value in cell supernatant（P<0.05）;Compared with the model group,gypsum and its compatibility can significantly reduce the increase of iNOS expression induced by lipopolysaccharide（P<0.05）;3.4 Compared with the model group,gypsum and its compatibility can significantly reduce the expression of NF-κB and IκKβ protein in lower inflammatory cells（P<0.05）,and the expression of IκBα protein is significantly increased（P<0.05）;3.5 The composition,structure and abundance of flora in fever model rats are significantly different from those in normal rats.After microzyme induced fever in rats,intestinal bacteria swarmed with structural changes,including Alpha-proteobacteria,Selenomonadales,Rhodospirillales,Akkermansiaceae,Burkholderiaceae,Acidaminococcaceae,Lachnospirace-ae_NK4 A136_group,Prevotella₉,Phascolarctobacterium have obvious changes.After administration of the tested drugs,each species has different degrees of callback,especially gypsum group,drug pair group and Baihu decoction group,almost all of the above species have callback effect.Conclusion: Gypsum and its compatibility can significantly reduce fever model rats body temperature,may be by intervening in the nf-kappa B signaling pathways regulating,maintain the balance of body fluid composition,adjusting intestinal flora biodiversity,improve the metabolism of amino acids,lipid metabolism disorders.