ObjectiveThis study aims to analyze the pharmacodynamic material basis and multi-target mechanism of action of Banxia Baizhu Tianmatang combined with Danggui Shaoyaosan in the treatment of Meniere's disease（MD）. MethodsUltra-performance liquid chromatography-high resolution mass spectrometry （UPLC-HRMS） （mobile phase： gradient elution with 0.1% formic acid aqueous solution-acetonitrile. Mass spectrometry scanning range： m/z 90-1 300） was used to identify the chemical components of the compound recipe and components absorbed into blood. The core mechanism was predicted by combining network pharmacology （target screening via SwissTargetPrediction and GeneCards databases， and construction of protein-protein interaction （PPI） network by STRING） and molecular docking （evaluated by Autodock， with binding energy ≤ -5.0 kcal·mol^-1）. For animal experiment validation， 36 Sprague Dawley （SD） rats were divided into a blank group， a model group （postauricular injection of lipopolysaccharide （LPS） at 1 mg·kg^-1）， low/medium/high-dose Chinese medicine groups （5.94， 11.88， and 23.76 g·kg^-1·d^-1， respectively）， and Western medicine group （betahistine at 0.1 mg·kg^-1·d^-1）. After eight weeks of intervention， the gene and protein expressions in cochlear tissue were detected. Results①A total of 2 831 chemical components and 173 components absorbed into blood were identified， with terpenoids showing the highest absorption rate into blood（10.28%）. ②60 common drug-disease targets were screened， with core targets including tumor necrosis factor-α（TNF-α），interleukin-6（IL-6）， Toll-like receptor 4（TLR4）， angiotensin-converting enzyme（ACE）， and endothelial nitric oxide synthase 3（NOS3）.These targets were enriched in the nuclear factor-κB（NF-κB） signaling pathway and renin-angiotensin system（P<0.05）. Molecular docking showed that the active component YC-1 had a strong binding ability to TNF（binding energy-9.66 kcal·mol^-1）. ③In animal experiments， the high-dose Chinese medicine group significantly down-regulated the expression of pro-inflammatory factors TNF mRNA（P<0.01）and up-regulated vascular regulatory factors NOS3 protein（P<0.01）， and alleviated cochlear pathological damage［hematoxylin eosin （HE） score： from 4 to 2］. ConclusionThis compound recipe synergistically regulates the TNF/NF-κB inflammatory pathway and ACE/NOS3 vascular homeostasis pathway through flavonoids， triterpenoids， and other components， thereby inhibiting endolymphatic hydrops and cochlear damage. It provides a scientific basis for the theory of "simultaneous treatment of phlegm and blood stasis" in traditional Chinese medicine.

OBJECTIVE: To investigate the regulatory effect of electroacupuncture (EA) at "Neiguan" (PC6) on mitochondrial autophagy in rats with myocardial ischemia-reperfusion injury (MIRI) at different phases (ischemia and reperfusion phases), and to explore the bidirectional regulatory effects of EA at "Neiguan" (PC6) and its potential mechanism. METHODS: Forty-five male SD rats were randomly divided into 6 groups according to the random number table method, namely, sham-operation group (n=9), model-A group (n=6), model-B group (n=9), EA-A1 group (n=6), EA-B1 group (n=6), and EA-B2 group (n=9). Except the rats in the sham-operation group, the MIRI model was established in the other groups with the physical ligation and tube pushing method. In the model-A group, the samples were collected directly after ligation, and in the model-B group, the samples were collected after ligation and reperfusion. In the EA-A1 group, EA was delivered while the ligation was performed, and afterwards, the samples were collected. In the EA-B1 group, while the ligation was performed, EA was operated at the same time, and after reperfusion, the samples were collected. In the EA-B2 group, during ligation and the opening of the left anterior descending branch of the coronary artery, EA was delivered, and after reperfusion, the samples were collected. EA was performed at bilateral "Neiguan" (PC6), with a disperse-dense wave, a frequency of 2 Hz/100 Hz, a current of 1 mA, and a duration of 30 min. HE staining was employed to observe the morphology of cardiomyocytes, TUNEL was adopted to detect the apoptosis of cardiomyocytes, transcriptome sequencing was to detect the differentially expressed genes in the left ventricle, JC-1 flow cytometry was to detect the mitochondrial membrane potential (MMP) of cardiomyocytes, Western blot was to detect the protein expression of phosphatase and tensin homolog-induced kinase 1 (Pink1), Parkin and p62 in the left ventricle of rats, and ELISA was to detect the levels of serum creatine kinase isoenzyme (CK-MB) and cardiac troponin I (cTn-I) in the rats. RESULTS: Compared with the sham-operation group, the cardiomyocytes of rats in the model-B group were severely damaged, with disordered arrangement, unclear boundaries, broken muscle fibers, edema and loose distribution; and the cardiomyocytes in the EA-B2 group were slightly damaged, the cell structure was partially unclear, the cells were arranged more regularly, and the intact cardiomyocytes were visible. Compared with the sham-operation group, the apoptosis of cardiomyocytes increased in the model-B group (P<0.001); and when compared with the model-B group, the apoptosis alleviated in the EA-B2 group (P<0.001). The differentially expressed genes among the EA-B2 group, the sham-operation group and the model-B group were closely related to cell autophagy and mitochondrial autophagy. Compared with the sham-operation group, MMP of cardiomyocytes was reduced (P<0.001), the protein expression of Pink1, Parkin, and p62 of the left ventricle and the levels of serum CK-MB and cTn-I were elevated in the model B group (P<0.001). In comparison with model-A group, the MMP of cardiomyocytes and the levels of serum CK-MB and cTn-I were reduced (P<0.001, P<0.05), and the protein expression of Pink1 in the left ventricle rose in the EA-A1 group (P<0.01). Compared with the model-B group, MMP of cardiomyocytes increased (P<0.001), the protein expression of Pink1, Parkin, and p62 of the left ventricle, and the levels of serum CK-MB and cTn-I decreased (P<0.001) in the EA-B1 group and the EA-B2 group. When compared with the EA-A1 group, MMP of cardiomyocytes increased (P<0.001), and the protein expression of Pink1, Parkin, and p62 of the left ventricle, and the levels of serum CK-MB and cTn-I decreased in the EA-B1 group (P<0.01). CONCLUSION EA at "Neiguan" (PC6) can ameliorate MIRI in rats, which may be achieved through the Pink1/Parkin-mediated mitochondrial autophagy pathway. EA can alleviate myocardial injury by enhancing mitochondrial autophagy at the ischemia phase, and it can reduce reperfusion injury by weakening mitochondrial autophagy at the reperfusion phase.
Animals ; Electroacupuncture ; Male ; Myocardial Reperfusion Injury/metabolism* ; Rats, Sprague-Dawley ; Rats ; Acupuncture Points ; Autophagy ; Humans ; Mitochondria/genetics*