acute myocardial infarction (acute myocardial infarction, AMI) is characterized by myocardial ischemia and hypoxia. Its incidence and mortality rate are high, which seriously endangers human health [1]. There are many factors that induce myocardial ischemia injury, including oxidative stress , calcium overload, inflammatory response, apoptosis, autophagy and necrosis, among which autophagy plays an important role in the occurrence and development of myocardial ischemia. Autophagy is an important process for the degradation and recycling of longevity proteins and cytoplasmic organelles. Inhibition of autophagy during ischemia and hypoxia of cardiomyocytes is conducive to the survival of cardiomyocytes and alleviates myocardial damage.
Kowloon Garcinia Garcinia (Bauhinia Championii flavones, BCF) is the main active ingredient of Guangxi national specialty herbal medicine Kowloon Garcinia Bauhinia championii (Benth.) Benth. Previous studies of this research group showed that BCF has the effects of anti-inflammatory and antioxidant, and can activate phosphatidylinositol-3-kinase (PI3K)/protein kinase B (protein kinase). B, Akt) signaling pathway, inhibits cell apoptosis and excessive autophagy, and anti-myoxia damage [4-6]; cell experiments found that upregulating miR-384-5p can enhance the effect of BCF inhibiting autophagy [4], but at the overall animal level, the effect of miR-384-5p on BCF is still unclear.
study shows that long non-coding RNA (LncRNA) can exert the role of microRNA (microRNA, miRNA) sponge, compete to bind miRNA reaction elements, and then negatively regulate its target genes [7]. LncRNA Myocardial infarction-related transcript (MIAT) is involved in various diseases such as myocardial infarction and atherosclerosis [8]. For example, combining miR-150 partially enhances myocardial hypertrophy, combined with miR-26a anti-atherosclerosis [9-10], but LncRNA The molecular mechanism of MIAT involved in myocardial infarction remains to be studied in depth. LncRNA MIAT can be combined with miR-384-5p to affect autophagy, and there are no relevant reports on myocardial ischemia injury, and its myocardial protective effect on BCF has not been clarified. This study explores the role and mechanism of BCF on myocardial hypoxia injury based on the LncRNA MIAT/miR-384-5p signal axis, providing experimental basis for clinical treatment of myocardial ischemia injury.
Results
BCF downregulates LncRNA MIAT and upregulates miR-384-5p gene expression
As shown in Figure 1, compared with the control group, LncRNA in OGD cells was compared with the control group. The expression of MIAT gene was significantly upregulated (P <0.01),>miR-384-5p gene was significantly downregulated (P <0.01);>LncRNA in cells in BCF group The expression of MIAT gene was significantly downregulated (P<0.01),>miR-384-5p gene was significantly upregulated (P<0.05).>
LncRNA MIAT targeted regulation of miR-384-5p
dual luciferase reporter gene experiment results showed (Figure 2), and miRNA-384-5p and LncRNA were transferred together. The luciferase activity was significantly reduced after MIAT-WT (P <0.001),>miRNA-384-5p interacted with LncRNA MIAT. RAP experiment further verified the relationship between the two (Figure 2-C). miRNA-384-5p was enriched by the LncRNA MIAT probe, indicating that LncRNA MIATLncRNA MIAT and miR-384-5p.As shown in Figure 2-D and E, compared with the sh-NC group, the expression of LncRNA MIATht gene was reduced after knocking down LncRNA MIAT (P <0.001),>miRNA-384-5p6 gene was increased (P <0.05).>miR-384-5p is the target gene of LncRNA MIAT and is negatively regulated by LncRNA MIAT.
knockdown LncRNA MIAT inhibits autophagy and relieves myocardial hypoxia damage
As shown in Figures 3-A and B, compared with the control group, the cell survival rate of OGD group was significantly reduced (P <0.001),>P <0.001);>LncRNA MIAT can improve the survival rate of H9c2 cells (P <0.01),>P <0.01),>LncRNA MIAT can alleviate OGD-induced myocardial hypoxia damage.
Further verification of LncRNA The effect of MIAT on the autophagy of H9c2 cells induced by OGD is shown in Figure 3-Ch~E. Compared with the control group, the number of autophagosomes and autophagolysosomes in the OGD group increased (P <0.01),>P <0.001),>P <0.001);>LncRNA After MIAT, the number of autophagosomes and autophagolysosomes decreased (P <0.01),>P <0.01,>P <0.001),>LncRNA MIAT can inhibit OGD-induced autophagy. Effects of
BCF on the survival of H9c2 cells induced by OGD and cTn-I levels in the supernatant
As shown in Figure 4, compared with the OGD group, the cell survival rate of BCF group was significantly increased (P <>P <>LncRNA MIAT can enhance the effect of BCF (P <>
BCF on the autophagosomes of OGD-induced H9c2 cells
As shown in Figure 5, compared with the OGD group, the number of autophagosomes in the BCF group was significantly reduced (P <0.01);>LncRNA MIAT can enhance the effect of BCF (P <0.01).>
BCF on the expression of autophagy-related genes and protein protein
as shown in Figure 6. Compared with the OGD group, the expression levels of Beclin1, Cathepsin D and LC3 LC3666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666666 The expression level of D protein was significantly reduced (P <>P <>LncRNA MIATh can enhance the inhibitory effect of BCF on OGD-induced autophagy of H9c2 cells (P <>
BCF on the autophagy flow of H9c2 cells induced by OGD
is shown in Figure 7. Compared with the OGD group, the number of autophagosomes and autophagolysosomes in the BCF group cells was significantly reduced (P <0.01);>LncRNA MIAT further decreased (P <0.05),>LncRNA MIAT inhibits the formation of autophagosomes and autophagolysosomes, thereby blocking autophagy flow.
knockdown LncRNA Effect of MIAT on miR-384-5p gene expression in rat heart
As shown in Figure 8, compared with the control group, the expression of miR-384-5p p p gene in rat hearts was significantly downregulated (P <0.001);>LncRNA MIAT significantly increased the expression of miR-384-5p gene in the heart (P <0.05).>
Silencing miR-384-5p gene expression reverses BCF anti-myocardial ischemia injury effect
As shown in Figure 9, compared with the AMI+BCF group, BCF was given after silencing miR-384-5p, the myocardial infarction area increased and the infarction rate was significant in rats. (P<0.01);>P<0.01),>P<0.01),>
Discussion
AMI is a serious fatal disease, and myocardial ischemia injury caused by coronary artery occlusion is one of the causes of death in patients [13]. At present, the main method for clinical treatment of AMI is percutaneous coronary intervention (PCI). PCI can restore blood flow reperfusion faster and safely, but the onset blood vessels still have the possibility of acute reocclusion [14]. Some blood circulation and stasis removal Chinese medicines have the effect of preventing and treating cardiovascular diseases. In-depth research on its mechanism of action is expected to provide a basis for the development of drugs to prevent and treat AMI.
autophagy in AMI has been a hot topic in recent years [2]. Autophagy is a dynamic process, including three consecutive steps: autophagosome formation (usually marked by Beclin1 and LC3II), autophagosome fusion (usually marked by Cathepsin D), and autophagosome degradation (usually marked by p62). Under normal circumstances, cells rarely experience autophagy, while endoplasmic reticulum stress, ischemia and hypoxia often induce autophagy [3]. Kowloon Teng is a traditional Chinese herbal medicine and functional food, with low toxicity, and BCF is the main active ingredient of its stem extract. Research has found that BCF can antioxidant, inhibit cell apoptosis and autophagy, and alleviate myocardial ischemia and hypoxia damage [5-6], but its specific molecular mechanism is still unclear. Previous studies of this research group showed that miR-384-5p negatively regulates Beclin1 expression to inhibit autophagy, and overexpression of miR-384-5p can enhance the myocardial protective effect of BCF [4]. This study also found that after BCF pretreatment by ODG-induced H9c2 cells, the expression of miR-384-5p gene was upregulated, suggesting that miR-384-5p may be involved in the inhibition of autophagy by BCF.
used dual luciferase reporter gene experiment and RAP experiment to analyze the upstream target of miR-384-5p. The results showed that LncRNA MIAT negatively targeted regulation miR-384-5p. As a risk allele of AMI, LncRNA MIAT highly expressed [15-16] in plasma of AMI patients and mice, but the role of LncRNA MIAT in OGD-induced H9c2 cells has not been reported. This study found that the expression of LncRNA MIAT in OGD-induced H9c2 cells was upregulated. Knocking down the LncRNA MIAT can improve cardiomyocyte viability, reduce cTn-I levels, reduce the number of autophagosomes and autophagolysosomes, reduce Beclin1 protein expression and LC3-II/LC3-I value, increase p62 protein expression, and inhibit myocardial hypoxia damage.In order to further verify whether BCF plays a myocardial protective role by affecting LncRNA MIAT, BCF pretreatment was given in the steady-transplant of cardiomyocytes knocking down the LncRNA MIAT gene. The results showed that compared with BCF pretreatment alone, the effect of BCF inhibiting autophagy and alleviating myocardial hypoxia damage was significantly enhanced, indicating that LncRNA MIAT may be an important target for BCF to play a myocardial protective role.
To further verify the effect of miR-384-5p on BCF, an AMI animal model was constructed, and the expression of miR-384-5p gene was silenced by in vivo transfection. The results showed that silencing miR-384-5p reversed the effect of BCF inhibiting autophagy and aggravated myocardial ischemia injury in rats, suggesting that the anti-myocardial ischemia injury effect may be related to upregulating miR-384-5p gene expression.
In summary, this study found that BCF inhibits autophagy and alleviates myocardial hypoxia damage through LncRNA MIAT/miR-384-5p3 (Figure 10). Since the full-length sequence of LncRNA MIAT is 8474 bp, and the sequence is too long, and it is very likely to cause cell death after overexpression, this study did not analyze the overexpression of LncRNA MIAT. In the future, BCF will be further verified in vivo that the effect of BCF downregulating LncRNA MIAT inhibits autophagy and alleviates myocardial ischemia injury.
Conflict of Interest All authors declare that there is no conflict of Interest
References (omitted)
Source: Su Wenxiao, Lu Jun, Gan Xiaowen, Chen Huibin, Jian Jie. Research on the mechanism of action of Kowloon Garcinoside in inhibiting autophagy against myocardial hypoxia damage based on LncRNA MIAT/miR-384-5p [J]. Chinese Herbal Medicine, 2022, 53(18): 5701-5711.
