The Mechanism Of Emotional Stimulation-induced Liver Injury And Protective Effects Of Chaihu Shugan San

Background:Exploring the emotion-induced diseases is one of the most important parts of the etiological theories of traditional Chinese medicine.Strong emotional stimulation can affect the physiological balance of Yin,Yang,Qi and blood and the function of viscera,leading to various diseases.As one of the most common emotions,anger is closely related to the liver organ,as indicated by proverbs such as "Anger is the will of the liver" and "Anger hurts the liver" in Chinese medicine.Thus,anger is believed to cause the dysfunction and lesions of the liver organ.By establishing a susceptible model of liver injury caused by restraint stress,our group preliminarily found that the level of lipid peroxidation in liver of mouse increased significantly under a constrained condition.However,the detailed mechanism is still unknown.In this study,we expected and found that detained stress loading could induce the change of biological rhythm of liver tissues in mice.To clarify the biological mechanism of "Anger hurts the liver",therefore,we plan to further investigate the role of biorhythm factors such as Brain and muscle Arnt-like protein-1(BMAL1)in emotional stimulation-induced liver injury from the perspective of phospholipid peroxidation.As a result,we are able to objectively evaluate the medical effect of Chaihu Shugan San,a traditional Chinese medicine compound which be used in the treatment of depression.This work intends to enrich the theory of "emotional-pathogenicity" and provide an experimental basis for applying the modern biological theory to further explain the mechanism of “Emotion-induced diseases”,which could promote the use of traditional Chinese medicines as auxiliary treatments of the liver injury.Methods:1)Firstly,restraint stress-induced liver injury model was established for mice.Aspartate aminotransferase(AST)and alanine aminotransferase(ALT)kits were used to detect the level changes of AST and ALT in serum of mice.Mice with liver injury were adopted and the pathological and morphologic characteristics of their livers were captured by hematoxylin-eosin staining(H&E).The levels of malondialdehyde(MDA),4-hydroxynonenal(4-HNE),and Glutathione(GSH)were detected by MDA kits,Western Blot and LC-MS techniques,respectively.MDA and 4-HNE are two typical products of phospholipid peroxidation in liver tissues,while GSH is an antioxidant for phospholipid peroxidation.Meanwhile,the changes of phospholipid peroxidation degrees and the composition of oxidized phospholipids in the liver of restraint mice were analyzed by using LC-MS/MS technique.Furthermore,Western Blot and q-PCR were used to detect the clock genes in the liver of mice with restraint stress to determine the relationship between emotional stimulation-induced liver injury and circadian rhythm.2)The physiological rhythm disturbance(circadian disruption)model was further established.The correlation between circadian rhythm and phospholipid peroxidation can be illustrated by applying MDA kits,Western Blot and LC-MS to detect the content of MDA,4-HNE and GSH in the liver tissues of mouse,respectively.Subsequently,BMAL1 was overexpressed in Hepa RG cells derived from human hepatic cell line.The relationship between the BMAL1 and phospholipid peroxidation levels was also explored on a cellular level by using C11-BODIPY dyes and the LC-MS/MS method.Besides,the expression levels of genes and proteins related to phospholipids peroxidation were examined by q-PCR and Western Blot techniques and it was confirmed that BMAL1 and phospholipids of lipoxygenase,a key enzyme in 15-lipoxygenase(Arachidonate 15-lipoxygenase,ALOX15)affected each other directly.Dual luciferase reporter gene assay and gel electrophoresis migration assay were used to further verify the interaction and regulatory mechanism of BMAL1 and ALOX15.3)Using the emotional stress-induced liver injury model,we evaluated and discussed the effect and mechanism of Chaihu Shugan San,a classic Chinese medicine compound,in relieving emotional stress-induced liver injury.Results:In this study,we firstly found that restraint stress significantly increased ALT(p< 0.05)and AST(p < 0.001)levels in plasma,with an obvious liver injury in histopathological analysis when compared to control.In the meantime,the levels of MDA(p < 0.001)and 4-HNE in liver were remarkably increased in restraint stress mice,while GSH level(p < 0.05)were inhibited.To confirm that restraint stress-induced liver injury is contributed by oxidized phospholipids,liver tissues were analyzed by LC-MS/MS method and the results uncovered that restraint stress dramatically accumulated peroxidized phospholipids in liver.Unexpectedly,restraint stress also disrupted the rhythmicity of clock genes,including Bmal1,Rev-erbα and Per2 in liver of mice.In particular,BMAL1 expression increased significantly(p <0.01)in restraint stress when compared to control.Based on these results,we therefore infer that peroxidized phospholipids and the rhythmicity of clock genes might be critical for stress-induced liver injury.In order to further study how biorhythms involving in the process of phospholipid peroxidation in liver injury,we developed a biorhythm disorder model.The results showed that the level of 4-HNE(p < 0.01)and MDA(p < 0.001)in the liver tissues of mice with circadian disruption both greatly increased compared to mice in the control group,while the content of GSH(p < 0.001)was significantly decreased.These results indicated that circadian disruption indeed leads to an increased incidence of hepatic phospholipid peroxidation.On the other hand,in vitro cell model,we found that overexpression of BMAL1 dramatically increased the levels of phospholipid peroxides(p < 0.01)as well as the content of 4-HNE(p < 0.05)in Hepa RG cells.LC-MS/MS results also showed that the content of oxidized phospholipid increased and peroxidized phospholipids accumulated in Hepa RG cells with the overexpressed BMAL1.The phospholipid peroxidation related genes were screened,and found that the expression of ALOX15,an inducer of phospholipid peroxidation,were remarkably increased in BMAL1 overexpressed Hepa RG cells(p< 0.01).That is the reason why the overexpression of BMAL1 could induce the accumulation of peroxidized phospholipids.More importantly,BMAL1 was found to combine with the E-BOX sequence in the promoter sequence of ALOX15 and directly participate in transcriptional regulation of ALOX15 to increase its expression level as supported by double luciferase reporter gene assay and gel electrophoresis migration assay.Finally,the hepatoprotective effect and its related mechanism of Chaihu Shugan San were carefully evaluated and investigated by using the emotional stress-induced liver injury model.Base on this model,it was found that both low and high doses of Chaihu Shugan San effectively reduced the levels of ALT(p < 0.05,p < 0.001)and AST(p < 0.01,p < 0.001)in the serum of restraint mice.At the same time,Chaihu Shugan San could ameliorate the pathological injury and reduce the inflammatory infiltration in the liver tissues of mice.In addition,the results of the MDA kit and Western Blot characterizations showed that low and high doses of Chaihu Shugan San obviously reduced the levels of MDA(p < 0.001,p < 0.001)in restraint mice.Especially,high doses of Chaihu Shugan San could even simultaneously reduce the expression of 4-HNE(p < 0.01).Moreover,the LC-MS data further showed that low and high doses of Chaihu Shugan San similarly significantly increased the level of GSH(p < 0.001,p < 0.001).Lastly,the q-PCR results showed that Chaihu Shugan San were able to greatly reduce the expression levels of Bmal1 and Alox15 genes(p <0.01,p < 0.01)in the liver tissues of restraint mice.In conclusion,the hepatoprotective mechanism of Chaihu Shugan San might be to inhibit phospholipid peroxidation by down-regulating the expression levels of Bmal1 and Alox15 genes in mouse livers.Conclusion:Restraint stress-induced liver injury might be involved in obvious phospholipid peroxidation.The possible mechanism of this kind of liver injury is that the restraint stress increases the expression of the rhythm factor BMAL1,which directly regulates the expression of the key enzyme ALOX15 of phospholipid peroxidation,then the level of phospholipid peroxidation in liver tissue increases,leading to liver injury.Interestingly,Chaihu Shugan San can reduce restraint stress-induced liver injury by down-regulating the expression levels of Bmal1 and Alox15 genes in mouse livers;contributing to the inhibition of phospholipid peroxidation in damaged livers,which provides an experimental basis for the development and utilization of Chaihu Shugan San in protecting liver.