Study On The Parallel Regulation Mechanism Of Suanzaoren Decoction On Central SCN Circadian Rhythm And Energy Metabolism Based On PPARγ And PGC-1α

Objective:To investigate the relationship between the circadian rhythm of central SCN system and mitochondrial energy metabolism in different parts of brain,heart and liver in senile insomnia model,and to clarify the pathogenesis of"liver blood deficiency,yin deficiency and internal disturbance"To explore the parallel regulation mechanism of Suanzaoren Decoction on circadian rhythm and energy metabolism.Method:1.Methods:80 male C57BL/6 mice were randomly divided into blank control group,positive control group(0.26mg·kg^-1·d^-1),high-dose and low-dose Suanzaoren Decoction groups(18.72g·kg^-1·d^-1,9.36g·kg^-1·d^-1).The dosage of Suanzaoren Decoction was 0.2m L·10g^-1,once aday for 7days.The blank control group was given the samevolume of normal saline,30 minutes after the last administration,To observe the sedative and hypnotic effects of Suanzaoren Decoction,the subthreshold and suprathreshold sleep experiments of pentobarbital sodium were carried out.2.Methods:50 male Wistar rats were randomly divided into blank control group,model control group,positive control group(0.18mg·kg^-1·d^-1),high and low dose Suanzaoren Decoction groups(12.96g·kg^-1·d^-1,6.48g·kg^-1·d^-1).Except for blank control group,D-galactose was injected subcutaneously into other groups.The dosage of each group was 0.125g·kg^-1·d^-1,and the dosage was 0.5ml·100g^-1,after the last administration,the rats were deprived 18 hours a day for 21 consecutive days to establish the elderly chronic REM sleep deprivation model.After the end of the model,the rats in each group were given 2m L·100g^-1 by gavage,once a day for 7consecutive days,the model control group was given the same volume of normal saline.The effects of Suanzaoren Decoction on the autonomic activitiesof the aged rats with chronic REM sleep deprivation were detected by autonomic activity instrument.Objective To observe the effect of Suanzaoren Decoction on the circadian rhythm of elderly rats with chronic REM sleep deprivation,the clock gene Clock,Bmal1 and clock control gene Rev-erbα、Rorαof hypothalamus were detected by Real-Time PCR and immunohistochemistry.3.Real time PCR and Western blot were used to detect the m RNA and protein expression levels of PPARγand its coactivator PGC-1αin hypothalamus of rats in each group,and to observe the effect of Suanzaoren Decoction on PPARγand its coactivator PGC-1αm RNA in hypothalamus of rats with chronic REM sleep deprivation.4.The mitochondrial morphology of central hypothalamus was observed by transmission electron microscope,the content of ATP in hypothalamus was detected by colorimetry,the activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in central hypothalamus were detected by spectrophotometry,and the activities of Cyt c,Bcl-2,Bax and Caspase 3 in Central hypothalamus were detected by Western blot,objective To observe the effect of Suanzaoren Decoction on mitochondrial energy metabolism in central hypothalamus of elderly rats with chronic REM sleep deprivation.5.Transmission electron microscope was used to observe the morphology of heart mitochondria,colorimetric method was used to detect the changes of myocardial ATP content,and spectrophotometr was used to detect the content of malondialdehyde（MDA）and theactivity of superoxide dismutase（SOD）in each group Real Time PCR was used to detect the m RNA expression of SIRT3 and SOD2.Western blot was used to detect the m RNA expression of SIRT3 and SOD2 The protein expression levels of SIRT3 and SOD2 were detected by blot,and the localization expression of SIRT3 was detected by immunofluorescence staining,objective To observe the effect of Suanzaoren Decoction on energy metabolism of myocardial mitochondria in aged rats with chronic REM sleep deprivation.6.The morphology of liver mitochondria was observed by transmission electron microscope,the content of ATP in liver was detected by colorimetry,and the activity of respiratory chain complex I-IV in liver mitochondria was detected by colorimetry The expression levels of CS,IDH and ATP5F1 protein in liver of rats were detected by blot method,objective To observe the effect of Suanzaoren Decoction on energy metabolism of liver mitochondria in elderly rats with chronic REM sleep deprivation.Result:1.Compared with the blank control group,the sleep rate,sleep latency and sleep time of high-dose Suanzaoren Decoction group and positive control group were significantly increased（P<0.01）,but there was no significant difference in low-dose Suanzaoren Decoction group（P>0.05）.2.Compared with the blank control group,the activity distance and time in light environment,dark environment,total activity distance and time in model control group were significantly increased（P<0.01）,the m RNA and protein expression levels of Clock、Bmal1、Rev-erbα、Rorαwere significantly decreased（P<0.01）;compared with the model control group,the distance and time of light environment activity,dark environment activity,total activity distance and time in the positive control group and high-dose Suanzaoren Decoction group were significantly reduced（P<0.05,P<0.01）,the m RNA and protein expression levels of Clock、Bmal1、Rev-erbα、Rorαin the positive control group and high-dose Suanzaoren Decoction group were significantly increased（P<0.01）,the m RNA expression levelsof Bmal1 and Rorαin the low-dose Suanzaoren Decoction group were significantly increased（P<0.05,P<0.01）,the m RNA expression leves of Clock、Rev-erbα,activity distance and time in light environment,activity distance and time in dark environment,total activity distance and time and the protein expression levels of Clock、Bmal、Rev-erbα、Rorαwere not significantly different in the low-dose Suanzaoren Decoction group（P>0.05）.3.Compared with the blank control group,the m RNA and protein expression levels of PPARγand PGC-1αin hypothalamus of the model control group were significantly decreased（P<0.01）;compared with the model control group,the m RNA and protein expression levels of PPARγand PGC-1αin hypothalamus of the positive control group and high-dose Suanzaoren Decoction group were significantly increased（P<0.05,P<0.01）,the m RNA expression level of PGC-1αin low-dose Suanzaoren Decoction group were significantly increased（P<0.01）,but there was no significant difference in the expression levels of PGC-1αprotein、PPARγm RNA and protein（P>0.05）.4.Under the transmission electron microscope,there was no obvious pathological change in the hypothalamic mitochondria of the blank control group,with moderate size,oval or spindle shape,and regular cristae.In the model control group,mitochondrial morphology was abnormal,mitochondria swelled obviously with vacuolation,appeared"myelin sheath like",cristae ruptured and the number of cristae decreased;compared with the model control group,mitochondrial damage in the positive control group and the high-dose Suanzaoren Decoction group was reduced,vacuolation phenomenon was reduced,some cristae ruptured,and no obvious swelling was found,while the low-dose Suanzaoren Decoction group had no obvious improvement.Compared with the blank control group,the hypothalamic ATP content,Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase activity and Bcl-2 protein in the model control group were significantly decreased（P<0.01）,while Cyt c,Bax and Caspase 3 were significantly decreased（P<0.01）Compared with the model control group,ATP content,Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase activity and Bcl-2 protein expression in hypothalamus of positive control group and high-dose Suanzaoren Decoction group were significantly increased（P<0.05,P<0.01）,while Cyt c,Bax and Caspase 3 activity were significantly increased（P<0.05,P<0.01）The expression level of Bax protein was significantly decreased（P<0.05）,the activity of Na⁺-K⁺-ATPase and the expression level of Bcl-2 protein were significantly increased（P<0.05,P<0.01）,the expression level of Bax protein was significantly decreased（P<0.05）,the activity of Ca²⁺-Mg²⁺-ATPase and the expression level of Cyt C and Caspase 3 protein were not significantly different（P>0.05）.5.Under the transmission electron microscope,the structure of myocardial cells in the blank control group was clear,the thick andthin myofilaments were arranged in order,the nuclear morphology was regular,the relatively uniform mitochondria were densely distributed between myofilaments,the shape of mitochondria were mostlyround or oval,and the mitochondrial membrane was intact,In the model control group,the arrangement of myofilament was disordered,accompanied with myofilament rupture and dissolution,the swelling of mitochondria was obvious,and the arrangement of mitochondria was disordered,Compared with the model control group,the positive control group and the high-dose Suanzaoren Decoction group had more orderly arrangement of myofilaments,less mitochondrial damage,only part of the cristae were broken,and the swelling degree was relatively light,but the low-dose Suanzaoren Decoction group had no obvious improvement.Compared with the blank control group,the myocardial ATP content,SOD activity,SIRT3,SOD2 m RNA and protein expression levels in the model control group were significantly decreased（P<0.01）,and the MDA content was significantly increased（P<0.01）;compared with the model control group,the myocardial ATP content,SOD activity,SIRT3,SOD2 m RNA and protein expression levels in the high-dose Suanzaoren Decoction group were significantly decreased（P<0.01）The expression levels of m RNA and protein were significantly increased（P<0.05,P<0.01）,and the content of MDA was significantly decreased in the positive control group The expression levels of m RNA and protein were significantly increased（P<0.05,P<0.01）,the content of MDA was significantly decreased（P<0.01）,the content of ATP and the average fluorescence intensity of SIRT3 had no significant difference（P>0.05）,the activity of SOD and the expression level of SOD2 protein were significantly increased（P<0.05,P<0.01）,the content of ATP and MDA,the content of SIRT3 and SOD2 were significantly increased（P<0.05,P<0.01）in the low-dose Suanzaoren Decoction group,there was no significant difference in m RNA expression and the average fluorescence intensity of SIRT3（P>0.05）.6.Under the transmission electron microscope,the mitochondriain the liver of the blank control group were regular in shape,moderate in size,round or oval in shape,and arranged in order.In the model control group,the mitochondria were abnormal,swollen,deformed,cristae broken,and the number decreased to a certain extent In the low-dose group,mitochondrial damage,swelling,deformation and cristae breakage were reduced.Compared with the blank control group,the contents of ATP,the activities of mitochondrial respiratory chaincomplexes I,II,III and IV,and the expression levels of IDH,CS and ATP5F1 protein in the model control group were significantly decreased（P<0.01）;Compared with the model control group,the liver ATP content,mitochondrial respiratory chain complex I,II,III,IV activity,IDH,CS,ATP5F1 protein expression in the high-dose Suanzaoren Decoction group were significantly increased（P<0.01）,and the liver ATP content,mitochondrial respiratory chain complex I,III activity,CS,ATP5F1 protein expression in the positive control group were significantly increased（P<0.05,P<0.01）,There was no significant difference in the expression of IDH protein（P>0.05）.The activities ofmitochondrial respiratory chain complexes I and III and the expression of ATP5F1 protein in the low-dose Suanzaoren Decoction group weresignificantly increased（P<0.01,P<0.05）,while the activities of mitochondrial respiratory chain complexes II and IV and the expressionof IDH and CS protein in the low-dose Suanzaoren Decoction group were not significantly different（P>0.05）.Conclusion:1.Suanzaoren Decoction has good sedative and hypnotic effects.2.Suanzaoren Decoction can improve the circadian rhythm of elderly rats with chronic REM sleep deprivation.3.Suanzaoren Decoction can increase the expression of PPARγand PGC-1αin hypothalamus.4.Suanzaoren Decoction can improve the abnormal energy metabolism of hypothalamus,inhibit the apoptosis of nerve cells and protect the nerve cells of hypothalamus induced by chronic REM sleep deprivation.5.Suanzaoren Decoction can improve mitochondrial damage and abnormal energy metabolism induced by chronic REM sleep deprivation in aged rats,and protect myocardial cells.6.Suanzaoren Decoction can improve the abnormal liver energy metabolism induced by chronic REM sleep deprivation,restore the normal function of mitochondria and protect the liver.7.Suanzaoren Decoction may improve the circadian rhythm induced by chronic rapid eye movement sleep deprivation in aged rats based on the increased expression of PPARγand PGC-1αin hypothalamic nucleus,and then regulate the energy metabolism of brain,heart and liver,so as to improve sleep.