Erianin mitigates diabetic cardiomyopathy via adenosine monophosphate-activated protein kinase-nuclear factor erythroid 2-related factor 2-heme oxygenase-1 pathway activation

Abstract

BACKGROUND

Erianin is a natural bibenzyl compound extracted from Dendrobium chrysotoxum and is known for its anti-inflammatory and antioxidant properties.

AIM

To explore the possible therapeutic mechanisms of erianin and determine if it can reduce cardiac damage in mice with type 2 diabetes.

METHODS

High-fat diet and intraperitoneal injections of streptozotocin were used to induce type 2 diabetes mellitus in C57BL/6 mice. Mice were divided into different groups including control, model, and treatment with various doses of erianin (10, 20, and 40 mg/kg) as well as ML-385 + erianin group.

RESULTS

Erianin reduced oxidative stress and inflammation and alleviated diabetic cardiomyopathy through the activation of the adenosine monophosphate-activated protein kinase (AMPK)-nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) pathway. Treatments with erianin-M and erianin-H promoted weight stabilization and normalized fasting glucose levels relative to diabetic controls. Echocardiographic assessment demonstrated that erianin dose-dependently enhanced left ventricular systolic function (left ventricular ejection fraction, left ventricular fractional shortening) and mitigated ventricular remodeling (left ventricular internal diameter at end-diastole, left ventricular internal diameter at end-systole; P < 0.05 vs model group). No significant differences were observed between the ML-385 + erianin and placebo-treated groups. Histopathological examination through hematoxylin-eosin staining indicated that erianin ameliorated myocardial fiber fragmentation, structural disorganization, inflammatory cell infiltration, and cytolytic damage. Furthermore, it significantly reduced the serum levels of cardiac troponin I, creatine kinase, and its MB isoenzyme. However, the ML-385 + erianin co-treatment failed to alleviate myocardial injury. Metabolic profiling revealed erianin-mediated improvements in glycemic regulation (glycated hemoglobin: P < 0.001), plasma insulin homeostasis, and lipid metabolism (total cholesterol, triglycerides, low-density lipoprotein cholesterol reduction, and high-density lipoprotein cholesterol restoration; P < 0.05 vs model group). Proinflammatory cytokines including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 were markedly suppressed in the erianin-M and erianin-H groups compared with the model group, whereas no significant differences were detected between the model and ML-385 + erianin groups. Oxidative stress parameters showed decreased malondialdehyde levels accompanied by elevated superoxide dismutase and catalase activities in erianin-treated groups, with the most pronounced effects in the erianin-H group (P < 0.05). Western blot analysis confirmed the significant upregulation of proteins associated with the AMPK/Nrf2/HO-1 pathway in erianin-M and erianin-H groups. These protective effects were abolished in the ML-385 + erianin co-treatment group, which showed no statistical differences from the model group.

CONCLUSION

Erianin can effectively alleviate myocardial injury in type 2 diabetic mice by activating the AMPK-Nrf2-HO-1 pathway.

Key Words: Erianin; Diabetic cardiomyopathy; Adenosine monophosphate-activated protein kinase pathway; Nuclear factor erythroid 2-related factor 2; Cardioprotection; Oxidative stress

Core Tip: This study investigates the cardioprotective effects of erianin, a natural compound from Dendrobium chrysotoxum, in a type 2 diabetic mouse model. Erianin administration significantly reduced myocardial damage and improved metabolic parameters by activating the adenosine monophosphate-activated protein kinase/nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway. These findings suggest that erianin may serve as a potential therapeutic agent to mitigate cardiac injury and inflammation in diabetes, offering new insights into managing diabetic cardiomyopathy.

INTRODUCTION

Diabetes is a leading cause of cardiovascular diseases worldwide, and diabetic cardiomyopathy (DCM) is a significant complication that has limited therapeutic options. More than 450 million individuals are affected by diabetes worldwide, and this number is projected to reach 693 million by 2045 according to the International Diabetes Federation[1]. This growing epidemic poses a serious threat to public health because it can cause extensive damages to multiple target organs, including the heart. In Framingham’s study, when other confounding factors are excluded, people with diabetes are two to five times more likely to develop heart failure than normal people[2]. More than 90% of all diabetes cases in China are type 2 diabetes mellitus (T2DM). Data from the Chinese Medical Association’s Diabetes Branch revealed that the incidence rates of complications in T2DM are as follows: Cardiovascular disease at 17.1%, cerebrovascular disease at 12.6%, hypertension at 34.2%, and lower limb vascular disease at 5.2%. DCM refers to a condition in which patients with diabetes experience impaired cardiac contraction and relaxation functions independently of coronary artery disease, hypertension, and other heart-related ailments. The development of DCM progresses through early-stage diastolic dysfunction and late-stage systolic impairment, and it culminates in the eventual onset of heart failure[3]. Despite nearly five decades of advancements in our understanding of DCM in modern medicine, effective treatment strategies remain elusive, with glycemic control still being the primary approach. Cardiovascular benefits vary widely among numerous medications used in clinical diabetes management[4]. Traditional Chinese medicine boasts a rich heritage in addressing diabetes and its associated complications. In recent years, research has identified several Chinese herbal medicines and their constituents with potential therapeutic effects on DCM[5-7].

Nuclear factor erythroid 2-related factor 2 (Nrf2), as a potent endogenous antioxidant factor, plays a major role in the defense of the body against the damaging effects of diabetes. Numerous studies have proven that Nrf2 can ameliorate diabetic myocardial injury. Chen et al[8] reported that diabetic people and animal models exhibited a considerable reduction in Nrf2 expression. The reduced expression of Nrf2 may exacerbate insulin resistance, abnormal angiogenesis, and endothelial dysfunction, leading to cardiac injury. Conversely, increasing Nrf2 expression can protect myocardial cells and the heart from the effects of a high-glucose environment[9]. Therapeutic strategies targeting Nrf2 can significantly enhance Nrf2 expression in the heart, increase myocardial heme oxygenase-1 (HO-1) expression, and alleviate myocardial hypertrophy and cardiac dysfunction[10]. In addition, the Nrf2 signaling pathway can potentially reverse diabetes progression by regulating glucose utilization and insulin secretion[11]. Moreover, Nrf2 is implicated in regulating the pathological processes of DCM, including inflammation, fibrosis, and cell apoptosis. The high glucose state in patients with diabetes can activate signaling pathways, such as nuclear factor kappa-B (NF-κB), resulting in pro-inflammatory cytokine release, myocardial hypertrophy, and fibrosis and promoting DCM development[12]. Nrf2 activation can reduce intracellular reactive oxygen species (ROS) production, thereby inhibiting pro-inflammatory signals. It can also counteract the inflammation process driven by NF-κB, exert an anti-inflammatory effect, and thus reduce myocardial damage in DCM.

Erianin, derived from Dendrobium orchid, is a lignan compound known for its diverse biological effects, including anti-inflammatory, antioxidant, and anti-tumor properties[13]. Dendrobium can decrease fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) levels, enhance insulin sensitivity, and improve insulin resistance in patients with T2DM[14-17]. Dendrobium not only has a therapeutic effect on diabetes but also shows considerable therapeutic potential for DCM. Iron-bark Dendrobium possesses potential to ameliorate DCM through mechanisms involving enhanced lipid transportation, insulin resistance inhibition, and epithelial-mesenchymal transition suppression[18]. Considering the protective effects of Dendrobium on T2DM and cardiovascular health, its main component, erianin, may serve as an adjuvant therapeutic agent for myocardial injury. This study presents an initial exploration of how erianin alleviates myocardial damage in T2DM mice and its potential mechanism.

MATERIALS AND METHODS

Animal model

Under license number SCXK (Beijing, China) 2019-0010, 51 male C57BL/6 mice with specific pathogen-free grade and a weight of 18-22 g were acquired from Sipeifu (Beijing, China) Biotechnology Co., Ltd. The mice were allowed to and had unrestricted access to food and water at 25 ± 2 °C for 1 week. All experimental procedures were approved by the Animal Ethics Committee of Jinzhou Medical University (No. 2023062) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Forty-three C57BL/6 mice were given a diet heavy in fat and sugar (MS1606, MKBio, China) containing 10% lard, 20% sucrose, 2.5% cholesterol, 1% cholic acid, and 66.5% basic diet for 3 weeks to induce insulin resistance and hyperglycemia. Plasma insulin levels were measured using an ELISA kit (ab277390, Abcam, United Kingdom) to confirm the presence of insulin resistance. Hyperlipidemia was assessed by measuring total cholesterol (TC) (RF8213, Ruifan, China) and triglyceride (TG) (RF8037, Ruifan, China) levels using commercial kits. After a 12-hour fast, the mice were injected intraperitoneally with 1% streptozotocin (STZ) (40 mg/kg body weight) (No. 20131211, Sigma-Aldrich, United States) solution for 3 consecutive days[19]. Following another 12-hour fast, FBG level of ≥ 11.1 mmol/L and appearance of symptoms such as polydipsia, polyphagia, and polyuria confirmed the success of the model. Three mice were selected randomly, and their heart tissues were sampled and observed by hematoxylin-eosin (HE) staining (No. C0105S, Beyotime, China). Our results confirmed that the mice had symptoms of myocardial injury. The successfully modeled mice were randomized into five groups: Three erianin intervention groups of eight mice each, receiving oral administration of erianin (10, 20, and 40 mg/kg, dissolved in corn oil; No. YT62752, Ita Biological Technology Co., Ltd., China) for 6 weeks. The ML-385 (a specific Nrf2 inhibitor) intervention group was given ML-385 (20 mg/kg; No. 846557-71-9, Abcam, the United Kingdom) and erianin (40 mg/kg) for 6 weeks. The model group received an equivalent volume of corn oil through oral administration for 6 weeks. The control group was normally fed and received intraperitoneal injection of an equivalent volume of citrate solution for 3 days, following the same dosing schedule as the erianin intervention groups. They were also orally administered with an equivalent volume of corn oil for 6 weeks. Following STZ injections, all the mice remained on high-fat diet until study termination to sustain diabetic metabolic derangements. Metabolic parameters including insulin resistance and lipid profile alterations were monitored longitudinally.

Animal groups are summarized in Supplementary Table 1. The research design flowchart for this study is shown in Supplementary Figure 1.

Body weight and blood glucose measurement and cardiac tissue collection

The mice were observed at different intervals, and blood glucose and body weight were recorded each week. The mice were then given 1% pentobarbital sodium intraperitoneally to induce anesthesia. Blood samples were obtained from the orbital sinus, and the serum was separated and stored at -80 °C. The heart was removed and stored in 4% paraformaldehyde at -80 °C.

Echocardiography

Cardiac function was assessed using the Vevo 2100 high-resolution ultrasound system (VisualSonics, Canada) under 2% isoflurane anesthesia. Left ventricular dimensions and functional parameters including left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal dimension in diastole (LVIDd), and left ventricular internal dimension in systole (LVIDs) were measured in parasternal long-axis views following established protocols.

Metabolic parameter measurements

HbA1c levels were quantified using a commercial kit (No. A056-2, Nanjing Jiancheng Bioengineering Institute, China). Plasma insulin concentrations were measured using an ELISA kit (ab 285341, Abcam, United States). TC (ab 65390, Abcam, United States) and TG (MAK266, Sigma-Aldrich, United States) levels were determined by enzymatic assays. Low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were analyzed using enzymatic assay kits (specific catalog numbers for LDL-C and HDL-C should be provided here if available). High-fat diet administration continued throughout STZ injection and subsequent interventions to maintain metabolic challenge.

Metabolic and biochemical analyses

Commercial kits (No. A032-1-1, No. H197-1-1, No. H149-2-2, No. H002-1-2, No. H052-1-2, No. H007-1-1, No. A003-1-2, No. A001-1-2, No. A007-1-1, Nanjing Jiancheng Bioengineering Institute, China) were acquired for analyses. The levels of creatine kinase (CK), CK isoenzymes (CK-MB), cardiac troponin I (cTnI), interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were determined as directed in the kits.

HE staining

The cardiac tissues were embedded in paraffin and treated in 4% paraformaldehyde to create 4 μm-thick slices. For light microscopy viewing and photography, the paraffin slices from each group were deparaffinized to water, stained with HE, dried, and cover slipped.

RNA isolation and quantitative reverse transcription-polymerase chain reaction

Cardiac tissues were subjected to RNA extraction using Thermo Fisher Scientific’s Trizol reagent (AM9738, United States). Reverse transcription was carried out using a reverse transcription-polymerase chain reaction test kit (RR014A and RR037A, Takara, Japan) and the β-actin gene as an internal control. The 2^ΔΔct technique was employed to calculate the relative levels of mRNA. Table 1 lists the primer sequences.

Table 1 Primer sequences used in qPCR analysis.

	Forward sequence	Reverse sequence
TNF-α	ACCACGCTCTTCTGTCTACT	AGGAGGTTGACTTTCTCCTG
IL-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT
IL-6	CACATGTTCTCTGGGAAATCGTGGA	TCTCTCTGAAGGACTCTGGCTTTGT
β-actin	CGTGGGCCGCCCTAGGCACCA	TTGGCCTTAGGGTTCAGGGGG

TNF-α: Tumor necrosis factor-α; IL: Interleukin.

Western blot analysis

The frozen cardiac tissues from each group were retrieved, minced, and added to tissue lysis buffer (P0013, Beyotime, China). After being thoroughly homogenized and centrifuged, the supernatant was collected as the total protein solution. Protein concentration was determined using the kit (042820200917, Beyotime, China). Subsequently, 5 × loading buffer was added and thoroughly mixed. The mixture was boiled to denature the proteins, which were electrophoretically separated and transferred onto a membrane. The primary antibody [1:1000, p-adenosine monophosphate-activated protein kinase (AMPK)-α ab92701, AMPKα ab32047, Nrf2 ab62352, HO-1 ab305290, NAD(P)H:quinone oxidoreductase 1 (NQO1) ab80588, Abcam, United States) and secondary antibody (1:10000, ab205718, Abcam, United States) were used for incubation. After washing the membrane, enhanced chemiluminescence solution was used for visualization.

Cardiac function assay

A priori power analysis (α = 0.05, power = 0.8) was conducted using GPower 3.1.9.7 based on preliminary data from cardiac function parameters (LVEF and LVFS). The calculated effect size (Cohen’s f = 0.35) indicated that a minimum sample size of six mice per group would be sufficient to detect significant differences. Our final sample size (n = 8 per group) exceeded the requirement to ensure adequate statistical power. The Shapiro-Wilk test confirmed the normal distribution of data, thereby justifying the use of parametric tests (one-way ANOVA with LSD post-hoc analysis). These methodological choices enhanced the validity of our conclusions by minimizing Types I and II errors.

Statistical analysis

Statistical analysis was conducted by GraphPad Prism 5, and results were expressed as mean ± SE. The Shapiro-Wilk test was used for continuous variable to confirm normal distribution. One-way analysis of variance was performed for comparisons among multiple groups, whereas least significant difference method was used for pairwise comparisons. Statistical significance was set at P < 0.05.

RESULTS

Effects of erianin on body weight and FBG in type 2 diabetic mice

The weight of mice in the model group increased dramatically following high-fat diet administration and then gradually decreased following STZ injection. The body weight of mice in the erianin group increased slowly compared with that in the model group (Figure 1A). The FBG level in the model group considerably increased compared with that in the control, whereas the FBG level in the erianin group significantly decreased compared with that in the model (Figure 1B). The findings indicate that erianin effectively mitigates the negative impact of a high-fat diet and STZ induction on body weight and FBG levels in type 2 diabetic mice. The erianin-treated groups exhibited a controlled rate of weight gain and significantly reduced FBG levels than the model group, which showed substantial increases. Hence, erianin might contribute to the regulation of glucose metabolism and weight management under diabetic conditions.

Figure 1 Effect of erianin on body weight and fasting blood glucose in type 2 diabetes mellitus mice. A: Body weight; B: Fasting blood glucose. Three independent biological repetitions were used. L: Low; M: Middle; H: High.

Effects of erianin on myocardial tissue in type 2 diabetic mice

The myocardial fibers of control mice were arranged neatly and uniformly, and infiltration of inflammatory cells or significant cellular degeneration or necrosis were not observed (Figure 2). In the model group, mouse heart muscle fibers were disorganized and irregularly arranged, with localized occurrences of inflammatory cell infiltration, cellular degeneration, and necrosis. Compared with the model, the erianin-L and erianin-H groups exhibited well-organized cell arrangement in mouse cardiac tissues and improved myoplasmic dissolution. In the erianin-H group, the myocardial tissue pathology improved. As illustrated in Figure 3, the erianin-treated groups demonstrated significant reductions in the levels of serum markers of myocardial damage compared with the model group (CK: 1214.0 ± 71.7 U/L; CK-MB: 937.7 ± 39.2 U/L; cTnI: 2.5 ± 0.2 mg/mL). Specifically, the erianin-H group exhibited marked decreases in CK (868.9 ± 80.6 U/L, P < 0.001), CK-MB (726.8 ± 39.2 U/L, P < 0.001), and cTnI (1.7 ± 0.2 mg/mL, P < 0.001). Erianin confers protective effects against myocardial damage in type 2 diabetic mice. The improvements in myocardial fiber arrangement and the reductions in serum markers of myocardial injury in the erianin-treated groups highlight its potential to ameliorate heart tissue pathology and reduce myocardial damage. Hence, erianin may play a beneficial role in maintaining cardiac structure and function under diabetic conditions.

Figure 2 Hematoxylin-eosin staining of myocardial tissue in type 2 diabetes mellitus mice (× 200). L: Low; M: Middle; H: High.

Figure 3 Indices of myocardial damage in type 2 diabetes mellitus mice. A: Creatine kinase (CK) (U/L); B: CK-isoenzymes (U/L); C: Cardiac troponin I (mg/mL). Bar charts represent the mean ± SEM, n = 8. ^cP < 0.001 vs control group; ^dP < 0.05 vs model group; ^eP < 0.01 vs model group; ^fP < 0.001 vs model group; CK: Creatine kinase; CK-MB: Creatine kinase-isoenzymes; cTnI: Cardiac troponin I.

Effects of erianin on cardiac function in type 2 diabetic mice

As shown in Figure 4, echocardiographic assessment revealed significant cardiac dysfunction in the model group compared with the controls (P < 0.001 for all parameters). Treatment with erianin dose-dependently improved left ventricular systolic function, particularly in the high-dose group where LVEF and LVFS approached normal levels (P < 0.001 vs model). Ventricular remodeling parameters such as LVIDd and LVIDs were significantly restored in the erianin-treated groups (P < 0.05 vs model).

Figure 4 Echocardiographic parameters in type 2 diabetic mice. A: Left ventricular ejection fraction (LVEF) (%); B: Left ventricular fractional shortening (LVFS) (%); C: Left ventricular internal dimension at diastole (LVIDd) (mm); D: Left ventricular internal dimension at systole (LVIDs) (mm). Bar charts represent the mean ± SEM, n = 8. ^cP < 0.001 vs control group; ^dP < 0.05 vs model group; ^eP < 0.01 vs model group; ^fP < 0.001 vs model group.

Effects of erianin on improving metabolic parameters in type 2 diabetic mice

Comprehensive metabolic profiling demonstrated that erianin intervention significantly ameliorated diabetes-associated dysregulation. Glycemic control improved progressively across treatment groups, with high-dose erianin achieving near-physiological HbA1c levels (P < 0.001 vs model; Figure 5A). Plasma insulin levels increased dose-dependently (P < 0.05), indicating enhanced β-cell function (Figure 5B). Lipid metabolism analysis revealed that erianin decreased the levels of TC, TG, and LDL-C (P < 0.05 vs model) and restored the level of HDL-C (P < 0.01; Figure 5C-F).

Figure 5 Metabolic parameters in type 2 diabetic mice. A: Glycated hemoglobin (%); B: Plasma insulin concentration (μIU/mL); C: Total cholesterol (mmol/L); D: Triglyceride (mmol/L); E: Low-density lipoprotein cholesterol (mmol/L); F: High-density lipoprotein cholesterol (mmol/L). Bar charts represent the mean ± SEM, n = 8. ^cP < 0.001 vs control group; ^dP < 0.05 vs model group; ^eP < 0.01 vs model group; ^fP < 0.001 vs model group; HbA1c: Glycated hemoglobin; TC: Total cholesterol; TG: Triglyceride; LDL-C: Low-density lipoprotein cholesterol; HDL-C: High-density lipoprotein cholesterol.

Effects of erianin on proinflammatory factors in type 2 diabetic mice

The serum and cardiac tissue mRNA levels of IL-1β, TNF-α, and IL-6 in the model group (Figure 6, respectively) were higher than those in the control (P < 0.05). However, their expression was significantly reduced in the erianin group (P < 0.05), and the effect was dose dependent. Our observations suggest that erianin significantly attenuates inflammatory responses in the myocardium of type 2 diabetic mice. The marked decrease in the serum and cardiac tissue levels of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 after erianin treatment underscores its anti-inflammatory potential. Hence, erianin may be integrated into therapeutic strategies aimed at reducing inflammation-associated cardiac damage in diabetes.

Figure 6 Comparison of proinflammatory factor expression in type 2 diabetes mellitus mice. A: Level of tumor necrosis factor-α (TNF-α) in serum; B: Level of interleukin (IL)-1β in serum; C: Level of IL-6 in serum; D: mRNA level of TNF-α in heart tissues; E: mRNA level of IL-1β in heart tissues; F: mRNA level of IL-6 in heart tissues. Bar charts represent the mean ± SEM, n = 8. ^bP < 0.01 vs control group; ^cP < 0.001 vs control group; ^dP < 0.05 vs model group; ^eP < 0.01 vs model group; ^fP < 0.001 vs model group; TNF-α: Tumor necrosis factor-α; IL: Interleukin.

Effects of erianin on oxidative stress in type 2 diabetic mice

The MDA levels and the activities of SOD and CAT in mouse cardiac tissues were measured using assay kits (Figure 7). Compared with the control group (MDA: 5.48 ± 1.26 nmol/mg; SOD: 12.54 ± 1.93 U/mg; CAT: 7.69 ± 1.26 U/mg), the model group (MDA: 9.11 ± 2.08 nmol/mg; SOD: 6.60 ± 1.34 U/mg; CAT: 4.58 ± 0.63 U/mg) exhibited a significant increase in MDA levels and a decrease in the activities of SOD and CAT (P < 0.05). The MDA levels and the activities of SOD and CAT in the erianin-L group (MDA: 7.64 ± 1.01 nmol/mg; SOD: 7.75 ± 1.90 U/mg; CAT: 5.31 ± 0.80 U/mg) had no statistically significant difference compared with the model group. In the erianin-M group (MDA: 7.14 ± 0.96 nmol/mg; SOD: 9.18 ± 2.16 U/mg; CAT: 6.31 ± 0.95 U/mg) and the erianin-H group (MDA: 5.26 ± 1.03 nmol/mg; SOD: 10.30 ± 1.51 U/mg; CAT: 7.37 ± 0.89 U/mg), the MDA levels decreased (P < 0.05) and the activities of SOD and CAT increased to some extent (P < 0.05). The erianin-H group showed the most pronounced changes (P < 0.001). Erianin shows promising antioxidative properties in the cardiac tissues of type 2 diabetic mice, as evidenced by the decrease in MDA levels and the increase in SOD and CAT activities, particularly in the higher dose groups. Hence, erianin effectively reduces oxidative stress, contributing to the preservation of myocardial integrity and function in diabetic environments.

Figure 7 Comparison of malondialdehyde contents and superoxide dismutase and catalase activities in mice with type 2 diabetes mellitus. A: Malondialdehyde; B: Superoxide dismutase; C: Catalase. Bar charts represent the mean ± SEM, n = 8. ^cP < 0.001 vs control group; ^dP < 0.05 vs model group; ^eP < 0.01 vs model group; ^fP < 0.001 vs model group; MDA: Malondialdehyde; SOD: Superoxide dismutase; CAT: Catalase.

Effects of erianin on the AMPK/Nrf2/HO-1 signaling pathway in myocardium of type 2 diabetic mice

The model group exhibited significantly lower protein levels of p-AMPKα, Nrf2, HO-1, and NQO1 than the control (P < 0.05; Figure 8). The protein levels of p-AMPKα, Nrf2, HO-1, and NQO1 in all intervention groups with erianin were significantly higher than those in the model, and this increase indicated a dose-dependent response to erianin (P < 0.05). As such, erianin has a notable effect on enhancing the AMPK/Nrf2/HO-1 signaling pathway in the myocardium, thereby inducing an improved adaptive response to oxidative and inflammatory stresses in type 2 diabetic mice. Hence, erianin might serve as a modulator of protective cellular pathways. The results offer a mechanistic insight into the cardioprotective function of erianin in diabetes.

Figure 8 Expression of the nuclear factor erythroid 2-related factor 2 signaling pathway in myocardial tissue of mice with type 2 diabetes mellitus. A: Western blot analysis of phosphorylated adenosine monophosphate-activated protein kinase (pAMPK), adenosine monophosphate-activated protein kinase, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H:quinone oxidoreductase 1 (NQO1); B: pAMPK protein levels; C: Nrf2 protein levels; D: HO-1 protein levels; E: NQO1 protein levels. Bar charts represent the mean ± SEM, n = 8. ^cP < 0.001 vs control group; ^dP < 0.05 vs model group; ^eP < 0.01 vs model group; ^fP < 0.001 vs model group; AMPK: Adenosine monophosphate-activated protein kinase; pAMPK: Phosphorylated adenosine monophosphate-activated protein kinase; Nrf2: Nuclear factor erythroid 2-related factor 2; HO-1: Heme oxygenase-1; NQO1: NAD(P)H:quinone oxidoreductase 1; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Nrf2 is involved in the treatment of erianin

Myocardial tissues from the model group, erianin group (40 mg/kg), and ML-385 (20 mg/kg) + erianin group (40 mg/kg) were stained by HE. Mice in the ML-385+erianin group had improved myocardial pathological damage compared with those in the model group; nevertheless, myocardial fiber disarray, irregularity, and inflammatory cell infiltration were not observed (Figure 9A). The serum markers of myocardial injury were significantly elevated in the ML-385 + erianin group compared with those in the erianin group (P < 0.05; Figure 9B). Proinflammatory cytokines TNF-α, IL-1β, and IL-6 had higher blood levels (Figure 9C) and myocardial tissue mRNA levels (Figure 9D) in the ML-385+erianin group than in the erianin group (P < 0.05). The ML-385 + erianin group showed significantly higher levels of MDA (P < 0.05) and lower SOD and CAT activities (P < 0.05) in the cardiac tissues than the erianin group (Figure 9E).

Figure 9 Inhibition of nuclear factor erythroid 2-related factor 2 reverses the effect of erianin on type 2 diabetes mellitus mice. A: Hematoxylin-eosin-stained myocardial section; B: Markers of myocardial damage; C: Serum proinflammatory factors; D: mRNA levels of proinflammatory factors; E: Oxidative stress. Bar charts represent the mean ± SEM, n = 8. ^aP < 0.05 vs control group; ^bP < 0.01 vs control group; ^cP < 0.001 vs control group; ^dP < 0.05 vs model group; ^eP < 0.01 vs model group; ^fP < 0.001 vs model group; CK: Creatine kinase; CK-MB: Creatine kinase-isoenzymes; cThI: Cardiac troponin I; TNF-α: Tumor necrosis factor-α; IL: Interleukin; MDA: Malondialdehyde; SOD: Superoxide dismutase; CAT: Catalase.

Nrf2 is involved in the erianin-mediated AMPK/Nrf2/HO-1 signaling pathway

Western blot detection revealed significantly higher levels of p-AMPK α, HO-1, and NQO1 in the erianin group than in the ML-385 + erianin group (Figure 10).

Figure 10  Inhibition of nuclear factor erythroid 2-related factor 2 reverses the effect of erianin on the expression of the nuclear factor erythroid 2-related factor 2 signaling pathway in myocardial tissue of mice with type 2 diabetes mellitus. A: Western blot analysis of phosphorylated adenosine monophosphate-activated protein kinase (pAMPK), adenosine monophosphate-activated protein kinase, heme oxygenase-1 (HO-1), and NAD(P)H:quinone oxidoreductase 1 (NQO1) in type 2 diabetes mellitus mice; B: pAMPK protein levels; C: HO-1 protein levels; D: NQO1 protein levels. Bar charts represent the mean ± SEM, n = 8. ^cP < 0.001 vs control group; ^fP < 0.001 vs model group; AMPK: Adenosine monophosphate-activated protein kinase; pAMPK: Phosphorylated adenosine monophosphate-activated protein kinase; Nrf2: Nuclear factor erythroid 2-related factor 2; HO-1: Heme oxygenase-1; NQO1: NAD(P)H:quinone oxidoreductase 1; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

DISCUSSION

Adverse outcomes in patients with diabetes are mainly associated with various complications caused by long-term disturbances in glucose and lipid metabolism and can lead to damage and dysfunction in multiple organs such as the heart, kidneys, and brain[20]. Diabetic myocardial injury is one of the deadliest complications for people with diabetes. Overall, studies involving type 2 diabetic mice indicated that erianin may mitigate the detrimental effects of T2DM. Animals treated with erianin demonstrated a consistent weight response, alleviated myocardial pathological damage, and reduced levels of FBG, myocardial damage markers, proinflammatory factors, and oxidative stress. These changes were probably linked to the increased expression of Nrf2, HO-1, NQO1, and p-AMPKα and the activation of Nrf2 regulatory mechanisms. Importantly, these findings highlight the potential of erianin as an adjuvant therapy for managing DCM. Further clinical validation is needed to translate preclinical benefits into human applications.

In the T2DM model, mice subjected to the high-fat diet exhibited a notable weight gain and a slight increase in blood glucose levels; on the basis of obesity in mice, low-dose STZ injection induced a late-stage T2DM model, characterized by symptoms such as weight loss and high blood sugar[21]. The present study demonstrated that erianin could alleviate the rapid decline in mouse body weight and reduce blood glucose levels. The results suggested that erianin exerted a regulatory effect on the body weight and blood glucose levels of mice. The improvement in metabolic parameters including HbA1c, lipid profiles, and insulin levels reflects the function of erianin in dual moderation of AMPK-mediated glucose uptake and lipid metabolism[21], thereby restoring cardiac energy homeostasis and function.

The functional decline of the heart in DCM is commonly associated with structural changes in the myocardium and is characterized by the accumulation of extracellular collagen and remodeling of the matrix. Excessive collagen formation and the progression of myocardial fibrosis contribute to increased myocardial stiffness and subsequently affect cardiac contractile function[22,23]. The serum levels of cardiac markers including CK, CK-MB, and cTnI are sensitive indicators of myocardial tissue damage. The elevated levels of CK, CK-MB, and cTnI in the body indicate myocardial tissue injury[24]. Pathological examination of cardiac tissue slices revealed varying degrees of deformation, myolysis, and disruptions in cardiac fibers in mice subjected to a prolonged high-fat diet[25]. A high-fat diet can induce a certain degree of myocardial fibrosis, and the degree of fibrosis increases in individuals with T2DM[26]. Erianin dose-dependently decreased the myocardial infarct area and the levels of serum indicators of myocardial damage (CK, CK-MB, and cTnI) in a rat ischemia-reperfusion injury model[27]. This finding is supported by the present echocardiographic data, which indicated that erianin improved left ventricular systolic function (LVEF, LVFS) and attenuated ventricular remodeling (LVIDd, LVIDs) possibly through AMPK/Nrf2/HO-1-mediated suppression of oxidative stress and fibrosis[25]. Erianin may have a protective impact on cardiac tissue because the activities of CK, CK-MB, and cTnI in mouse serum were lower in the erianin treatment groups than in the model. The HE staining results revealed that erianin improved fiber rupture caused by T2DM. Myocardial fibers exhibited organized alignment, devoid of edema and infiltration by inflammatory cells, with the tissue maintaining its structural integrity. Thus, erianin had a significant therapeutic effect on myocardial damage in T2DM.

Inflammation plays a crucial role in the progression of diabetes and the development of its complications. The high expression of inflammatory factors leads to the aggravation of the disease and the increase in the incidence of complications[28]. IL-1β is the main circulating form of IL-1 and, along with TNF-α, is a hallmark inflammatory factor associated with vascular inflammation. Individuals with diabetes have considerably higher expression levels of TNF-α, IL-1β, and IL-6 than their normal counterparts[29,30]. In diabetic animal models, alleviating inflammatory responses can lead to myocardial protection[31]. Erianin exhibits certain anti-inflammatory effects, namely, reducing NF-κB transcription activation[32] and increasing TNF-α expression[33]. The present study demonstrated that inflammatory factor levels were elevated in DCM mice, and erianin reduced the levels of IL-1β, IL-6, and TNF-α. The interplay between inflammation and oxidative stress is bidirectional, that is, inflammatory cytokines, such as TNF-α and IL-6, can exacerbate ROS production by impairing mitochondrial function[34], while oxidative stress amplifies NF-κB-driven inflammation[35]. The role of erianin in simultaneous suppression of both pathways likely disrupts this vicious cycle, as evidenced by reduced MDA levels (a lipid peroxidation marker) and restored SOD/CAT activity, which can mitigate myocardial cell apoptosis and fibrosis. Inflammation can damage endothelial cells, enhance autophagy, accelerate apoptosis, and promote fibrosis in myocardial cells. The long-term presence of chronic inflammation in the body stimulates metabolic disorders in cells, thereby exacerbating insulin resistance, forming a vicious cycle. Erianin possibly alleviates myocardial damage caused by inflammation by inhibiting autophagy, apoptosis, and metabolic disorders.

Oxidative stress is caused by the excessive production of ROS, and this phenomenon is one of the main mechanisms underlying the occurrence of DCM. Lipid peroxidation products and advanced glycation end-products accumulate as a result of imbalances in the metabolism of fat and glucose. This disturbance throws off the balance between the production and elimination of ROS, which then damages cardiac cells through oxidative stress[34]. In type 2 diabetic mice, the disruption of glucose metabolism leads to a large amount of ROS through processes, such as glucose auto-oxidation and metabolic stress. Moreover, the antioxidant enzyme activity in the body decreases, and the ability to clear free radicals significantly declines. These changes are evident in the markedly increased content of MDA and the significant decrease in the levels of SOD and CAT[35]. Chen et al[36] found that erianin may act as an antioxidant by inhibiting ROS/MAPK/NF-κB signaling, thereby reducing oxidative damage induced by high glucose in renal tubular epithelial cells. In the present experiment, the content of the lipid metabolism product MDA in the erianin group was significantly reduced. As such, erianin intervention helped improve pancreatic islet cell function and reduced oxidative stress in type 2 diabetic mice. The activity levels of SOD and CAT significantly increased, suggesting that erianin may exert its antioxidant stress effects by enhancing the activity levels of antioxidant enzymes. This antioxidant effect likely synergizes with anti-inflammatory actions because SOD and CAT not only neutralize ROS but also suppress NLRP3 inflammasome activation, thereby attenuating IL-1β and IL-18 production[35].

AMPK, a protein kinase activated by adenosine monophosphate, comprises a heterotrimeric complex composed of catalytic subunits α and β as well as regulatory subunit γ. The biological activity of AMPK is triggered when serine 72 on the α subunit is phosphorylated to form p-AMPKα, which regulates downstream signaling molecules. AMPK plays a key role in regulating glucose, lipid, and energy metabolism in the body. AMPK activation can suppress inflammatory responses and oxidative stress reactions[37]. Nrf2, a pivotal regulator of antioxidant factor transcription, plays a vital role in the Nrf2/HO-1 signaling pathway, which is essential for sustaining antioxidant response. Nrf2 can be activated by AMPK[38-40]. Previous studies indicated that erianin regulated Nrf2, NQO1, and HO-1 in human lung cancer A549 cells[41]. In the present experimental study, we observed that p-AMPKα, Nrf2, HO-1, and NQO1 were significantly reduced in the heart of mice in the model group. In T2DM mice, AMPK activation was inhibited, and the Nrf2/HO-1 signaling pathway was suppressed. After erianin intervention, the levels of p-AMPKα, Nrf2, HO-1, and NQO1 were significantly upregulated, which indicated that the AMPK/Nrf2/HO-1 pathway inhibited by T2DM was activated by erianin. This mechanistic interplay explains the observed improvements in metabolic parameters (e.g., HbA1c, lipid profiles) and echocardiographic indices (e.g., LVEF, LVIDd); AMPK activation enhances myocardial glucose uptake and mitochondrial biogenesis, while Nrf2/HO-1 signaling reduces oxidative injury[25,40].

Nrf2 is a crucial regulator of oxidative stress and a transcription factor that primarily exerts antioxidant and protective effects in various cells by transcriptionally regulating the expression of antioxidant stress proteins[42]. In the present experiment, Nrf2-inhibited mice treated with erianin showed no significant decrease in serum blood urea nitrogen and serum creatinine; HE staining revealed persistent swelling, congestion, and severe damage to kidney tissues. Hence, Nrf2 may be a crucial mechanism for erianin to exert its protective and therapeutic effects on myocardial injury. After Nrf2 inhibition in mice treated with erianin, the levels of p-AMPKα and AMPKα did not significantly increase, confirming the involvement of Nrf2 in the upregulation of p-AMPKα and AMPKα expression induced by erianin. Nevertheless, further comprehensive investigations are necessary to determine the precise mechanism by which Nrf2 controls p-AMPKα and AMPKα.

This study provides robust evidence for the cardioprotective effects of erianin via the AMPK/Nrf2/HO-1 pathway, but it is limited by its focus on a single signaling axis. Other pathways implicated in DCM, such as NF-κB, MAPK, and PI3K/Akt, were not explored, leaving potential cross-talk mechanisms unaddressed. Future studies should employ multi-omics approaches to map the broad regulatory snetwork of erianin. In clinical settings, the findings position erianin as a promising candidate for adjunctive therapy in diabetic patients with early-stage cardiac dysfunction. The dual anti-inflammatory and antioxidant properties of erianin as well as its metabolic benefits could complement existing glucose-lowering regimens. However, rigorous pharmacokinetic and safety studies in larger animal models (e.g., diabetic primates) and human trials are imperative to validate its translational potential.

CONCLUSION

Erianin has the potential to mitigate the effects of oxidative stress and inflammation on cardiac tissues, which might lead to a reduction in myocardial injury in mice with T2DM. The underlying mechanism could be connected to the activation of the AMPK/Nrf2/HO-1 signaling pathway. This work offers a novel approach to the therapeutic prevention and management of cardiac damage in T2DM.