Geniposide effect on cardiac zinc level, oxidative stress, inflammation, and apoptosis in rats with obesity-linked heart injury

Introduction

Obesity rate has been dramatically increased throughout the world during the last 50 years. Recent reports show that nearly 30 % of the global population is either obese or overweight [1], 2]. It is globally well-elucidated that obesity significantly enhances the risk of various diseases or chronic conditions such as diabetes mellitus (DM), hypertension, nonalcoholic fatty liver disease (NAFLD), hyperlipidemia, and certain types of cancer that are correlated to mortality [3], 4]. Obesity is also highly associated with atherosclerosis and cardiovascular disease (CVD), as the main cause of death [5]. Previous studies showed that obesity contributes directly to a spectrum of cardiovascular changes ranging from cardiac diastolic dysfunction to atrial/ventricular dysfunction and heart failure [6]. It is proposed that obesity may increase the risk of CVD through several mechanisms such as hormonal changes, increase in oxidative stress, pro-inflammatory mediators and cardiomyocytes loss (apoptosis) [5]. Thus, inhibiting the obesity-induced excessive oxidative stress, apoptosis and inflammatory mediators may be an effective target for preventing cardiac injury.

Since inflammation and oxidative stress are proposed linked to cardiomyocyte apoptosis, pretreatment with antioxidants may be helpful in ameliorating/preventing obesity-induced cardiac injury. Geniposide (GNP) is a naturally occurring iridoid glycoside that features a cyclopentanoid monoterpene skeleton bonded to a glucose unit. It is predominantly extracted from the fruit of Gardenia jasminoides Ellis, a medicinal plant commonly used in traditional East Asian herbal practices. This compound exhibits a variety of biological activities, including anti-inflammatory, antioxidant, and liver-protective effects [7]. GNP exerts its protective effect via GLP-1R/AMP-activated protein kinase α (AMPKα) pathway [8]. Previous studies reported its high anti-inflammatory and antihyperlipidemia properties [9], 10]. Many studies reported that GNP protects against a spectrum of cardiovascular changes such as cardiac remodeling [11], myocardial ischemia [12], myocardial ferroptosis [13] and myocardial dysfunction [8] by suppressing the oxidative stress and inflammation, indicating GNP can effectively prevent free radical-directed lipid peroxidation in heart tissue. For example, Ma et al. [14] found that GNP improved cardiac function in the obese mice by attenuating the myocardial inflammation, oxidative stress and apoptosis. However, the potential effects of GNP on oxidative stress, inflammation and cardiomyocyte apoptosis in obesity-induced cardiac injury are still unknown. Therefore, we aimed to consider the protective role of GNP against obesity-induced inflammation, oxidative stress and apoptosis in cardiac tissue of Wistar rats.

Materials and methods

Animals and study design

Eighteen 4-week-old male Wistar rats with body weight of 75±8 g were assigned into the study. From 4 to 21 week of age, 12 rats received a high fat diet (HFD) with 40 % kilocalories (∼15 % proteins, ∼40 % carbohydrate, and ∼40 % fat) to induce obesity and cardiac dysfunction [15]. Rats with HFD were subsequently subdivided into two experimental groups, including HFD alone (n=6) and HFD+GNP (n=6). From the last 3 weeks, rats in HFD+GNP group were administered orally with GNP (50 mg/kg daily). The control group (n=6) received ∼10 % kilocalories (∼15 % protein, ∼70 % carbohydrate, and ∼10 % fat) during the same period. Figure 1 displays the study design. All rats were kept under a 12-h:12-h light-dark cycle, with a temperature of 22±2 °C, 55±5 % humidity and free access to the relevant diet and water). Geniposide (Sichuan Weikeqi Biological Technology Co., Ltd.) concentration was selected based on a recent study [16].

Figure 1:

Flow chart showing the experimental design of the studies (left side) and histopathological examination of heart tissue in (A) control, (B) HFD and (C) HFD+GNP groups (right side). Cardiac tissue sections of normal group exhibited normal structure, while HFD group exhibited more injuries, including inflammatory cell infiltration, cardiomyocyte vacuolization, and myocardial tissue separation. Arrows indicate areas of inflammatory cell infiltration. GNP administration attenuated cardiomyocyte vacuolization and infiltration of mononuclear cells. X20 magnification.

Results

Histopathological analysis

Histological evaluation of cardiac tissues from all experimental groups is presented in Figure 2. Heart tissue sections from the normal group exhibited preserved architecture with no observable pathological alterations (Figure 2A). Compared to the control group, HFD rats showed more injuries, including inflammatory cell infiltration, cardiomyocyte vacuolization, and myocardial tissue separation (Figure 2B). Treatment with GNP in HFD rats attenuated the histopathological changes in heart tissue such as cardiomyocyte vacuolization and decreased infiltration of mononuclear cells (Figure 2C).

Figure 2:

TUNEL assay of cardiac cells in different animals. Treatment with GNP significantly decreased the number of apoptotic cells in the cardiac tissue of HFD rats. (A) Control, (B) HFD, (C) HFD+GNP groups. Arrows indicate TUNEL-positive apoptotic cells. (D) Percentage of cardiac apoptotic cells in different groups. *p<0.001; ***p<0.05 compared to control.

Results of transcriptomic analysis

The expression of study genes in the cardiac tissues of all groups is summarized in Table 4 and Figure 3. HFD group exhibited significantly higher level of Bax (3.56-fold), Caspase-3 (4.27-fold), TNF-α (3.68-fold), Caspase-9 (3.27-fold), and lower expression of Bcl2 (2.41-fold), IL-10 (2.63-fold), SOD (2.52-fold) and CAT (2.35-fold) compared to the normal group (p<0.001). However, GNP administration significantly downregulated Bax (1.97-fold), Caspase-3 (1.81-fold), Caspase-9 (1.78-fold), TNF-α (2.12-fold), and upregulated Bcl2 (2.07-fold), IL-10 (2.0-fold), SOD (1.95-fold), CAT (1.63-fold) compared to rats treated with HFD alone (Figure 3).

Table 4:

Fold change ratio of the genes expression in each group.

	Bax	Bcl2	Casp3	Casp9	IL-10	TNF-α	SOD	CAT
Control vs. HFD	−3.56	+2.41	−4.27	−3.27	+2.63	−3.68	+2.52	+2.35
Control vs. HFD+GNP	−1.84	+1.16	−2.36	−1.84	+1.26	−1.75	+1.29	+1.44
HFD+GNP vs. HFD	−1.97	+2.07	−1.81	−1.78	+2.00	−2.12	+1.95	+1.63

1. GNP, geniposide; HFD, high fat diet; ⁽⁺⁾means up-regulation; ⁽⁻⁾means down-regulation.

Figure 3:

Comparison of the normalized expression of different genes between control, patient and geniposide treated groups. GNP, geniposide; HFD, high fat diet; ^*p<0.001; ^**p<0.01; ^***p<0.05 compared to control group.

Discussion

Recent studies have demonstrated that inflammatory reactions and oxidative stress that linked to cells apoptosis are main mechanisms of obesity-induced cardiac injury [5]. A growing number of animal studies reported that GNP supplementation attenuates cellular apoptosis through suppressing of oxidative stress and inflammatory reactions, and compensating of antioxidant pools in different organs [20], [21], [22]. For this reason, we designed this study to consider the protective role of GNP administration on obesity-induced cardiac injury. The findings of our study revealed that HFD not only linked to overweight and obesity, but also it caused morphological and structural changes in cardiac tissue. We also observed hyperlipidemia and increased CRR and AC values in HFD group, indicating elevated risk of CVD and cardiac dysfunction in obese animals. Previous studies reported a wide range of structural/morphological abnormalities such as myocardial structure disorganization, cardiac hypertrophy, myocardial tissue separation, inflammatory cell infiltration, and cardiomyocyte vacuolization in HFD animals [17], 23], 24]. These data highlight that obesity is a serious risk factor for cardiac injury and CVD.

The present study showed that HFD was strongly associated with depletion of zinc and TAC contents, and downregulation of SOD and CAT enzymes in cardiac tissue, whereas MDA level was elevated. Similarly, Feriani et al. [17] found that HFD significantly reduced cardiac contents of glutathione, SOD, CAT and glutathione peroxidase (GPX), while levels of MDA, proteins carbonyl and reactive oxygen species (ROS) were remarkably increased. These findings mean that HFD declines cardiac antioxidant pools and disturbs cellular redox capacity that promotes cardiac cells susceptibility to free radicals and, consequently, causes cardiac cells apoptosis and injury. To support this hypothesis, we found higher rate of cardiac cells apoptosis, as well as overexpression of apoptosis-related genes (Bax, Caspsaes-3, -9) in heart tissue of HFD rats. Besides, HFD was significantly linked to TNF-α upregulation in cardiac tissue, but IL-10 was significantly downregulated. A recent study reported upregulation of apoptosis proteins (Bax and Caspase-3) and inflammatory cytokines (IL-6 and TNF-α) in heart tissues of HFD rats [17]. These data explain the role of inflammatory mediators and oxidative stress as potential mechanism of obesity-induced cardiac cells apoptosis and injury.

Our findings might make a wise basis for antioxidants therapy that could protect heart cells against obesity-induced cardiac injury. Here, we examined the therapeutic effect of GNP administration on morphological/structural changes, hyperlipidemia, oxidative stress, depletion of antioxidants, and apoptosis of cardiac cells in HFD rats. Our results showed that GNP pretreatment significantly reversed the adverse effects of HFD on heart tissues. These improvements were associated with a significant enhancement in zinc and TAC values and upregulation of SOD and CAT. Also, GNP therapy significantly diminished MDA level in cardiac tissue. Interestingly, GNP administration reduced TNFα expression, but increased IL-10 expression in cardiac tissue. It also balanced the expression level of apoptosis-related genes, Bax, Bcl2, Caspase-3 and Caspase-9 in the heart tissue of HFD rats. Although expression levels of apoptosis and oxidative stress biomarkers in the cardiac tissue of rats fed with GNP+HFD were still somewhat high, GNP supplementation significantly balanced the level of these mediators when compared to animals received HFD alone. These results support the idea that pretreatment with GNP mitigates oxidative stress, inflammation and apoptosis of cardiac cells in obese rats. As support to these results, some studies reported that pretreatment with GNP protects different organs or tissues through suppressing of free radicals’ production, inflammatory reactions, oxidative stress and apoptosis. Ma et al. [14] indicated that GNP administration exerted a protective effect against obesity-induced cardiac injury by ameliorating myocardial inflammation and myocyte apoptosis. A recent study has reported that GNP treatment not only enhanced SOD and GPX activities, but also it ameliorated levels of IL-6, IL-1β, and TNF-α, as well as decreased apoptosis of hepatocytes and downregulated expression of Bax, Caspase-3 and -9 in mouse model with liver fibrosis [16]. Fang et al. [25] demonstrated that GNP supplementation improved skin wound healing through inhibiting pro-inflammatory mediators such as TNF-α, IL-1β and IL-6, and cellular apoptosis in diabetic rats. Therefore, we propose that depletion of cellular antioxidants and subsequent enhancement of inflammatory cytokines, oxidative stress, and dysregulation of apoptotic mediators are the major underlying mechanisms by which obesity triggers cardiac injury. GNP treatment reversed all of these adverse effects of obesity-related cardiac injury. Our findings support the idea that the adverse effects of HFD on cardiac tissue are mediated through the depletion of cardiac antioxidant pools, oxidative stress, and apoptosis of heart cells. Therefore, antioxidant therapy with GNP can be valuable to alleviate the detrimental effects of obesity on the cardiac tissues.

Therefore, our findings indicate that GNP exerts significant protective effects against obesity-induced cardiac injury through modulation of several key molecular pathways. The observed increase in antioxidant enzyme expression, including CAT and SOD, along with a reduction in MDA levels, suggests that GNP enhances the cardiac antioxidant defense system and reduces lipid peroxidation. These effects are likely mediated through activation of the nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway, which has been previously reported as a primary mechanism underlying GNP antioxidant activity. In addition to mitigating oxidative stress, GNP also modulated the inflammatory response in cardiac tissue. The downregulation of pro-inflammatory TNF-α and upregulation of the anti-inflammatory cytokine IL-10 point to its anti-inflammatory role. These alterations may involve inhibition of the NF-κB pathway, which plays a central role in obesity-related chronic inflammation and cardiac remodeling. Furthermore, GNP was found to regulate apoptosis-related gene expression by reducing Bax, Caspase-3, and Caspase-9 levels, while increasing Bcl-2 expression. This shift toward cell survival suggests inhibition of the intrinsic apoptotic pathway, potentially through PI3K/Akt or MAPK signaling pathways, as supported by other experimental models. Taken together, our results support the notion that GNP protects against high-fat diet–induced cardiac damage by orchestrating antioxidant, anti-inflammatory, and anti-apoptotic responses. These findings align with and expand upon previous studies, highlighting GNP as a promising candidate for preventing cardiovascular complications associated with obesity (Figure 4). Nevertheless, an important limitation of this study is the absence of protein-level confirmation for the gene expression results. Although real-time PCR provides valuable data on transcriptional changes, it does not necessarily reflect the abundance or functional activity of the corresponding proteins. In particular, analysis of oxidative stress markers (such as SOD and CAT), inflammatory mediators (including TNF-α and IL-10), and apoptotic regulators (such as Caspase-3, Bax, and Bcl-2) at the protein level using Western blotting would have offered stronger mechanistic evidence. Incorporating such assays in future studies will be essential to verify and support the proposed pathways modulated by Geniposide.

Figure 4:

Proposed mechanistic pathway of geniposide (GNP) protective effects against high-fat diet (HFD)-induced cardiac injury in rats. HFD induces oxidative stress by depleting antioxidant enzymes (SOD, CAT) and zinc levels, leading to increased lipid peroxidation (↑MDA), inflammation (↑TNF-α, ↓IL-10), and apoptosis (↑Bax, Caspase-3, Caspase-9; ↓Bcl-2) in cardiac tissue. GNP treatment mitigates these adverse effects through antioxidant, anti-inflammatory, and anti-apoptotic mechanisms. GNP likely activates Nrf2/ARE signaling to restore antioxidant defense, inhibits NF-κB to suppress inflammation, and modulates PI3K/Akt or MAPK pathways to balance apoptosis-related gene expression. Together, these actions protect the heart from HFD-induced structural and functional damage.

In conclusion, our data revealed that HFD is strongly associated with cardiac injury, structural/morphological changes in heart tissue, inflammation, oxidative stress, depletion of zinc and antioxidants and consequently apoptosis of cardiac cells. Surprisingly, pretreatment with GNP protected cardiac tissue against HFD-induced cardiac injury by elevating antioxidants pools, declining inflammation and oxidative stress, and down-regulating apoptosis-related genes.