Role And Mechanism Of IL-1β In The Model Of Deficiency Cold Syndrome And Deficiency Heat Syndrome In Rats

Objective:The animal models of deficiency cold syndrome and deficiency heat syndrome were replicated to investigate the role and mechanism of interleukin-1β（IL-1β）in the formation of animal models of deficiency cold syndrome and deficiency heat syndrome.Methods:SPF male Wistar rats were used to duplicate the animal models of deficiency cold syndrome and deficiency heat syndrome according to the previous animal model preparation method of the research group.The animal model of deficiency cold syndrome was intragastrically administered with gypsum,gentian,anemarrhena asphodeloides,and phellodendron amurense aqueous decoction for 14 d;the animal model of deficiency heat syndrome was intragastrically administered with aconite root,cinnamon,and dried ginger aqueous decoction for 14 d.Partial least squares regression analysis（PLS-DA）was used to analyze and identify the model and select the best rat model of deficiency cold syndrome and deficiency heat syndrome.The animal model of deficiency cold syndrome with successful replication was randomly divided into deficiency cold group（DC）,deficiency cold+IL-1βgroup（DC+IL-1β）,and deficiency cold+Anakinra（DC+Anakinra）group.The animal model of deficiency heat syndrome with successful replication was randomly divided into deficiency heat group（DH）,deficiency heat+IL-1βgroup（DH+IL-1β）,and deficiency heat+Anakinra group（DH+Anakinra）.Among them,the DC+IL-1βgroup and DH+IL-1βgroup were intraperitoneally injected with IL-1β（0.0000125 g/kg）,the DC+Anakinra group and DH+Anakinra group were intraperitoneally injected with IL-1receptor antagonist Anakinra（0.1 g/kg）,and the control group and DC group were intraperitoneally injected with an equal volume of normal saline.The general condition,body temperature,cold and heat tendency of the rats were observed;Water intake,food intake,urine output and feces of rats were collected by metabolic cage for the detection of energy intake,digestible energy and metabolizable energy.The spontaneous activity ability of rats in each group was measured by open field test;the respiratory gas exchange rate（RER）of rats was measured using the TSE animal metabolic measurement and analysis system;and the triglyceride（TG）in rat serum was measured using a microplate reader,i.e.Pyruvate,lactate（LD）,glycogen,ATPase activity,lactate dehydrogenase（LDH）,and succinate dehydrogenase（SDH）in liver tissue;HE staining was used to observe the pathological changes in liver tissue of rats with deficiency heat syndrome;Elisa method was used to detect the serum free triiodothyronine（FT3）,free tetraiodothyronine（FT4）,thyrotropin-releasing hormone（TRH）,thyroid-stimulating hormone（TSH）,growth hormone（GH）,interleukin-2（IL-2）,interleukin-4（IL-4）,interferon-γ（IFN-γ）,interleukin-6（IL-6）,and tumor necrosis factor-α（TNF-α）contents;immunofluorescence technique was used to detect the expression of nuclear factorκB（NF-κB）and activator protein-1（AP-1）in liver tissue of rats with deficiency heat syndrome;q RT-PCR was used to detect the expression of NF-κB and AP-1 m RNA in liver tissue of rats;Western blot was used to detect NF-κB and AP-in liver tissue and adipose tissue of rats Protein expression of 1.Results:1.Compared with the control group,the anal temperature（P<0.001）,body surface temperature（P<0.01）,toe temperature（P<0.05）,the number of tending to cold box in cold and heat tending instrument（P<0.05）,total distance of autonomous activity（P<0.05）,and energy intake（P<0.05）were significantly increased in the DH group;compared with the DH group,the toe temperature was significantly increased（P<0.001）,and the total distance of activity（P<0.01）,RER（P<0.05）,energy intake（P<0.001）,metabolizable energy（P<0.05）and the number of tending to cold box in cold and heat tending instrument（P<0.001）were significantly reduced in the DH+IL-1βgroup.Biochemical results showed that TG in serum was significantly increased（P<0.01）and LD and liver glycogen content in liver tissue were significantly decreased（P<0.05）in DH group compared with control group;LD and glycogen content in liver tissue were significantly increased（P<0.001）in DH+IL-1βgroup and Na⁺-K⁺-ATPase activity,Ca²⁺-Mg²⁺-ATPase activity and LDH content were significantly decreased（P<0.05）in DH+Anakinra group.Compared with the DH+IL-1βgroup,LD（P<0.001）,liver glycogen（P<0.01）,LDH（P<0.01）,pyruvate（P<0.05）,and Ca²⁺-Mg²⁺-ATPase activities（P<0.05）were significantly lower in the DH+Anakinra group.The results of HE staining showed that a clear and complete structure of the hepatic lobule was observed in the liver tissue of rats in each group,the central vein was located in the central part of the hepatic lobule,the hepatocyte cords were arranged neatly around the central vein,and no obvious abnormalities such as hepatocyte degeneration,hepatocyte nuclear fragmentation,or deformation were observed.Liver sinusoids were clearly visible,and no cellular infiltration such as monocytes,lymphocytes,eosinophils and fibroblasts were observed in the sinusoids.Elisa results showed that compared with the control group,the serum GH content was significantly lower（P<0.05）,and the FT4（P<0.01）and TRH（P<0.05）contents were significantly higher;compared with the DH group,the GH（P<0.01）IL-6（P<0.05）and TNF-α（P<0.05）contents in serum were significantly higher,and the FT4（P<0.001）,FT3（P<0.05）,TSH（P<0.05）,and TRH（P<0.05）contents were significantly lower in the DH+IL-1βgroup.Immunofluorescence results showed that NF-κB（P<0.05）and AP-1（P<0.01）expression was significantly increased in the liver tissue of rats in the DH group compared with the control group;NF-κB（P<0.05）and AP-1（P<0.01）expression was significantly down-regulated in the DH+IL-1βgroup compared with the DH group.The results of q RT-PCR experiments showed that the expression of NF-κB（P<0.05）and AP-1（P<0.01）m RNA in the liver tissue of rats in the DH group was significantly increased compared with the control group;the expression of NF-κB and AP-1 m RNA in the DH+IL-1βgroup was significantly down-regulated compared with the DH group（P<0.01）,and the expression of NF-κB m RNA in the DH+Anakinra group was also significantly reduced（P<0.05）.The results of Western blot experiments showed that the protein expression of NF-κB and AP-1 in the adipose tissue of rats in the DH group was significantly increased compared with the control group（P<0.05）,while the protein expression of NF-κB and AP-1 in the liver tissue of rats in the DH group tended to increase,but it was not statistically significant（P>0.05）;the protein expression of AP-1 in the adipose tissue of rats in the DH+IL-1βgroup was significantly lower compared with the DH group（P<0.05）.2.Compared with control group,the anal temperature and body surface temperature of rats in the DC group were significantly decreased（P<0.05）,the number of rats tending to the hot box was significantly increased（P<0.01）,and the total distance of activity was significantly decreased（P<0.01）.IL-1βincreased the spontaneous activity（P<0.05）,energy intake（P<0.05）,digestible energy（P<0.01）and metabolizable energy（P<0.01）of rats with deficiency cold syndrome.Biochemical results showed that compared with the control group,the TG content in the serum of rats in the DC group was significantly reduced（P<0.05）,the LDH（P<0.05）,Ca²⁺-Mg²⁺-ATPase activity（P<0.05）,and pyruvate（P<0.01）contents in the liver tissue were significantly increased,the LD,SDH content,and Na⁺-K⁺-ATPase activity tended to increase,and the liver glucose originally decreased,but there was no statistically significant difference.Compared with the DC group,the TG content in the serum of rats in the DC+IL-1βgroup was significantly increased（P<0.05）,the LD（P<0.05）and pyruvate（P<0.01）contents in the liver tissue of rats were significantly reduced,and the liver glycogen content was significantly increased（P<0.05）.Elisa results showed that TSH（P<0.01）and TRH（P<0.001）contents in serum were significantly increased and IL-4（P<0.05）,IFN-γ（P<0.05）,IL-6（P<0.01）and TNF-α（P<0.01）were significantly decreased in the DC group compared with the control group;FT4（P<0.05）and FT3（P<0.01）contents were significantly increased and TRH（P<0.05）,IL-4（P<0.05）,TNF-α（P<0.05）,and IL-6（P<0.01）were significantly decreased in the DC+IL-1βgroup compared with the DC group.The results of q RT-PCR experiments showed that the expression of NF-κB and AP-1 m RNA in the liver tissue of rats in the DC group was significantly down-regulated compared with the control group（P<0.05）;the expression of NF-κB（P<0.01）and AP-1（P<0.05）m RNA in the DC+Anakinra group was also significantly down-regulated;the expression of NF-κB and AP-1 m RNA in the DC+IL-1βgroup was significantly up-regulated compared with the DC group（P<0.01）;and the expression of NF-κB and AP-1 m RNA in the DC+Anakinra group was significantly down-regulated compared with the DC+IL-1βgroup（P<0.01）.The results of Western blot experiments showed that the protein expression of NF-κB（P<0.01）and AP-1（P<0.05）in the liver tissue of rats in the DC group was significantly reduced compared with the control group,and the protein expression of AP-1 in the adipose tissue was also significantly reduced（P<0.01）;the NF-κB protein expression in the liver tissue of rats in the DC+IL-1βgroup was significantly increased compared with the DC group（P<0.05）.Conclusions:IL-1βis an important cytokine affecting the occurrence and development of deficiency cold syndrome and deficiency heat syndrome.Its mechanism in deficiency heat syndrome may be related to aerobic oxidation of sugars and regulation of thyroid function,and is closely related to NF-κB and AP-1 signaling pathways.Its mechanism in asthenia cold syndrome may be achieved by increasing body energy supply,promoting the secretion of thyroid hormones and activating NF-κB and AP-1signaling pathways.