Suxiao Jiuxin Pill alleviates myocardial ischemia–reperfusion injury through the ALKBH5/GSK3β/mTOR pathway

Abstract

Background Many studies have shown effective protection from myocardial ischemia–reperfusion injury (MIRI) in animal models, but few, if any, treatments have yielded a substantial reduction in clinical. Several studies showed significant therapeutic effects for the Chinese patent medicine Suxiao Jiuxin Pill (SJP) in MIRI, although the specific molecular mechanisms remain undefined. Recently, increasing evidence indicates an important role for m6A modification in autophagy regulation in MIRI, and SJP has not been investigated in this regard. Methods In vivo experiments were performed in a Wistar rat MIRI model. In vitro assays were conducted in hypoxia/reoxygenation (H/R)-treated H9c2 cells. H9c2 cells with ALKBH5 and GSK3β silencing were constructed by lentivirus transfection. TUNEL and Annexin V/PI assays were carried out for apoptosis detection. Then, m6A modification was detected with the EpiQuik m6A RNA methylation quantification kit, and GFP-RFP-LC3B was used to observe dynamic changes in autophagy. The autophagosome structure was assessed by Transmission electron microscopy. qPCR and immunoblot were performed for mRNA and protein analyses, receptively. Results SJP significantly mitigated MIRI in rats, reducing infarct size and myocardial apoptosis, and improving left ventricular function. In addition, SJP inhibited autophagy through the GSK3β/mTOR pathway in MIRI rats. In cultured H9c2 cells, SJP significantly inhibited H/R- related apoptosis and autophagic activity through the GSK3β/mTOR pathway. Additionally, SJP enhanced ALKBH5 expression in H/R cardiomyocytes, which is important in impaired m6A modification. Interestingly, ALKBH5 knockdown enhanced autophagy and apoptosis in H/R-induced cells, whereas SJP reversed these effects. Further experiments showed that autophagic activity and apoptosis enhanced by ALKBH5 deficiency are GSK3β/mTOR pathway dependent in H/R-treated H9c2 cells. After SJP administration the above effects were alleviated, suggesting SJP inhibited autophagy through the ALKBH5/GSK3β/mTOR pathway in H/R-induced cardiomyocytes. These effects of SJP were common to its two main constituents, including tetra-methylpyrazine (TMP) and borneol (BOR). Conclusion SJP improves MIRI in rats and alleviates autophagy and apoptosis in H9c2 cells through the ALKBH5/GSK3β/mTOR pathway, thanks to its two major constituents TMP and BOR.

Oleuropein alleviates myocardial ischemia–reperfusion injury by suppressing oxidative stress and excessive autophagy via TLR4/MAPK signaling pathway

“…Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/treptomycin (P/S), and 0.25% Trypsin–EDTA (1 ×) were obtained from Gibco (California, USA). Tert-Butyl hydroperoxide (tBHP) and 3-[ 4 , 5 ]-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) were purchased from Sigma-Aldrich (298-93-1, Taufkirchen, Germany). Chloroquine (CQ) and TAK242 were purchased from MedChemExpress (Shanghai, China).…”

“…Thus, myocardial ischemia/reperfusion injury (MIRI) seriously affects patient recovery and prognosis [ 2 ]. Currently, research shows that a number of natural products or traditional Chinese medicine compounds have the potential to treat MIRI, which may open up new avenues for MIRI treatment [ 3 , 4 ].…”

Tongxinluo capsule as a multi-functional traditional Chinese medicine in treating cardiovascular disease: A review of components, pharmacological mechanisms, and clinical applications

“…The main active ingredients of traditional Chinese medicine borneol include borneol, isoproterenol, etc. A large number of studies have found that its active ingredients have anti-inflammatory, antioxidant, anti apoptotic, anticoagulant activity, improving energy metabolism, relaxing blood vessels, regulating blood pressure, improving intracellular lipid accumulation, sedation and pain relief, protecting myocardial tissue, and alleviating autophagy and apoptosis of myocardial cells [ [96] , [97] , [98] , [99] , [100] , [101] , [102] ].…”

Novel insights into the regulatory role of N6-methyladenosine methylation modified autophagy in sepsis

“…Then, we develop the discussion of positive regulation. ALKBH5-mediated reduction of m 6 A methylation indirectly inhibits the development of autophagy by regulating the expression of GSK3β/mTOR signaling pathway [ 166 ]. Earlier, study has observed that miR-199a impairs autophagy in thick heart muscle cells in a cell-autonomous way through targeted GSK3β/mTOR signal pathway [ 167 ].…”