Study On The Mechanism Of Tongyang Huoxue Recipe Regulating β-tubulin Skeleton Protein And Mitochondrial Homeostasis In The Treatment Of Sick Sinus Syndrome

Sick sinus syndrome(SSS)is a comprehensive manifestation of a variety of arrhythmias caused by sinus node disease leading to sinus node dysfunction.Sinus syndrome is one of the most difficult cardiovascular diseases to treat clinically.The anatomical and pathological basis of sick sinus syndrome is complete or nearly complete destruction of the sinus node,discontinuity of the sinus-atrial region,inflammation or degeneration of the surrounding tissue of the sinus node,and pathological changes in the atrium,with fibrosis and fatty infiltration.The sinoatrial and atrioventricular nodes and their branches often have sclerosing degenerative lesions.According to previous clinical studies,the onset time of sinoatrial node syndrome tends to be aging.In the heart,fibrotic tissue blocks the hyperpolarizing effect of the sinoatrial node and the atrium.Sinus node fibrosis may lead to tachycardia-bradycardia arrhythmias and cardiac arrest,a process that Possibly due to SAN reentry and exit blocking.Tongyang Huoxue Recipe is a classic and original prescription of Chinese medicine master Liu Zhiming for the treatment of sick sinus syndrome.In the early stage,extensive research has been carried out in the field of various types of arrhythmia diseases.The treatment of nodal syndrome has unique advantages.In the early stage,the research group conducted systematic and comprehensive basic and clinical research on the treatment of sick sinus syndrome with Tongyang Huoxue Recipe from the perspectives of mitochondrial energy metabolism,calcium homeostasis regulation and skeletal protein homeostasis regulation.However,the related research on the treatment mechanism of sinus node fibrosis and the protection mechanism of sinoatrial node cells is still insufficient.This experimental study is based on the pathogenesis theory of "Yang deficiency and blood stasis" in the sick sinus and the aging pathogenesis of sinus node fibrosis.The model of atrial node fibrosis confirmed the mechanism of Tongyang Huoxue Recipe in improving sinus node fibrosis through oxidative stress/calcium homeostasis regulation and autophagy.Through in vitro(cell)experiments,it was confirmed that Tongyang Huoxue Recipe protects sinoatrial node cells under hypoxia/reoxygenation stress state(simulating ischemia-reperfusion,I/R)by regulating β-tubulin skeleton protein and mitochondrial homeostasis mechanism.mechanism of action.This study explored the in-depth mechanism of Tongyang Huoxue Recipe in treating sinus syndrome and protecting sinoatrial node cells,in order to provide preliminary experimental basis and theoretical connotation for the treatment of sick sinus.Objective:In this experiment,the aging sinoatrial node fibrosis model of SD rats and primary sinoatrial node cells were studied.The objective of the experiment is to clarify the improvement mechanism of Tongyang Huoxue Recipe on sinoatrial node fibrosis in aging rats,and further clarify the protective mechanism of Tongyang Huoxue Recipe on sinoatrial node cells under stress through in vitro experiments.To explore the material basis and targeting mechanism of Tongyang Huoxue recipe from the macro and micro perspectives of sinoatrial node histopathology,cell morphology and molecular biology.Methods:1.A total of 60 SD rats(SPF grade)were taken,including 20 3-month-old rats(the rat age is equivalent to 10-12 years old in humans)and 20 14-month-old rats(the rat age is equivalent to 40-50 years old in humans).Years),20 20-month-old rats(the age of the rats is equivalent to the human age of 55-65 years).Then the 3-month-old/14-month-old/20-month-old rats were randomly divided into blank control group(3-month-old/14-month-old/20-month-old)and Tongyang Huoxuefang intervention group(3-month-old/14-month-old/20-month-old)20 months old),10 in each group.Rats in Tongyang Huoxue Recipe intervention group(3 months old/14 months old/20 months old)were given 2ml of Tongyang Huoxue Recipe regularly every day;The rats in the blank control group(3 months old/14 months old/20 months old)were given the same volume of normal saline 2ml.30 days in total.Masson and Sirius red staining was used to observe the histopathological and fibrin changes of sinoatrial node;The expression of fibrosis related proteins was detected by immunohistochemistry.The expression of fibrosis related indexes was detected by tissue immunofluorescence.The morphological and structural changes of mitochondria in sinoatrial node were detected by tissue transmission electron microscope.The expressions of fibrosis,oxidative stress and autophagy related proteins were detected by Western blot;1.New Zealand suckling rabbits within 24 hours were selected and divided into normal control group,model group,Tongyang Huoxue treatment group and Tongyang Huoxue+si-β-tubulingroup,Tongyang Huoxue Recipe+ad-β-tubulin group.2.The sinoatrial node cells of suckling rabbits were isolated by "single enzymatic hydrolysis+differential adhesion" method.In the Model group and Tongyang Huoxue Recipe+si-β-tubulin group/Tongyang Huoxue+ad-(3-tubulin group,the sinoatrial node cell injury model was prepared by simulated ischemia-reperfusion(hypoxia/reoxygenation).Utilize P-tubulin siRNA lentivirus vector was transfected into sinoatrial node cells of suckling rabbits to obtain Tongyang Huoxue+si-β-tubulin group(β-tubulin knockdown group).Utilize RNA lentivirus vector was transfected into sinoatrial node cells of suckling rabbits to obtain Tongyang Huoxue recipe+ad-β-tubulin Group(β-tubulin overexpression group);CCK-8,flow cytometry and TUNEL fluorescence technique were used to detect cell activity,apoptosis level,ROS production and mitochondrial membrane potential.The level of ROS and NCX were detected by immunofluorescence.The structure of β-tubulin protein,the expression of JC-1 mitochondrial membrane potential,mitochondrial length and mitochondrial fragmentation were detected by laser confocal technology.The expression of mitochondrial fission kinesin Drpl and mitophagy regulatory protein TOM20 were detected by fluorescence co-localization technique.The morphology and structure of mitochondria in sinoatrial node cells and the formation of mitophagolysosomes were detected by transmission electron microscopy.ELISA kits were used to detect the expression levels of oxidative stress marker MDA,antioxidant enzymes SOD/GSH/TrxR and mitochondrial respiratory chain enzyme complex Ⅰ/Ⅲ.RT-PCR technique was used to detect the mRNA expression levels of mitochondrial fission and fusion-related protein(Fisl/Mfn1),mitochondrial biosynthesis-related protein(Tfam/PGC1α)and mitophagy-related protein(Parkin/ATG5).Results:1.In-vivo studies:There were no obvious pathological changes in the 3-month-old blank control group and the administration group.The 14-month-old and 20-month-old model control rats had more severe fibrosis in the sinus node tissue(P<0.01).Especially in 20-month-old mice,the pathological changes of the sinus node and surrounding myocardial tissue were more obvious,and the level of fibrinogen agglutination was significantly increased(P<0.01).Immunohistochemistry showed that the expression levels of TGF-β and Smad3 were significantly increased(P<0.01).Immunofluorescence detection showed that tissue ROS production was significantly increased,Smad3 and P62/caspase-9 fluorescence expression levels were significantly increased,WGA staining showed that myocardial cell hypertrophy was significantly increased,oxidative stress markers were significantly increased,and antioxidant enzymes were significantly decreased(P<0.01).The expression levels of TGF-β/Smad3/α-SMA and caspase-3/caspase-12/IL-18 were significantly increased(P<0,01).The autophagy-related fluorescence expression and protein expression levels were significantly changed,the autophagy level was significantly decreased,the expression levels of COX2 and GPX4 were significantly changed,and the lipid peroxidation level was significantly increased(P<0.01).The arrangement of mitochondria in tissues is irregular,and the morphological structure of autophagolysosomes is abnormal.Compared with the rats in the model group,the 14-month-old and 20-month-old Tongyang Huoxue Recipe intervention group significantly decreased the degree of fibrosis in the sinus node tissue,and the level of fibrinogen agglutination was significantly decreased(P<0.01).Immunohistochemistry showed that the expression of TGF-β and Smad3 were significantly decreased,immunofluorescence detection showed that the production of ROS in tissues was significantly decreased,and the fluorescence expression of Smad and P62/caspase-9 were significantly decreased(P<0.01).WGA staining showed that cardiomyocyte hypertrophy was significantly decreased,a marker of oxidative stress(MDA)was significantly decreased,and the antioxidant enzymes SOD/GSH were significantly increased(P<0.01).The expression levels of TGF-β/Smad3/α-SMA and caspase-3/caspase-12/IL-18 were significantly decreased,and the level of autophagy increased significantly.The expression levels of COX2 and GPX4 were significantly changed,the level of lipid peroxidation was significantly decreased(P<0.01).The arrangement of tissue mitochondria is relatively regular,and the morphological structure of autophagolysosome is relatively stable(P<0.05).2.In-vitro study:After ischemia-reperfusion treatment(hypoxia/reoxygenation simulated),the activity of primary sinoatrial node cells decreased significantly,and the level of apoptosis and production of ROS increased significantly.Accompanied by the expression of β-tubulin skeleton protein decreased and calcium overload.After hypoxia/reoxygenation treatment,sinoatrial node cells also showed oxidative stress injury,mitochondrial respiratory chain dysfunction,mitochondrial membrane potential loss and mitochondrial fragmentation.Drp1/Fis1/Mfn1 mediated mitochondrial dynamics imbalance,increased mitochondrial fission,parkin/ATG5/Tom20 mediated mitophagy imbalance,accompanied by mitochondrial quality control and mitochondrial biosynthesis disorder.After the intervention treatment of Tongyang Huoxue recipe,the cell activity was significantly improved,the production of ROS and oxidative stress injury were reduced,and the level of mitochondrial fragmentation was reduced.The intervention of Tongyang Huoxue recipe can also restore the function of mitochondrial respiratory chain and the level of mitochondrial membrane potential,and maintain the level of mitochondrial quality control and mitochondrial biosynthesis.However,after treatment with siRNA(si-β-tubulin),the regulatory mechanisms of Tongyang Huoxue Recipe on mitochondrial homeostasis and oxidative stress were inhibited,and the protective effect on sinoatrial node cells was also counteracted.β-tubulin over-express could improve the regulatory effect of Tongyang Huoxue Recipe on mitochondrial homeostasis and oxidative stress,while enhancing the protective effect of Tongyang Huoxue Recipe on sinoatrial node cells.Conclusion:1.Tongyang Huoxue Recipe can improve the fibrosis in the sinus node of ageing rats,and reduce the expression of fibrin in the sinus node and the level of fibrinogen agglutination in the aging rat.Its regulatory mechanism may be related to autophagy regulatory mechanisms,as well as oxidative stress and dys-regulation of mitochondrial homeostasis/calcium homeostasis.2.Tongyang Huoxue Recipe can significantly improve the activity of sinoatrial node cells in the simulated I/R state,inhibit cell apoptosis,and maintain the structure and function of mitochondria,and it is further found that the improvement mechanism may be by β-tubulin.3.The improvement mechanism of Tongyang Huoxue Recipe on sinus node cells in simulated I/R state may be through β-tubulin skeleton protein to restore mitochondrial membrane potential,increase the level of mitochondrial respiratory chain,regulate the oxidative stress and mitochondrial homeostasis mechanism.4.Tongyang Huoxue Recipe may improve the sinus node fibrosis and increase the activity of sinoatrial node cells through the perspective of "warming the kidney-tongyang-activating blood",which is the material basis for the syndrome of sick sinus syndrome.This is of great significance for the basic experimental research of sick sinus syndrome and the clinical treatment of sick sinus syndrome.Innovation:1.This study is based on the theory of the original Fang Tongyang Huoxue Recipe that treats sick sinus syndrome with the heart and kidney of the original Chinese medicine master Professor Liu Zhiming.The application of Tongyang Huoxue Recipe to intervene on sinus node fibrosis in rats of different ages found that Tongyang Huoxue Recipe can increase the level of autophagy through the PI3K-AKT-mTOR signaling pathway,regulate mitochondrial homeostasis and "inflammation-apoptosis"Mechanisms in turn improve sinoatrial node fibrosis.This provides an experimental basis for basic research and targeted pharmacological research on sick sinus syndrome.2.This study clarified the mechanism by which Tongyang Huoxue Recipe protects sinoatrial node cells through β-tubulin microtubule skeleton protein,regulates redox balance,improves sinoatrial node cell damage under stress,and inhibits sinoatrial node cell apoptosis..Further clarified the scientific basis of Tongyang Huoxue Recipe for the treatment of sick sinus syndrome.