*Article* **Protective Effect of** *Ganoderma atrum* **Polysaccharide on Acrolein-Induced Apoptosis and Autophagic Flux in IEC-6 Cells**

**Yudan Wang, Xinxin Chang, Bing Zheng, Yi Chen, Jianhua Xie, Jialuo Shan, Xiaoyi Hu, Xiaomeng Ding, Xiaobo Hu and Qiang Yu \***

> State Key Laboratory of Food Science and Technology, China-Canada Joint Laboratory of Food Science and Technology (Nanchang), Key Laboratory of Bioactive Polysaccharides of Jiangxi Province, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China; W15698331381@163.com (Y.W.); cxx13870698052@163.com (X.C.); ncuspyzhengbing@163.com (B.Z.); chenyi15@ncu.edu.cn (Y.C.); jhxie@ncu.edu.cn (J.X.); jialuoa1998@163.com (J.S.); huxiaoyi0101@126.com (X.H.); dxm19970128@163.com (X.D.);

hxbxq2005@163.com (X.H.)

**\*** Correspondence: yuqiang8612@ncu.edu.cn

**Abstract:** This study was designed to explore the beneficial effect and mechanism of *Ganoderma atrum* (G. atrum) polysaccharide (PSG-1) on acrolein-induced IEC-6 cells. Our results indicated that PSG-1 significantly reduced the impairment of acrolein on cell viability, decreased oxidative stress, and enabled normal expression of tight junction (TJ) proteins that were inhibited by acrolein in IEC-6 cells. Furthermore, PSG-1 attenuated the elevation of microtubule-associated proteins light chain 3 (LC3) and Beclin 1-like protein 1 (Beclin 1) and increased the protein levels of phospho-mTOR (p-mTOR) and phospho-akt (p-akt), indicating that PSG-1 activated the mammalian target of rapamycin (mTOR) signaling pathway and alleviated acrolein-induced autophagy in IEC-6 cells. Moreover, PSG-1 markedly attenuated the acrolein-induced apoptosis, as evidenced by the increase in mitochondrial membrane potential (MMP) and B-cell lymphoma 2 (Bcl-2) expression, and the decrease in cysteine aspartate lyase (caspase)-3 and caspase-9. In addition, autophagy the inhibitor inhibited acroleininduced TJ and apoptosis of IEC-6 cells, while the apoptosis inhibitor also inhibited acrolein-induced TJ and autophagy, suggesting that autophagy and apoptosis were mutually regulated. Taken together, the present study proved that PSG-1 could protect IEC-6 cells from acrolein-induced oxidative stress and could repair TJ by inhibiting apoptosis and autophagic flux, where autophagy and apoptosis were mutually regulated.

**Keywords:** polysaccharide; acrolein; IEC-6 cells; autophagy; apoptosis

**1. Introduction**

Acrolein is an electrophilic, unsaturated aldehyde, a colorless, flammable, volatile, pure liquid, known for its pungent odor and strong irritation of mucous membranes [1], which comes from food, the environment, and the human body itself [2,3]. Acrolein in food is mainly produced by oil fumes or fried and baked foods, which produce acrolein mainly through the Maillard reaction and lipid oxidation [4]. Acrolein is neurotoxic, genotoxic, and potentially carcinogenic, and the question of how to mitigate the side effects of acrolein has generated scholars' attention [5]. Therefore, finding active substances that can reduce or antagonize the toxic effects of acrolein in food is important to protect humans from its harm.

Programmed cell death (PCD) is a self-regulatory mechanism of the organism, mainly including autophagy, apoptosis, and necrosis, the first two of which can play an indispensable role in the life activities of the organism by working closely together. Autophagy is a highly conserved intracellular degradation pathway, a protective mechanism for cells in response to various of hazardous stimuli, which maintains intracellular homeostasis

**Citation:** Wang, Y.; Chang, X.; Zheng, B.; Chen, Y.; Xie, J.; Shan, J.; Hu, X.; Ding, X.; Hu, X.; Yu, Q. Protective Effect of *Ganoderma atrum* Polysaccharide on Acrolein-Induced Apoptosis and Autophagic Flux in IEC-6 Cells. *Foods* **2022**, *11*, 240. https://doi.org/ 10.3390/foods11020240

Academic Editor: Antonio Cilla

Received: 3 December 2021 Accepted: 10 January 2022 Published: 17 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

by degrading damaged macromolecular proteins or organelles within the cell [6]. Autophagy is a multi-step regulated process with highly complex signaling. In this process, microtubule-associated proteins light chain 3 (LC3), Beclin 1-like protein 1 (Beclin 1), and so on, play remarkably important roles [7]. Apoptosis is an orderly and autogenous cell death that is regulated by the involvement of genes, acting in the extrinsic and intrinsic pathways [8]. Both pathways stimulate cysteine aspartate lyase (caspase), which is the basis of apoptosis [9]. The endogenous pathway surrounding mitochondria is mainly mediated by the proteins of B-cell lymphoma 2 (Bcl-2) family. Several structural features are altered during apoptosis: membrane blistering, cytoplasmic crumpling, DNA breaks, chromatin condensation, and DNA degradation of chromosomes [10].

The intestine is the body's largest immune system, which mainly relies on the intestinal mucosal barrier to resist the invasion of foreign toxic substances and prevent the invasion of adverse substances and displacement in the intestine [11]. Thus, the integrity of the intestinal mucosal screen is essential to maintain the normal physiological function of the intestine [12]. Many studies have found that autophagy and apoptosis act as vital players in the intestinal barrier and work together to sustain the complexity of the intestinal barrier [13].

Numerous studies have revealed that polysaccharides possessed great promises for food and health applications [14–16]. *Ganoderma atrum* (*G. atrum*) has been used as a medicine or diet with high nutritional value for thousands of years [17]. In the last decade, our laboratory has identified a polysaccharide from *G. atrum* (PSG-1), with purity > 99.8%. It is composed of glucose (Glc), mannose (Man), galactose (Gal), and galacturonic acid (GalA) in molar ratio of 4.91:1:1.28:0.71, and is rich in 17 proteins, including glutamic acid, asparagic acid, alanine, glycine, threonine, and serine; in addition, it is formed as a main chain of 1,3-linked and 1,6-linked β-Glcp residues, substituted at O-3 and O-6 position as the branch points, with the molecular weight of 1013 kDa. The residues of α-1,4-Galp, α-1,2-Manp, and α-1,4-Manp were also found in the backbone. Side chains were terminated by β-Glcp, with the composition of α-1,4-Galp, α-1,4-GalpA, β-1,3-Glcp, and β-1,6-Glcp [18]. PSG-1 have exhibited a broad range of beneficial health effects, including immunomodulatory, chemo-protective, antioxidant, and hypoglycemic activities [19–22]. In particular, latest studies have also proved that PSG-1 could modulate the mucosal immunity through epithelial cells [23,24]. However, there are few reports on acroleininduced intestinal effects. Therefore, in this study, an acrolein-induced IEC-6 cells injury model was established, aiming to examine the protective characteristics of PSG-1 against acrolein-induced intestinal injury and its intrinsic mechanism.

#### **2. Materials and Methods**

#### *2.1. Materials*

Acrolein was purchased from Shandong Xiya Chemical Industry Co., Ltd. (Linyi, China). 3-Methyladenine (3-MA) (PI3K inhibitor) and Z-DEVD-FMK (caspase-3 inhibitor) were purchased from MedChem Express Co., Ltd. (Monmouth Junction, NJ, USA). Annexin V-FITC apoptosis detection kits were purchased from Beyotime Biotechnology (Nanjing, China). Mitochondrial membrane potential (JC-1) assay kit was obtained from Solarbio Science and Technology Co., Ltd. (Beijing, China). The Cell Counting Kit-8 (CCK-8) was from Dojindo Molecular Technologies, Inc. (Shanghai, China).

#### *2.2. Cell Culture*

IEC-6 cells (rat-derived intestinal epithelial cell line) were cultured in high glucose Dulbecco's Minimal Essential Medium (DMEM) (Solarbio Science and Technology Co., Ltd. Beijing, China), supplemented with 10% fetal bovine serum (FBS) (BI, 04-001-1ACS), 5% CO2, temperature 37 ◦C and humidity saturation conditions. The passages 5–20 were used in this study.
