Traditional Chinese Medicine Ginseng Dingzhi Decoction Ameliorates Myocardial Fibrosis and High Glucose-Induced Cardiomyocyte Injury by Regulating Intestinal Flora and Mitochondrial Dysfunction

Abstract

Myocardial fibrosis refers to the pathological changes of heart structure and morphology caused by various reasons of myocardial damage. It has become an important challenge in the later clinical treatment of acute myocardial infarction/ischemic cardiomyopathy or diabetes complicated with heart failure. Ginseng Dingzhi Decoction (GN), a Chinese herbal medicine, can reduce heart failure and protect cardiomyocytes. We infer that this may be related to the interaction with intestinal microbiota and mitochondrial homeostasis. The regulatory mechanism of GN on gut microbiota and mitochondria has not yet been elucidated. The intestinal microbiota was analyzed by the 16S rRNA gene; the fecal samples were sequenced and statistically analyzed to determine the changes of microbiota in the phenotype of heart failure rats. In addition, GN can regulate the microbial population that increases the proportion of short-chain fatty acids and anti-inflammatory bacteria and reduces the proportion of conditional pathogens to diabetic phenotype. The results suggest that GN may improve myocardial injury by regulating intestinal flora. Our data also show that stress-type heart failure caused by TAC (transverse aortic constriction) is accompanied by severe cardiac hypertrophy, reduced cardiac function, redox imbalance, and mitochondrial dysfunction. However, the use of GN intervention can significantly reduce heart failure and myocardial hypertrophy, improve heart function and improve myocardial damage, and maintain the mitochondrial homeostasis and redox of myocardial cells under high glucose stimulation. Interestingly, through in vitro experiments after TMBIM6 siRNA treatment, the improvement effect of GN on cell damage and the regulation of mitochondrial homeostasis were eliminated. TMBIM6 can indirectly regulate mitophagy and mitochondrial homeostasis to attenuate myocardial damage and confirms the regulatory effect of GN on mitophagy and mitochondrial homeostasis. We further intervened cardiomyocytes in high glucose through metformin (MET) and GN combination therapy. Research data show that MET and GN combination therapy can improve the level of mitophagy and protect cardiomyocytes. Our findings provide novel mechanistic insights for the treatment of diabetes combined with myocardial injury (myocardial fibrosis) and provide a pharmacological basis for the study of the combination of Chinese medicine and conventional diabetes treatment drugs.

Figure 1

GN improves cardiac function and reduces myocardial fibrosis after TAC: (a) echocardiographic detection; (b) EF(%), FS(%), LVESV(μl), LVEDV(μl), E/A ratio, LVD-D(mm), LVD-S(mm), and myocardial fibrosis area (%); (c) hematoxylin-eosin staining; (d) Masson staining; (e) Sirius red staining. ∗p < 0.05.

Figure 2

GN can inhibit the synthesis of collagenase in damaged myocardium and improve myocardial hypertrophy. (a, b) The level of collagenase synthesis was detected by immunohistochemistry. (c, d) TGF-β 1 and TGF-α transcriptional level was detected by Q-PCR. (e, f) The hypertrophic and structure of cardiomyocytes were detected by WGA immunofluorescence. ∗p < 0.05.

Figure 3

GN inhibits NLRP3-mediated inflammatory response and improves cardiomyocyte death of mitochondrial apoptotic pathway: (a, b) immunofluorescence detection of NLRP3 in myocardial tissue, (c, g) TUNEL fluorescence detection of myocardial tissue, and (d–f) transcriptional level of caspase-3/-9/-12 in myocardial tissue. ∗p < 0.05.

Figure 4

GN changed the father of heart failure mice and changed the composition of intestinal microbiota community. (a–h) Differences in the abundance of specific intestinal microbial KO produced by TMAO. ∗p < 0.05.

Figure 5

GN changed the father of heart failure mice and changed the composition of intestinal microbiota community. (a–e) GN-induced functional changes of intestinal microbiota in mice with heart failure. ∗p < 0.05.

Figure 6

GN changed the father of heart failure mice and changed the composition of intestinal microbiota community. (a–e) Differential enrichment of specific gut microbial KOs for SCFAs production among four groups. ∗p < 0.05.

Figure 7

TMBIM6 contributes to GN-induced protection against cardiomyocyte injury through regulation of redox balance. (a) The morphology of mitochondria in myocardial tissue was detected by transmission electron microscope. (b) NLRP3 fluorescence intensity of cardiomyocytes was detected by immunofluorescence. (c, d) The activity of cardiomyocytes was detected by MTT assay. (e) The level of apoptosis was detected by flow cytometry. (f) The level of ROS production was detected by flow cytometry. ∗p < 0.05.

Figure 8

TMBIM6 contributes to GN-induced protection against cardiomyocyte injury through regulation of mitochondrial homeostasis: (a, b) ROS fluorescence intensity of cardiomyocytes was detected by immunofluorescence; (c) detection of antioxidant enzyme SOD activity; (d) detection of oxidative stress marker malondialdehyde (MDA); (e–g) detection of mitochondrial energy metabolism. ∗p < 0.05.

Figure 9

TMBIM6 contributes to GN-induced protection against cardiomyocyte injury through mitophagy and mitochondrial dynamics: (a) protein expression level of PINK/Parkin/Drp1/Mff/Fis1 and Bax was detected by western blot; (b) protein expression level of PINK; (c) protein expression level of Parkin; (d) protein expression level of Drp1; (e) protein expression level of Mff; (f) protein expression level of Fis1; (g) protein expression level of Bax; ∗p < 0.05.

Figure 10

TMBIM6 contributes to GN-induced protection against cardiomyocyte injury through mitophagy and mitochondrial biogenesis: (a) transcription level of PINK; (b) transcription level of Parkin; (c) transcription level of ATG5; (d) transcription level of ATG12; (e) transcription level of PGC1α; (f) NLRP3 fluorescence intensity of cardiomyocytes was detected by immunofluorescence. ∗p < 0.05.

Figure 11

GN can enhance the regulatory effect of metformin on high glucose-induced cardiomyocyte injury and mitochondrial homeostasis/mitophagy dysfunction: (a) CCK8 was used to detect cell activity; (b) MTT was used to detect cell activity; (c) transcription level of PINK; (d) immunofluorescence detection of ROS; (e) transcription level of Parkin; (f) transcription level of ATG5; (g) transcription level of ATG12. ∗p < 0.05.

Figure 12

In the state of heart failure and stress-stimulated cardiomyocyte injury after TAC, NLRP3 can be activated and further affect mitochondrial homeostasis and inhibition of mitophagy, increase mitochondrial oxidative stress, and reduce mitochondrial energy metabolism; it is accompanied by the disorder of intestinal flora and finally leads to myocardial fibrosis/cardiomyocyte hypertrophy and aggravation of cardiomyocyte injury. GN can lead to improve cardiac function, regulate the distribution and abundance of intestinal flora, maintain mitochondrial homeostasis, improve mitochondrial function, improve mitophagy, inhibit oxidative stress, and improve mitochondrial energy metabolism.