Suanzaoren Tang Treats Insomnia by Affecting Brain， Heart， and Liver Functions： A Review / 中国实验方剂学杂志

Sleep has been widely concerned by the medical field all over the world. Sleep deprivation can cause damage to organs of the human body， which is related to the occurrence of a variety of diseases. Besides， the pathological change in different organs of the human body is also a key factor that causes or aggravates insomnia. When treating insomnia and its complications， traditional Chinese medicine （TCM） focuses on the homology of the brain and heart， and insomnia is mainly treated from the five internal organs， especially the heart and liver. Sleep duration and structure change with age. The prevalence of insomnia is higher in older individuals susceptible to complications than in the younger population. In TCM， insomnia of blood deficiency and Yin deficiency is common among the elderly. Suanzaoren Tang is a classic prescription for nourishing blood and calming the mind and it is critical in the treatment of "sleeplessness due to consumptive disease and dysphoria"， with the effects of nourishing liver blood to calm the mind and clearing internal heat to relieve dysphoria. It has good efficacy on the insomnia of the elderly caused by deficiency of Qi and blood and abnormal operation of nutrient Qi and defense Qi. Furthermore， it also shows a certain therapeutic effect on insomnia combined with cardiovascular and cerebrovascular diseases. The present study revealed the damage to the brain， heart， and liver caused by sleep deprivation and the effect of Suanzaoren Tang on the brain， heart， and liver， and clarified the facts that Suanzaoren Tang inhibited the damage to organs caused by sleep deprivation and regulated energy metabolism， thereby exploring the sedative and hypnotic mechanism of Suanzaoren Tang to provide new ideas for Suanzaoren Tang in the treatment of sleep disorders and other diseases.

Effect of Suanzaoren Tang on Energy Metabolism of Liver Mitochondria in Aged Rats with Chronic Rapid Eye Movement Sleep Deprivation and Its Mechanism / 中国实验方剂学杂志

Objective:To study the effect of Suanzaoren Tang on energy metabolism of liver mitochondria in aged rats with chronic rapid eye movement （REM） sleep deprivation. Method:Fifty male Wistar rats were randomly divided into the control group， model group， estazolam group （0.18 mg·kg^-1·d^-1）， and low- （6.48 g·kg^-1·d^-1） and high-dose （12.96 g·kg^-1·d^-1） Suanzaoren Tang groups. Rats in all groups except for the control group received subcutaneous injection of <italic>D</italic>-galactose and then were deprived of sleep using the multiple platform method after the last administration. Following successful modeling， the rats in each group were treated with intragastric administration of the corresponding drugs for seven consecutive days. The morphology of liver mitochondria was observed under the transmission electron microscope. The content of adenosine triphosphate （ATP） in rat liver was detected by colorimetry， and the activities of ubiquinone oxidoreductase （complex Ⅰ）， succinate-ubiquinone oxidoreductase （complex Ⅱ）， ubiquinol-cytochrome c oxidoreductase （complex Ⅲ）， and cytochrome c oxidase （complex Ⅳ） in mitochondrial respiratory chain of rat liver were measured by colorimetry. The protein expression levels of citrate synthase （CS）， isocitrate dehydrogenase （IDH）， and ATP synthase， H⁺ transporting， mitochondrial F0 complex， subunit b， isoform 1 （ATP5F1） in rat liver were assayed by Western blot. Result:The mitochondrial damage in rat liver of the model group was more serious than that in the control group， manifested as mitochondrial swelling and deformation as well as cristae rupture and reduction. The comparison with the model group revealed that both the positive control and Suanzaoren Tang at the high dose obviously alleviated the mitochondrial swelling and deformation and reduced cristae rupture， with better improvements observed in the high-dose Suanzaoren Tang group. Compared with the control group， the content of ATP， the activities of mitochondrial respiratory chain complexes Ⅰ， Ⅱ， Ⅲ， and Ⅳ， and the protein expression levels of IDH， CS， and ATP5F1 in rat liver of the model group were all significantly decreased （<italic>P<</italic>0.01）. Compared with the model group， the content of ATP， the activities of mitochondrial respiratory chain complexes Ⅰ， Ⅱ， Ⅲ， and Ⅳ， and the protein expression levels of IDH， CS， and ATP5F1 in rat liver of the high-dose Suanzaoren Tang group were all significantly increased （<italic>P<</italic>0.05，<italic>P<</italic>0.01）. In the positive control group， the content of ATP， the activities of mitochondrial respiratory chain complexes I and Ⅲ， and the protein expression levels of CS and ATP5F1 in rat liver were significantly increased （<italic>P<</italic>0.05，<italic> P<</italic>0.01）. The activities of mitochondrial respiratory chain complexes Ⅰ and Ⅲ and the ATP5F1 protein expression in the low-dose Suanzaoren Tang group were significantly elevated （<italic>P<</italic>0.05， <italic>P<</italic>0.01）. Conclusion:Suanzaoren Tang alleviates the abnormal liver energy metabolism induced by chronic REM sleep deprivation in the elderly rats， which may be related to its enhancement of mitochondrial electron transport chain enzyme activities and the up-regulation of protein expression levels of CS， IDH and ATP5F1.

Effect of Suanzaoren Tang on Mitochondrial Damage and Apoptosis in Hypothalamus of Elderly Rats with Chronic Rapid Eye Movement Sleep Deprivation / 中国实验方剂学杂志

Objective:To investigate the effect of Suanzaoren Tang on mitochondria-mediated neuronal apoptosis. Method:Fifty male Wistar rats were randomly divided into the control group， model group， estazolam group （0.18 mg·kg^-1·d^-1）， and low- （6.48 g·kg^-1·d^-1） and high-dose （12.96 g·kg^-1·d^-1） Suanzaoren Tang groups. Rats in all groups except for the control group received subcutaneous injection of <italic>D</italic>-galactose and then were deprived of sleep using the multiple platform method after the last administration. Following successful modeling， the rats in each group were treated with intragastric administration of the corresponding drugs for seven consecutive days. The morphology of mitochondria in hypothalamus was observed under a transmission electron microscope. The activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in hypothalamus were detected by spectrophotometry. Western blotting was conducted to determine the protein expression levels of cytochrome C （Cyt C）， B cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax） and cysteinyl aspartate-specific protease-3 （Caspase-3） in hypothalamus. Result:In the control group， there was no obvious pathological change in mitochondria， which were moderate in size and oval or spindle in shape， with the cristae arranged orderly. Compared with the control group， the model group exhibited abnormal mitochondrial morphology， manifested as obvious swelling， vacuolation， myelin figures， and cristae rupture and reduction. The comparison with the model group revealed that both the estazolam group and high-dose Suanzaoren Tang group alleviated the mitochondrial damage and reduced the vacuolation and swelling. Only some cristae rupture was present. The improvements were more obvious in the high-dose Suanzaoren Tang group. Compared with the control group， the activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase and the Bcl-2 protein expression in the model group were significantly decreased （<italic>P<</italic>0.01）， whereas the protein expression levels of Cyt C， Bax， and Caspase-3 were significantly increased （<italic>P</italic><0.01）. Compared with the model group， the activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase and the Bcl-2 protein expression in the estazolam group and the high-dose Suanzaoren Tang group were significantly elevated （<italic>P<</italic>0.01）， while the protein expression levels of Cyt C， Bax， and Caspase-3 were significantly lowered （<italic>P<</italic>0.05， <italic>P<</italic>0.01）. The activity of Na⁺-K⁺-ATPase and the Bcl-2 protein expression in the low-dose Suanzaoren Tang group were increased significantly （<italic>P<</italic>0.01）， but the Bax protein expression was down-regulated （<italic>P<</italic>0.05）. Conclusion:Suanzaoren Tang is able to improve the mitochondrial function of hypothalamic nerve cells and inhibit their apoptosis.

Effect of Suanzaoren Tang on Energy Metabolism of Myocardial Mitochondria in Aged Rats with Chronic Rapid Eye Movement Sleep Deprivation / 中国实验方剂学杂志

Objective:To study the mechanism of Suanzaoren Tang in regulating the energy metabolism of myocardial mitochondria in aged rats with chronic rapid eye movement （REM） sleep deprivation through the sirtuin 3 （SIRT3）/superoxide dismutase2 （SOD2） signaling pathway. Method:Fifty male Wistar rats were randomly divided into the control group， model group， estazolam group （0.18 mg·kg^-1·d^-1）， and low- （6.48 g·kg^-1·d^-1） and high-dose （12.96 g·kg^-1·d^-1） Suanzaoren Tang groups. Rats in all groups except for the control group received subcutaneous injection of <italic>D</italic>-galactose and then were deprived of sleep using the multiple platform method after the last administration. Following successful modeling， the rats in each group were treated with intragastric administration of the corresponding drugs for seven consecutive days. The morphology of myocardial mitochondria was observed under a transmission electron microscope. The content of adenosine triphosphate （ATP） in rat hypothalamus was detected by colorimetry， while the malondialdehyde （MDA） content and the SOD activity in myocardium were measured by spectrophotometry. Real-time polymerase chain reaction （Real-time PCR） and Western blot were conducted to determine the mRNA and protein expression levels of SIRT3 and SOD2 in rat myocardium. The localization of SIRT3 was detected by immunofluorescence staining. Result:Compared with the control group， the model group exhibited a disordered arrangement of myocardial filaments， accompanied by filament rupture and dissolution， obviously swollen mitochondria arranged in disorder， and blurring and even rupture of most mitochondrial cristae. Besides， the content of ATP and SOD activity in the myocardium decreased significantly （<italic>P<</italic>0.01）， whereas that of MDA increased significantly （<italic>P<</italic>0.01）. The mRNA and protein expression levels of SIRT3 and SOD2 were down-regulated significantly （<italic>P<</italic>0.01）， and the average fluorescence intensity of SIRT3 protein declined significantly （<italic>P<</italic>0.01）. The comparison with the model group revealed that high-dose Suanzaoren Tang enabled the myocardial filaments to be neatly arranged， relieved the mitochondrial damage and swelling， only manifested as partial mitochondrial cristae rupture， significantly increased ATP content， SOD activity， as well as SIRT3 and SOD2 mRNA and protein expression levels （<italic>P<</italic>0.01）， reduced the content of MDA （<italic>P<</italic>0.01）， and enhanced the average fluorescence intensity of SIRT3 protein （<italic>P<</italic>0.05）. The myocardial mitochondrial injury in the estazolam group was also alleviated. The activity of SOD and the SIRT3 and SOD2 mRNA and protein expression levels in the myocardium were significantly elevated （<italic>P<</italic>0.01）， while the activity of MDA was significantly lowered （<italic>P<</italic>0.01）. In the low-dose Suanzaoren Tang group， the improvement in myocardial mitochondrial injury was not obvious. However， both the SOD activity and SOD2 protein expression were significantly increased （<italic>P<</italic>0.05）. Conclusion:Suanzaoren Tang ameliorates the myocardial mitochondria injury and abnormal energy metabolism induced by chronic REM sleep deprivation in aged rats possibly by up-regulating the SIRT3 and SOD2 expression.

Effect of Shenghuitang on Bmal1 in Hypothalamus and IL-6 and TNF-α in Hippocampus of APP/PS1 Double Transgenic Dementia Model Mice / 中国实验方剂学杂志

Objective:To investigate the effect of Shenghuitang on learning and memory, biological clock gene[brain and muscle arnt-like 1 (Bmal1)] in hypothalamus and interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in hippocampus of APP/PS1 double transgenic dementia model mice, in order to explore the possible mechanism of Shenghuitang to improve learning and memory and sleep disorders. Method:The experimental mice were randomly divided into model group, blank control group, melatonin group, high-dose Shenghuitang group and low-dose Shenghuitang group. Autonomic activity analysis system was used to detect the autonomic activities of mice in each group. Morris water maze was used to detect the learning ability and spatial memory ability of each group. quantitative real-time fluorescence polymerase chain reaction(Real-time PCR) was used to detect the expression of Bmal1 mRNA in the hypothalamic area of mice. Western blot was used to detect the expression of Bmal1 protein in each group. The content of inflammatory factors IL-6 and TNF-α in hippocampus of mice was detected by enzyme-linked immunosorbent assay(ELISA). The correlation between inflammatory factors IL-6, TNF-α and Bmal1 gene was analyzed by pearson analysis. Result:The results of voluntary activities showed that compared with the control group, the number of activities and activity distance of the model group were significantly decreased (PPPPPPPPPPPPPPα in the model group were significantly higher than those in the control group (Pα in the drug group were significantly lower(Pα and Bmal1 were correlated and negatively correlated. Conclusion:Shenghuitang may reduce the levels of inflammatory factors IL-6 and TNF-α in hippocampus by up-regulating the expression of Bmal1 gene in hypothalamic region, thus improving Alzheimer' s disease(AD) and circadian rhythm disorders.

Effect of Shenghuitang on Learning Memory, Levels of IL-6, TNF-α and COX-2 in Hippocampus of Chronic Sleep Deprivation Mice / 中国实验方剂学杂志

Objective: To observe the effect of Shenghuitang on learning and memory and expressions of interleukin-6(IL-6), tumor necrosis factor-α(TNF-α) and cyclooxygenase-2(COX-2) in hippocampus of chronic sleep deprived mice, in order to explore the possible mechanism of Shenghuitang in improving learning and memory ability. Method: Mice were randomly divided into sleep deprivation group, blank group, melatonin group(7.8×10-4 g·kg-1·d-1), high, middle and low-dose Shenghuitang groups(54,27,13.5 g·kg-1·d-1). The model of chronic sleep deprivation in mice was established using the "multi-platform water environment method". 28 d sleep deprivation and intragastric administration were provided. Morris water maze was used to detect the learning and memory ability of mice in each group. Real time-PCR was used to detect mRNA expressions of IL-6, TNF-α and COX-2 in the hippocampus of each group. Result: The results of Morris water maze test showed that compared with the blank group, the total time spent on finding the platform and the total swimming distance of the model group were significantly prolonged (PPPPPPPPPα, and COX-2 were increased in the model group compared with the blank group. Compared with the model group, mRNA expressions of IL-6, TNF-α, and COX-2 were decreased in the treated group. COX-2 mRNA expression was down-regulated. Conclusion: Shenghuitang may improve the learning and memory ability of mice by decreasing mRNA expressions of IL-6, TNF-α and COX-2 in hippocampus.

Network pharmacology deciphering multiple mechanisms of volatiles of Wendan granule for treatment of senile dementia / 中国药理学与毒理学杂志

OBJECTIVE To explore the mechanisms of the volatiles of Wendan granule for the treatment of senile dementia,network pharmacology method integrating absorption,distribution,metab-olism, and excretion (ADME) screening, target fishing, network constructing, pathway analyzing, and correlated diseases prediction was applied. METHODS Twelve small molecular compounds of WDG were selected as the objects from 74 volatiles with the relative abundances above 2%,and their ADME parameters were collected from Traditional Chinese Medicine Systems Pharmacology platform (TCMSP), and then the corresponding targets, genes, pathways and diseases were predicted according to the data provided by TCMSP,DrugBank,Uniport and the Database for Annotation,Visualization and Integrated Discovery(DAVID).The related pathways and correlation analysis were explored by the Kyoto Encyclo-pedia and Genomes (KEGG) database. Finally, the networks of compound-target, target-pathway and pathway-disease of WDG were constructed by Cytoscape software. RESULTS Twelve compounds interacted with 49 targets, of which top three targets were Gamma-aminobutyric acid receptor subunit alpha-1 (GABRA1), Prostaglandin G/H synthase 2 (PGHS-2) and Sodium-dependent noradrenaline transporter.Interestingly,these targets were highly associated with depression,insomnia and Alzheimer′s disease that mainly corresponded to mental and emotional illnesses. CONCLUSION The integrated network pharmacology method provides precise probe to illuminate the molecular mechanisms of volatiles of WDG for relieving senile dementia related syndromes,which will also facilitate the application of traditional Chinese medicine in modern medicine,as well as follow-up studies such as upgrading the quality stan-dard of clinical medicine and novel drug development.