| Background:Sleep problems are troubling people in contemporary society and seriously threaten people’s physical and mental health.Circadian rhythm disturbance is one of the main causes of sleep disturbance in contemporary society.Increased work pressure,shift work system,and emotional factors are all interfering with the normal biological clock in the human body,causing disturbances in the circadian rhythm.Circadian rhythm disorders not only bring sleep problems to patients,but also cause functional and organic damages such as lipid metabolism disorders and liver cell damage.Traditional Chinese and Western medicine have their own advantages in the treatment of sleep disorders.For different causes of sleep disorders,modern medicine has different guidelines to deal with.Traditional Chinese medicine has a long history of dealing with rhythmic sleep disorders.From the "Huangdi Neijing" that first proposed the concept of time medicine to Zhang Zhongjing’s "Treatise on Febrile Diseases and Miscellaneous Diseases" who proposed the principle of time-selected treatment,these theories support the application and development of traditional Chinese medicine in rhythmic diseases.Not only that,Chinese medicine is still playing the core idea of the overall concept,while improving rhythmic sleep disorders,while taking into account other symptoms caused by circadian rhythm disorders,such as metabolic disorders,emotional disorders,digestive disorders,etc.Therefore,Chinese medicine can play an important role in the treatment of many physical and mental problems caused by circadian rhythm disorders,and it is also a major topic worthy of attention by Chinese medicine scholars.Although Chinese medicine has been described as chrono-medicine more than 2,000 years ago,the development of modern Chinese medicine lags behind in this aspect.At the same time,when there is a clinical problem between the emotions and the viscera and the pathogenesis and pathology cannot be found,or when there are symptoms related to emotions,the pathogenesis is dredging and disorder,and the treatment principle is dredging the liver and regulating qi.This makes the efficacy uncertain and increases the time and economic cost for the patient.After Professor Yang Shuyu’s in-depth research and in-depth summary,he believes that sleep-metabolismemotion-digestion and other functional problems can be attributed to the category of dredging,and put forward the concept that dredging is in the liver,not only in the liver,but also in the coordination of the five internal organs.Nourishing Blood and Rougan Compound is a self-prescribed formula of Professor Yang Shuyu,which is derived from Suanzaoren Decoction and Huanglian Wendan Decoction.In patients with emotional disorders and/or gastrointestinal disorders,the effect is definite,but the mechanism is unknown.Therefore,under the guidance of the TCM theory of "liver stores blood and controls dredging",this topic explores the mechanism by which Yangxue Rougan Fang can improve lipid metabolism abnormalities and liver damage caused by circadian rhythm disorder,aiming at the basis of the holistic view of traditional Chinese medicine and the mechanism of circadian rhythm.Complications caused by disorders provide clinical solutions and scientific research data support,in order to relieve the physical and mental burden and economic pressure of patients.Materials and Methods:(1)Preparation and quality control of Nourishing Blood and Rougan Compound and its medicated serum.The decoction pieces of traditional Chinese medicine were decocted and mixed twice and then concentrated to the high and low dose groups by rotary evaporator.The drug-containing serum was obtained by gavage of rats.Then,the mass spectrometry data of traditional Chinese medicine decoction pieces of Yangxue Rougan Compound,and prepared rat blank serum and drug-containing serum samples were collected by ultra-high performance liquid chromatography-high resolution mass spectrometry.(2)Animal experiments.240 male C57BL/6 mice aged 6-8 weeks were divided into 5 groups:control group,circadian rhythm disorder group,YR low-dose group,YR high-dose group,agomelatine group,48 mice in each group.Except for the control group,the other 4 groups were kept in a completely dark environment for 2 weeks,while the drug intervention group was given corresponding drug intervention,and the control group and model group were given an equal volume of distilled water by gavage.After 2 weeks,animals in each group were randomly selected and placed in metabolic cages and running wheel devices to collect basal metabolic data such as activity,24hour food intake,and oxygen consumption.Then,the mice in each group were frozen or fixed according to ZT3,ZT7,ZT11,ZT15,ZT19,ZT236 time points.(3)In vitro experiments.AML 12 cells were recovered and passaged and divided into plates for subsequent experiments.Cell proliferation ability was determined with CellCountingKit-8 to obtain the optimal concentration and time of drug-containing serum,thapsigargin,agomelatine and reduced glutathione.Bmall overexpression and knockdown plasmids were constructed and packaged into viruses,and then stably transfected into AML 12 cells to obtain stable overexpression or knockdown of Bmall cell lines.(4)Molecular biology detection.Hematoxylin&eosin staining,Oil red O staining,TUNEL and perilipinl immunofluorescence,etc.are used to observe cell morphology,apoptosis,perilipin,etc.in liver tissue,or to observe lipid droplets in cells.Triglyceride levels in cells and liver tissue were detected using a triglyceride assay kit.The serum ALT/AST levels of mice in each group were detected by an automatic biochemical analyzer.Real-time quantitative PCR and Western Blotting were used to detect mRNA and protein expression in cells,mouse hypothalamus and liver tissue.The mass spectrometry data processing software is Peakviewl.2.Statistical analysis of experimental data was performed with SPSS25 software.Results:(1)The main components of each drug in the decoction pieces of traditional Chinese medicine were detected,and the main components of the drug-containing serum were not matched,and the rest of the components were also detected.(2)Low-dose Yangxue Rougan compound could restore the circadian activity,24hour food intake,oxygen consumption,carbon dioxide emission and other regularities in mice with circadian rhythm disorder.Its mechanism was to increase the Bmall protein in the hypothalamus of circadian rhythm disordered mice.The amplitude of expression restored the phase and amplitude of the liver rhythm gene and protein BMAL1 in circadian rhythm disordered mice,and could significantly increase the expression of Bmal1 protein in circadian rhythm disordered mice at the ZT15 time point.Both the high-dose group and the agomelatine group could improve the circadian activity pattern,but there was no statistical significance;at the same time,the two groups did not significantly improve other basal rhythms,and the expression patterns of rhythm proteins in the hypothalamus and liver tissues were not significant.(3)In animal experiments,both high-dose and low-dose groups could improve liver cell damage in mice with circadian rhythm disorder;high-dose group can reverse the level of serum AST in ZT7 mice;the degree of cell edema in agomelatine group also decreased.improved,but not as effective as the high and low dose groups.The mechanism of Yangxue Rougan Compound to improve liver injury lied in Bmall and endoplasmic reticulum stress,but had nothing to do with oxidative stress,ischemia and hypoxia,inflammation and endothelial function.In the low-dose group,Yangxue Rougan Compound reduced the mRNA expression of Grp78/ATF4/Chop and the protein expression of ATF4/Chop at the time point of ZT11,and promoted the inhibition of the protein expression of the apoptosis gene Bcl2,indicating that the lowdose Yangxue Rougan Compound could alleviate the symptoms.Endoplasmic reticulum stress and protection of hepatocytes.Agomelatine reduced ATF4 mRNA and protein expression at the ZT11 time point,while inhibiting the protein expression of Chop.In vitro experiments,we verified that Yangxue Rougan Compound reduced the protein expression of thapsigargin-induced p-eIF2α/ATF4 pathway and increased the viability of AML 12 cells by increasing the expression of Bmall protein.Agomelatine also increased the expression of Bmall,but did not inhibit the effect of thapsigargininduced p-eIF2α/ATF4,nor did it significantly improve cell viability.Reducedglutathione had no significant effect on up-regulating Bmall,improving cell viability and inhibiting p-eIF2α/ATF4.(4)In animal experiments,the effect of Yangxue Rougan Compound in the lowdose group in improving lipid metabolism in mice with circadian rhythm disorder was better than that in the high-dose group.Although both high and low dose groups and agomelatine groups could reduce triglyceride levels in the liver of mice at the ZT15 time point,only the low dose was statistically significant in the immunofluorescence of perilipin1.However,we did not detect significantly altered proteins in lipid metabolism pathways.In vitro experiments,we found that Bmall was also the target of Yangxue Rougan Compound,and inhibited the accumulation of lipids in hepatocytes by increasing the expression of Bmall.Agomelatine increased Bmall expression but had no effect on reducing lipid accumulation.Reduced glutathione had no significant effect on up-regulating Bmall and inhibiting lipid accumulation.Conclusion:(1)The low-dose Yangxue Rougan group had a protective effect on improving endoplasmic reticulum stress and liver lipid metabolism in mice with circadian rhythm disorder.(2)The low-dose Yangxuerougan group could restore the circadian activity law,the amplitude of Bmal1 mRNA expression in the hypothalamus and liver,and the phase and amplitude of hepatic Bmall protein expression in mice with circadian rhythm disorder,and significantly up-regulated the expression of liver Bmall at ZT15 time point quantity.(3)Yangxue Rougan Compound relieved endoplasmic reticulum stress and improved cell viability by up-regulating the expression of Bmall protein in AML 12 hepatocytes and inhibiting the protein expression of p-eIF2α/ATF4.(4)Yangxue Rougan Compound reduced lipid accumulation in hepatocyte AML 12 by up-regulating Bmall. |
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