Study On The Intervention And Mechanism Of Modified Tianwang Buxin Dan On Metabolic-inflammation In Sleep Deprivation Model Rats

Object:Based on the theory of Chinese medicine,the main pathogenesis of insomnia,namely"Yin deficiency and internal heat,restlessness of the mind",was used as the starting point to systematically explain the connection between Yin deficiency in the five internal organs and the symptoms related to sleeplessness,the internal mechanism of restlessness of the mind due to internal heat,the corresponding treatment rules and methods and its representative formula.On the other hand,based on the previous work of our research team,we conducted an experimental study to investigate the effect of the modified formula Tianwang Buxin Dan on the improvement of metabolic inflammation in a sleep deprivation（SD）model rat,with a view to providing theoretical support and experimental ideas for the effective intervention of modified Tianwang Buxin Dan in metabolic-related diseases induced by insomnia.Method:1.Theoretical part:Through literature research and a review of ancient texts,we start from the concept of insomnia in Chinese medicine,based on the key pathogenesis of"yin deficiency and internal heat",and summarize in detail the etiology,mechanism and clinical manifestations of yin deficiency in various internal organs;summarize the internal logic of yin deficiency and internal heat and the pathological changes caused by it;elaborate the corresponding treatments for yin deficiency and internal heat in insomnia and their representative methods.It also summarises the internal logic of yin deficiency and internal heat and the pathological changes in the body caused by it.2.Experimental part:Forty SPF Sprague Dawley breed male rats with a mean body mass of 220±10g were used for the experimental animals.After a1-week acclimatization period,the animals were divided into four groups by random number method:control,model,Tianwang Buxin Dan modified（TWBXD）group and melatonin.Except for the control group,the SD rat model was constructed using a homemade sleep deprivation water tank using the multi-platform water environment modeling method.Sleep deprivation was administered by gavage once a day for 4 weeks starting from the third week,and the control group and the model group were given equal volumes of pure water.The expression levels of ATP,Ca²⁺-Mg²⁺-ATPase and Na⁺-K⁺-ATPase in the hippocampus and hypothalamus of rats were measured by ELISA kits.RT-PCR tested expression of MCP-1,TNF-α,IL-1β,IL-6 and IL-10 in hippocampal and hypothalamic tissues;immunohistochemical labeling of i NOS and Arg-1 to compare their positive expression in hippocampal and hypothalamic regions;Western blot to detect changes in protein levels of AMPK,p-AMPK and SIRT1 in hippocampal and hypothalamic tissues.Result:1.Glucose and lipid metabolism:compared with the control group,TC,TG,LDL,GLU and GSP were increased to different degrees（p<0.01）and HDL was decreased（p<0.01）in the model group.After administration,glucose and lipid indexes fell back（p<0.01）and HDL increased（p<0.01）in the TWBXD and Melatonin groups.2.ATP metabolism:ultramicroscopic observation showed that the mitochondria in the brain cells of rats in the control group were regular spindle-shaped,with an intact outer membrane,clearly arranged ridges and uniform density of the matrix,while in the cells of rats in the model group,huge mitochondria were found,the length of which was several times that of normal mitochondria,with an irregular appearance,increased number of ridges and very confusing arrangement,suggesting the occurrence of metabolic abnormalities,and the intracellular mitochondrial morphology was restored after drug administration.The expression levels of ATP,Ca²⁺-Mg²⁺-ATPase,Na⁺-K⁺-ATPase were reduced in the model group,when compared with the control group（p<0.01）.The imbalance of ATP metabolism was significantly relieved after the drug administration.Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase expression levels rebounded in the TWBXD and Melatonin groups（p<0.01,0.05）.3.Inflammatory expression:the m RNA levels of pro-inflammatory factors MCP-1,TNF-α,IL-1βand IL-6 in the hippocampal and hypothalamic regions of the rat brain in the model group were found to occur significantly higher and the expression of anti-inflammatory factor IL-10 was decreased by RT-PCR assay compared to the control group（p<0.01）,while the pro-inflammatory factors in the administration group showed significantly lower expression levels and the expression of anti-inflammatory factors The expression levels of pro-inflammatory factors were significantly decreased and the expression of anti-inflammatory factors were elevated in the administered group（p<0.01,0.05）.Immunohistochemical method of labeling inflammatory markers in brain cells revealed that i NOS positive expression was increased and Arg-1 positive expression was decreased in hippocampal and hypothalamic regions in the model group compared to the control group（p<0.01）,and after drug administration,i NOS positive expression was decreased and Arg-1 positive expression was increased in the TWBXD and melatonin groups（p<0.01）.4.pathway proteins:The expression levels of AMPK/SIRT1 signaling pathway proteins in hippocampus and hypothalamus regions were detected by Western blot method.The expression levels of p-AMPK and SIRT1 protein in the model group showed a significant decrease compared with the control group（p<0.01）.The expression levels of p-AMPK and SIRT1 in the TWBXD and Melatonin groups showed a significant increase compared with the model group（p<0.01）;there was no significant statistical difference between the groups in the expression levels of AMPK protein.Conclusion:1.Yin deficiency and internal heat is an important pathogenesis of insomnia,and pathological changes can be seen in heat,deficiency and agitation,with co-morbidities and transmutations occurring in multiple internal organs,eventually leading to restlessness of the mind and affecting sleep.At the same time,the mind and brain are also closely related to sleeplessness.2.The method of nourishing Yin and clearing heat,nourishing the heart and calming the mind is an effective treatment for this pathology,and one of the representative formulas is modified Tianwang Buxin Dan,which has a rigorous formula,a comprehensive effect and a high clinical use rate.3.Modified Tianwang Buxin Dan can alleviate the disorder of glucolipid metabolism and ATP metabolism and effectively improve the expression of brain inflammation in SD rats.