Protective effect and mechanism of ethanol extract of rapeseed bee pollen on isoproterenol induced myocardial cell hypertrophy
Heart failure (HF) is a common cardiovascular syndrome and the final stage of various heart diseases, with high disability and mortality rates. Cardiac hypertrophy (CH) is an early adaptive response in the process of heart failure, but prolonged CH can accelerate myocardial fibrosis and systolic dysfunction, thereby accelerating the progression of HF. CH not only causes congestive heart failure, but is also an independent risk factor for hypertension, heart valve disease, dilated cardiomyopathy, myocardial ischemia, myocardial infarction, arrhythmia, and sudden death. The control of CH can significantly delay the progression of cardiomyopathy. Delaying or avoiding the occurrence of CH is a major therapeutic goal.
Isoproterenol (ISO) belongs to adrenergic receptor agonists. Repeated or continuous injection of ISO to excite β 1-adrenergic receptors can cause toxic damage to the myocardium, increase myocardial contractility and oxygen consumption, leading to arrhythmia, increased afterload, and myocardial ischemic necrosis, resulting in myocardial remodeling and inducing compensatory CH. Therefore, ISO is often used to establish CH models. Research has shown that ISO induced CH is accompanied by an increase in myocardial cell area, excessive protein synthesis, and reactivation of CH marker genes such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β – myosin heavy chain (β – MHC).
Bee pollen is one of the typical representatives among many bee products, which contains abundant ingredients. According to Thakur et al.’s statistics, the main components of bee pollen include carbohydrates (54.22%), proteins (21.30%), lipids (5.31%), fibers (8.75%), ash content (2.91%), etc. It also contains phenolic compounds, flavonoids, polyamines, nucleosides, amino acids, fatty acids, vitamins, etc. Bee pollen has various pharmacological activities such as antioxidant, anti myocardial infarction, and liver and kidney protection, and has potential application value. The research team conducted preliminary studies on the protective effects and mechanisms of ethanol extract and its main components from Schisandra chinensis bee pollen on H9c2 myocardial cells damaged by H2O2, confirming that the antioxidant capacity of bee pollen has a protective effect on myocardial cell damage. Rapeseed bee pollen is one of the hot selling products in the bee product market. Zhang et al. compared the differences in total phenolic and flavonoid content and antioxidant capacity among different bee pollen, and found that the total phenolic and flavonoid content of rapeseed bee pollen is higher, and its antioxidant capacity is also stronger. This article takes the common bee pollen – rapeseed bee pollen ethanol extract (RBPEE) as the research object, identifies its main chemical components, and studies its intervention effect and possible molecular mechanism on ISO induced myocardial cell hypertrophy, laying the foundation for the deep development and utilization of bee pollen resources.

The characteristics of CH mainly include increased myocardial cell volume, increased protein synthesis, increased mass, and thickening of the ventricular wall. ANP, BNP, and β – MHC, as molecular markers of CH, are re expressed during the CH process, autonomously produced and released by myocardial cells, actively participating in compensation mechanisms and regulating cardiac function. In this study, the surface area of cardiomyocytes significantly increased, the total protein content significantly increased, and the mRNA expression levels of three cardiac hypertrophy markers significantly increased, indicating the success of the ISO induced cardiac hypertrophy model. After treatment with RBPEE, the cell surface area and total protein content were significantly reduced compared to the model group, and the expression levels of three cardiac hypertrophy markers mRNA were also significantly reduced, indicating that RBPEE has a good preventive effect on ISO induced cardiac hypertrophy.
Under normal circumstances, the body can timely eliminate excess free radicals produced by tissue oxidative metabolism through antioxidant enzymes such as SOD, as well as non enzymatic antioxidants such as GSH, carotenoids, and vitamin C, thereby ensuring dynamic balance of free radicals in the body and maintaining normal functioning. The main pathway of oxidative stress damage is through the peroxidation reaction between reactive oxygen species (ROS) and biomolecules such as lipids, proteins, and nucleic acids in cells, causing structural damage and metabolic disorders, and ultimately leading to various diseases. When the cell membrane is attacked by ROS, unsaturated fatty acids in phospholipid molecules on the membrane oxidize to produce lipid peroxides such as MDA. Therefore, the degree of oxidative damage to body cells is reflected by changes in intracellular SOD activity, GSH content, and MDA content. The NF kB pathway is considered a pro-inflammatory signaling pathway, and NF kB activated by oxidative stress can be transported from the cytoplasm to the nucleus, thereby promoting the transcription of pro-inflammatory mediators such as IL-6 and TNF – α in cells. Therefore, detecting the expression level of pro-inflammatory cytokines can determine the degree of influence of inflammatory response on myocardial cells. Our use of ISO induced cardiomyocyte hypertrophy resulted in a significant decrease in SOD activity and GSH content, a significant increase in MDA content, and a significant increase in mR-NA expression levels of IL-6, TNF-a, and IL-2 in cardiomyocytes, indicating that cardiomyocyte hypertrophy is accompanied by oxidative stress and inflammatory response. After treatment with RBPEE, SOD activity and GSH content significantly increased, MDA content significantly decreased, and gene expression levels of three inflammatory response factors significantly decreased, indicating that RBPEE can prevent ISO damage to myocardial cells by inhibiting oxidative stress and inflammatory response.
The main pathways of cell apoptosis include exogenous pathway (death receptor pathway), endogenous mitochondrial pathway, and endoplasmic reticulum stress pathway. In the death receptor pathway, Caspase-8 initiates a signaling cascade after being activated by the death inducing signaling complex (DISC), leading to automatic activation of end effectors such as Caspase-3 or Caspase-7, ultimately inducing cell apoptosis signals. In the mitochondrial pathway, when mitochondria are stimulated by external factors, the apoptosis promoting factor Bax in the Bcl2 family regulates the outer membrane permeability of mitochondria, promoting outer membrane permeability and causing cytochrome C in the mitochondrial membrane cavity to form an apoptotic polymer complex with deoxyadenosine triphosphate (dATP) and apoptosis protease activating factor (APAF1), which is released into the cytoplasm and recruits the precursor of cysteine protease 9 (pro-Caspase-9), activating Caspase-9 and initiating a signaling cascade reaction to activate downstream Caspase-3, inducing cell apoptosis. Bcl-2 is an anti apoptotic member of the Bcl-2 family, and the ratio of Bcl-2 to Bax is an effective indicator for evaluating myocardial cell apoptosis. In this experiment, the gene expression levels of Bcl-2, Bax, Caspase-8, Caspase-9, and Caspase-3 were measured in each treatment group to determine the extent of myocardial cell apoptosis. The experimental results showed that ISO induced myocardial cell hypertrophy injury was accompanied by a process of cell apoptosis. RBPEE pretreatment has a significant inhibitory effect on the gene expression levels of Caspase-8, Caspase-9, Caspase-3, and Bax. The gene expression level of Bcl-2 and the Bcl-2/Bax ratio are significantly increased compared to the model group, indicating that RBPEE has a preventive effect on ISO induced cardiomyocyte apoptosis.
This experiment used UOLC-QTFF MS to qualitatively analyze the chemical composition of RBPEE, and preliminarily identified seven main components in RBPEE, including one carbohydrate (gluconic acid), two nucleosides (uridine and guanosine), one polyamine (N ′, N ′ – bis (p-coumaroyl) spermidine), and three flavonoid disaccharide compounds (Quercetin-3-O – (2 ′ – O-glucopyranosyl) – glucopyranoside, Quercetin-3-O – (2 ′ – O-glucopyranosyl) – rhamnoside, and kaempferol-3-O – (2 ′ – O-glucopyranosyl) – rhamnoside. – O – (2 ′ – O-glucopyranosyl) – glucopyranoside. Research has shown that both uridine and guanosine have antioxidant, anti-inflammatory, and anti apoptotic effects; N1 and N5 bis (p-coumaroyl) spermidine have good antioxidant properties, which can increase the total antioxidant capacity in cells, enhance the enzymatic activity of antioxidant enzymes SOD, CAT, and GPx, and reduce lipid oxidation levels; Flavonoid glycosides such as quercetin and kaempferol have pharmacological effects such as antioxidant, anti-inflammatory, and anti apoptotic properties. Therefore, nucleosides, polyamines, and flavonoids may be effective components of RBPEE in exerting antioxidant, anti-inflammatory, and anti apoptotic effects.
In summary, RBPEE has a certain preventive effect on ISO induced myocardial cell hypertrophy, and its mechanism of action may be closely related to reducing oxidative stress levels, inhibiting inflammatory responses, and anti apoptotic effects.