Citrinin (CTN) is commonly found in animal feed and stored grains and poses a serious threat to human and animal health. Formation of the IP3R1-GRP75-VDAC1 complex has been shown to play a key role in intestinal defense against harmful stimuli, but the mechanism of its action in CTN-exposure-induced enterotoxicity is not clear. Therefore, the aim of this study was to investigate the role of the IP3R1-GRP75-VDAC1 complex in CTN-exposure-induced intestinal and IPEC-J2 monolayer cell damage in mice. It was shown that CTN exposure triggered intestinal cell pyroptosis and increased IP3R1-GRP75-VDAC1 complex formation as well as mitochondrial levels of calcium ions and mitochondrial reactive oxygen species (mtROS). And mtROS is considered to be a key factor in cellular pyroptosis. Therefore, the removal of mtROS by using Mito-Tempo was found to attenuate CTN-exposure-induced cellular pyroptosis but failed to attenuate mitochondrial calcium ion overload. However, silencing of GRP75 alleviated CTN-exposure-induced increases in the level of mtROS, mitochondrial calcium ions, and subsequent cellular pyroptosis. Therefore, this study confirms that CTN exposure induces cellular juxtaposition in intestinal tissues and points out that mitochondrial oxidative stress mediated by the IP3R1-GRP75-VDAC1 complex is a key mechanism by which CTN exposure triggers intestinal cellular pyroptosis.

Citrinin (CTN) is a mycotoxin that is widespread and can contaminate a wide range of food products, posing a threat to human and animal health. The spleen and thymus are important immune organs of the body, and the damaging effects of CTN on immune organs and their mechanism are still unclear. In this study, we induced spleen and thymus injury in mice by exposure to different doses of CTN (0, 1.25, 5, or 20 mg/kg) and preliminarily investigated the damage mechanisms. It was observed that CTN exposure caused immune damage to the thymus and spleen, which are immune organs in mice. In addition, CTN exposure decreased the content of glutathione (GSH), an antioxidant, and the activities of antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT); it also increased the levels of oxidized products such as reactive oxygen species (ROS)and malondialdehyde (MDA). These results suggested that CTN induced oxidative stress in the thymus and spleen. The present study also found that CTN exposure significantly increased the expression of endoplasmic reticulum (ER) stress signature proteins, including C/EBP homologous protein (CHOP) and glucose-regulated protein 78(GRP78). Notably, pretreatment with the ER stress inhibitor 4-phenylbutyric acid (4-PBA, 240 mg/kg, intraperitoneally) attenuated CTN-induced oxidative stress in the spleen and thymus of mice and partially alleviated histopathological damage, demonstrating that inhibition of ER stress may be a novel strategy to prevent or treat CTN-induced immune organ damage.

BACKGROUND:Citrinin (CTN) is a mycotoxin that is difficult to eliminate and easy to ingest. Chronic exposure to CTN can lead to inflammatory bowel disease (IBD). The herb Koumine has strong anti-inflammatory activity and is considered a candidate for the treatment of IBD. PURPOSE:To investigate the effect of Koumine on IBD induced by CTN exposure and its mechanism of action. RESULTS:This study demonstrated that Koumine effectively attenuates CTN-induced inflammatory damage in the mouse intestine and IPEC-J2 cells. Furthermore, Koumine suppressed CTN-induced upregulation of the IP3R1-GRP75-VDAC1 complex, mitochondrial calcium overload, elevated mitochondrial reactive oxygen species (mtROS) levels, and subsequent pyroptosis. Specific overexpression of mtROS counteracted the therapeutic effect of Koumine on CTN exposure-induced pyroptosis but did not alter mitochondrial calcium levels. Silencing GRP75 ameliorated CTN-induced mitochondrial calcium overload and pyroptosis. Notably, siGRP75 addition did not further enhance the therapeutic effect of Koumine. CONCLUSIONS:Koumine ameliorates CTN-induced intestinal inflammation by mediating mtROS production via the IP3R1-GRP75-VDAC1 complex. Koumine is a potential agent for the treatment of intestinal inflammation induced by mycotoxin exposure such as CTN.

Citrinin (CTN), a nephrotoxic mycotoxin with high environmental stability, is widely present in contaminated food sources, leading to chronic ingestion in humans and animals. Although CTN exposure is closely associated with Balkan endemic nephropathy (BEN), the molecular mechanisms underlying CTN-induced renal injury remain largely elusive. PANoptosis—a newly recognized form of regulated cell death that integrates pyroptosis, apoptosis, and necroptosis—has emerged as a key contributor to kidney pathology. In this study, we established both in vivo (KM mice) and in vitro (TCMK-1 cells) models of CTN exposure and observed concurrent activation of pyroptotic, apoptotic, and necroptotic pathways, indicating PANoptosis induction. Furthermore, CTN treatment triggered mitochondrial structural damage accompanied by DRP1 upregulation, consequently leading to elevated mitochondrial ROS generation and cytochrome c release. To clarify the role of mitochondrial dysfunction in PANoptosis, we used the DRP1 inhibitor Mdivi-1, which restored mitochondrial integrity, alleviated dysfunction, and significantly reduced CTN-induced apoptotic and necroptotic cell death. Furthermore, mtROS and cytochrome c were identified as essential mediators of necroptosis and apoptosis, respectively, as shown by treatment with Mito-TEMPO and BALI. Notably, disulfiram, a GSDMD-N inhibitor, also mitigated mitochondrial dysfunction and PANoptotic responses through the DRP1 pathway. Molecular docking and Co-IP assays confirmed a direct interaction between GSDMD-N and DRP1. Collectively, these findings reveal that CTN induces PANoptosis through GSDMD-N/DRP1-mediated mitochondrial dysfunction, offering new mechanistic insights into CTN-induced nephrotoxicity and highlighting potential therapeutic targets for mitigating mycotoxin-associated kidney injury.

Citrinin (CTN), a widespread food and feed contaminant, poses a significant health risk, yet its hepatic toxicity remains unclear. Here, we investigated the role of endoplasmic reticulum (ER) stress-mediated pyroptosis in CTN-induced liver injury using mice and HL-7702 cells. CTN exposure disrupted the hepatic cord structure, induced hepatocyte swelling with karyolysis, and promoted inflammatory infiltration. Liver injury markers and pro-inflammatory cytokines IL-1β and IL-18 were significantly elevated in both models, confirming inflammatory liver injury. Mechanistically, CTN activated pyroptosis-related proteins and triggered ER stress. In HL-7702 cells, CTN-induced inflammatory injury was mediated by NLRP3-dependent pyroptosis. Silencing CHOP alleviated injury by suppressing NLRP3 activation, while selective inhibition of PERK reduced CHOP expression and further attenuated pyroptosis. Collectively, these findings demonstrate that the PERK-CHOP pathway regulates NLRP3-dependent pyroptosis, contributing to CTN-induced hepatotoxicity. The PERK-CHOP-NLRP3 axis may represent a potential therapeutic target against CTN-related liver injury.

T-2 toxin, a mycotoxin found in foods and feeds, poses a threat to female reproductive health in both humans and animals. LncRNA CUFF.253988.1 (CUFF.253988.1), highly expressed in pigs, has an undisclosed regulatory role. This study aimed to establish a model of T-2 toxin-induced ovarian injury in sows, both in vivo and in vitro, and to explore the regulatory role and potential mechanisms of CUFF.253988.1. The results showed that feeding T-2 toxin-contaminated feed (1 mg/kg) induced ovarian follicle atresia and mitochondrial structural damage, accompanied by a significant upregulation of CUFF.253988.1 expression in the ovaries. Additionally, T-2 toxin inhibited the SIRT3/PGC1-α pathway associated with mitochondrial function. Moreover, T-2 toxin induced cell apoptosis by upregulating the expression of Cyt c, Bax, cleaved-caspase-9, and cleaved-caspase-3 proteins. In T-2 toxin-induced injury to the ovarian granulosa AVG-16 cells at concentrations of 10, 40 and 160 nM, not only were the previously mentioned effects observed, but also a decrease in mitochondrial membrane potential, ATP content, and an elevation in ROS levels. However, downregulating CUFF.253988.1 reversed T-2 toxin's inhibition of the SIRT3/PGC1-α pathway, alleviating mitochondrial dysfunction and reducing cell apoptosis. Notably, this may be attributed to the inhibition of T-2 toxin-induced enrichment of CUFF.253988.1 in mitochondria. In conclusion, CUFF.253988.1 plays a pivotal role in T-2 toxin-induced ovarian damage, operating through the inhibition of the SIRT3/PGC1-α pathway and promotion of cell apoptosis.

T-2 toxin, an unavoidable contaminant in animal feeds, can induce oxidative stress and damage immune organs. Melatonin (MT), a natural and potent antioxidant, has shown promise as a detoxifier for various mycotoxins. However, the detoxifying effect of MT on T-2 toxin has not been previously reported. In order to investigate the protective effect of MT added to diets on the immune system of T-2 toxin-exposed piglets, twenty piglets weaned at 28d of age were randomly divided into control, T-2 toxin (1 mg/kg), MT (5 mg/kg), and T-2 toxin (1 mg/kg) + MT (5 mg/kg) groups(n = 5 per group). Our results demonstrated that MT mitigated T-2 toxin-induced histoarchitectural alterations in the spleen and thymus, such as hemorrhage, decreased white pulp size in the spleen, and medullary cell sparing in the thymus. Further research revealed that MT promoted the expression of Nrf2 and increased the activities of antioxidant enzymes CAT and SOD, while reducing the production of the lipid peroxidation product MDA. Moreover, MT inhibited the NF-kappa B signaling pathway, regulated the expression of downstream cytokines IL-1 beta, IL -6, TNF-alpha, and TGF-beta 1. MT also suppressed the activation of caspase-3 while down-regulating the ratio of Bax/Bcl-2 to reduce apoptosis. Additionally, MT ameliorated the T-2 toxin-induced disorders of immune cells and immune molecules in the blood. In conclusion, our findings suggest that MT may effectively protect the immune system of piglets against T-2 toxin-induced damage by inhibiting oxidative stress, inflammatory response, and apoptosis in the spleen and thymus. Therefore, MT holds the potential as an antidote for T-2 toxin poisoning.

Citrinin (CTN) is a mycotoxin commonly found in contaminated foods and feed, posing health risks to both humans and animals. However, the mechanism by which CTN damages the intestine remains unclear. In this study, a model of intestinal injury was induced by administering 1.25 mg/kg and 5 mg/kg of CTN via gavage for 28 consecutive days in 6-week-old Kunming mice, aiming to explore the potential mechanisms underlying intestinal injury. The results demonstrate that CTN can cause structural damage to the mouse jejunum. Additionally, CTN reduces the protein expression of Claudin-1, Occludin, ZO-1, and MUC2, thereby disrupting the physical and chemical barriers of the intestine. Furthermore, exposure to CTN alters the structure of the intestinal microbiota in mice, thus compromising the intestinal microbial barrier. Meanwhile, the results showed that CTN exposure could induce excessive apoptosis in intestinal cells by altering the expression of proteins such as CHOP and GRP78 in the endoplasmic reticulum and Bax and Cyt c in mitochondria. The mitochondria and endoplasmic reticulum are connected through the mitochondria-associated endoplasmic reticulum membrane (MAM), which regulates the membrane. We found that the expression of bridging proteins Fis1 and BAP31 on the membrane was increased after CTN treatment, which would exacerbate the endoplasmic reticulum dysfunction, and could activate proteins such as Caspase-8 and Bid, thus further inducing apoptosis via the mitochondrial pathway. Taken together, these results suggest that CTN exposure can cause intestinal damage by disrupting the intestinal barrier and inducing excessive apoptosis in intestinal cells.

Pseudorabies virus (PRV), the cause agent of Aujeszky’s disease, is an infectious pathogen which greatly affects the heathy development of pig industry worldwide. The low specific host tropism of PRV allows this virus to infect a variety of animals, such as pigs, cattle, minks, dogs, and even possible humans. However, the occurrence of PRV natural infection in goats has never been documented. Herein we provided robust evidences demonstrating the first case of a variant PRV infection leading to the acute goat death in Yunnan Province, China, which might be resulted from mixed feeding with PRV-infected fattening pigs. Therefore, this report not only highlights the potential threat of newly emerging variant PRV strain(s) to goat industry, but also appeals the development of effective and safe vaccines against PRV variants for goats/ruminants in future.

Citrinin (CTN) has been reported to induce renal failure and structural damage, but its nephrotoxic effects and mechanisms are not fully understood. Therefore, we established a model by orally administering CTN (0, 1.25, 5, or 20 mg/kg) to mice for 21 consecutive days. Histological and biochemical analyses revealed that CTN caused structural damage to renal tubules, increased inflammatory cell infiltration, and elevated levels of serum markers of renal function (creatinine, urea, and uric acid). Moreover, mRNA transcript levels of the inflammatory factors TNF-α, IL-1β, and IL-6 were increased, indicating the occurrence of an inflammatory response. Furthermore, exposure to CTN induced renal oxidative stress by decreasing antioxidant GSH levels, antioxidant enzyme (SOD, CAT) activities, and increasing oxidative products (ROS, MDA). In addition, CTN increased the expression of proteins associated with endoplasmic reticulum (ER)stress and apoptotic pathways. ER stress has been shown to be involved in regulating various models of kidney disease, but its role in CTN-induced renal injury has not been reported. We found that pretreatment with the ER stress inhibitor 4-PBA (240 mg/kg, ip) alleviated CTN-induced oxidative stress, NF-κB pathway mediated inflammatory response, and apoptosis. Interestingly, 4-PBA also partially alleviated renal structural damage and dysfunction. Thus, ER stress may be a novel target for the prevention and treatment of CTN-induced renal injury.

Osteoarthritis (OA) is a degenerative joint disease, Increasingly, mitochondrial autophagy has been found to play an important regulatory role in the prevention and treatment of osteoarthritis. Koumine is a bioactive alkaloid extracted from the plant Gelsemium elegans. In previous research, Koumine was found to have potential in improving the progression of OA in rats. However, the specific mechanism of its action has not been fully explained. Therefore, the aim of this study was to investigate whether Koumine can alleviate OA in rats by influencing mitochondrial autophagy. In the in vitro study, rat chondrocytes (RCCS-1) were induced with IL-1β (10 ng/mL) to induce inflammation, and Koumine (50 μg/mL) was co-treated. In the in vivo study, a rat OA model was established by intra-articular injection of 2% papain, and Koumine was administered orally (1 mg/kg, once daily for two weeks). It was found that Koumine effectively reduced cartilage erosion in rats with osteoarthritis. Additionally, it decreased the levels of inflammatory factors such as IL-1β, IL-6, and extracellular matrix (ECM) components MMP13 and ADAMTS5 in chondrocytes and articular cartilage tissue, while increasing the level of Collagen II.Koumine inhibited the production of reactive oxygen species (ROS) in cartilage tissue and increased the number of autophagosomes in chondrocytes and articular cartilage tissue. Additionally, it upregulated the expression of mitochondrial autophagy proteins LC3Ⅱ/Ⅰ, PINK1, Parkin, and Drp1. The administration of Mdivi-1 (50 μM) reversed the enhanced effect of Koumine on mitochondrial autophagy, as well as its anti-inflammatory and anti-ECM degradation effects in rats with OA. These findings suggest that Koumine can alleviate chondrocyte inflammation and improve the progression of OA in rats by activating PINK1/Parkin-mediated mitochondrial autophagy.

Neuroinflammation is a key factor in cognitive dysfunction and neurodegenerative diseases such as Alzheimer’s disease (AD), so inhibiting neuroinflammation is considered as a potential treatment for AD. Epigallocatechin-3-gallate (EGCG), a polyhydroxyphenol of green tea, has been found to exhibit anti-oxidative, anti-inflammatory and neuroprotective effects. The aim of this study was to investigate the inhibitory effect of EGCG on inflammation and its mechanism. In this study, BV2 cells were simultaneously exposed to lipopolysaccharides (LPS) and the amyloid-β oligomer (AβO) to induce inflammatory microenvironments. Inflammatory cytokines and NLRP3 inflammasome-related molecules were detected by RT-PCR and Western Blot. The results show that EGCG inhibits LPS/AβO-induced inflammation in BV2 cells through regulating IL-1β, IL-6, and TNF-α. Meanwhile, EGCG reduces the activation of the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome and levels of intracellular ROS in BV2 cells treated with LPS/AβO by affecting the mitochondrial membrane potential (MMP). Further research found that EGCG inhibited MMP through regulating thioredoxin-interacting protein (TXNIP) in LPS/AβO-induced neuroinflammation. In conclusion, EGCG may alleviate LPS/AβO-induced microglial neuroinflammation by suppressing the ROS/ TXNIP/ NLRP3 pathway. It may provide a potential mechanism underlying the anti-inflammatory properties of EGCG for alleviating AD.

Ethnopharmacological relevance: Koumine, an indole alkaloid extracted from Gelsemium elegans Benth, exerts anti-inflammation and antioxidant activities. However, the effects of koumine on intestinal injury induced by H2O2 and its potential molecular mechanisms need larger studies. Aim of the study: We established an IPEC-J2 cell damage model induced by H2O2 to explore the protective mechanism of koumine on intestinal injury.Materials and methods: In the experiment, cell damage models were made with hydrogen peroxide. To assess the protective effect of koumine on H2O2-induced IPEC-J2 cell injury, CCK-8, the release of LDH and ROS, trans-mission electron microscopy and Annexin V-FITC/PI were employed. Western Blot and Quantitative Real-time PCR were used to determine the potential alleviated mechanism of koumine on H2O2-trigged IPEC-J2 cell damage.Results: The results of CCK-8 and LDH implied that koumine has a mitigative effect on H2O2-induced cell damage via upregulating cell viability and suppressing cell membrane fragmentation. Simultaneously, koumine notably inhibited the level of pro-inflammatory factors (IL-1 beta, IL-6, IL-8, TNF-alpha and TGF-beta), the over-production of ROS along with decreasing the injury of mitochondrion, endoplasmic reticulum and lysosome induced by H2O2. Moreover, koumine dramatically attenuated H2O2-triggered IPEC-J2 cell apoptosis and autophagy. Subse-quently, Western blot analysis identified NF-Kappa B, PI3K and ERS as possible pathway responsible for the protective effect of koumine on H2O2-stimulated IPEC-J2 cell inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE:Viola yedoensis Makino (VYM) is a traditional Chinese herbal medicine widely distributed in China. It has many pharmacological effects such as anti-inflammatory, immune regulation and anti-oxidation. However, the protective effect of VYM on the spleen and thymus of broilers induced by heat stress has rarely been reported. AIM OF THE STUDY:We established a heat stress model of broilers to explore the protective effect of VYM on spleen and thymus of broilers. MATERIALS AND METHODS:In this experiment, a heat stress model was made by adjusting the feeding temperature of broilers. The protective effect of VYM on the spleen and thymus of heat-stressed broilers were evaluated by detecting immune organ coefficient, histological observation, Enzyme-Linked Immunosorbent Assay, production of antioxidant enzymes and peroxides, TUNEL Staining, Quantitative Real-time PCR. RESULTS:In this study, 60 healthy male AA broilers were divided into 6 groups: Control, 4.5% VYM, HS, HS + 0.5% VYM, HS + 1.5% VYM, HS + 4.5% VYM. After 42 days of feeding, serum, spleen and thymus were collected for detection and analysis. The study revealed that heat stress can lead to pathological damage in the spleen and thymus of broilers, reduce the content of immunoglobulin and newcastle disease (ND), infectious bursal disease (IBD) antibody levels, increase the expression of inflammatory factors IL-1β, INF-γ, heat shock 70 kDa protein (HSP70), heat shock 90 kDa protein (HSP90). Heat stress inhibits the activity of antioxidant enzymes CAT and SOD, promotes the production of MDA, and then lead to oxidative damage of the spleen and thymus. In addition, apoptotic cells and the ratio of Bax/Bcl-2 was increased. However, the addition of VYM to the feed can alleviate the adverse effects of heat stress on the spleen and thymus of broilers. CONCLUSIONS:This study showed that the addition of VYM to the diet could inhibit oxidative stress and apoptosis, and reduce the inflammatory damage of heat stress on the spleen and thymus of broilers. This study provides a basis for further exploring the regulatory role of VYM in heat stress-induced immune imbalance in broilers. In addition, this study also provides a theoretical basis for the development of VYM as a feed additive with immunomodulatory effects.

旨在探讨内质网应激在桔青霉素诱导肠道屏障功能障碍中的作用.试验选用24只初始体重为(27.8+1.5)g的7周龄昆明小鼠,随机分为4组,分别为对照组(5％乙醇)和桔青霉素低(1.25 mg·kg-1)、中(5 mg·kg-1)、高(20 mg·kg-1)剂量组.适应性饲养1周后,对照组按0.1 mL·10g-1体重灌胃5％乙醇,毒素处理组分别灌胃不同剂量桔青霉素,持续14 d.试验结束后,采血并分离血清,测量小肠长度并收集小鼠空肠样本.HE染色观察肠道病理变化;ELISA 检测血清中二胺氧化酶(diamine oxidase,DAO)、D-乳酸(D-lactic acid,D-LA)、内毒素(endotox-in,ET)的含量;检测空肠组织抗氧化酶的活性及活性氧(reactive oxygen species,ROS)的释放.为进一步探究内质网应激在桔青霉素诱导肠道损伤中的作用,选用32只昆明小鼠随机分为4组,分别为对照组(5％乙醇)、桔青霉素中剂量(5 mg·kg-1)组、内质网应激抑制剂4-苯基丁酸苯基丁酸(4-Phenylbutyric acid,4-PBA)(240 mg·kg-1)组和桔青霉素(5 mg·kg-1)+4-PBA(240 mg·kg-1)组.4-PBA经腹腔注射预处理小鼠1 h后,用桔青霉素对小鼠进行灌胃处理.14 d后,检测空肠组织病理损伤,氧化损伤,细胞凋亡情况及内质网应激相关蛋白表达,并用Spearman相关性分析确定内质网应激信号通路与肠道氧化应激、肠道屏障功能障碍之间的相关性.结果显示,不同剂量的桔青霉素会造成肠道组织损伤、氧化应激及屏障功能障碍.与桔青霉素中剂量组相比,4-PBA预处理能提高小鼠空肠总超氧化物歧化酶(total superoxide dismutase,T-SOD)、过氧化氢酶(catalase,CAT)的活性和总抗氧化能力(total antioxidant capacity,T-AOC),降低 ROS 和丙二醛(malondialdehyde,MDA)含量,抑制细胞凋亡及内质网应激,降低血清中DAO、D-LA和ET的含量,升高空肠组织中紧密连接蛋白ZO-1、Occludin和Claudin-1 mRNA表达,缓解桔青霉素诱导的空肠组织损伤,且内质网应激信号通路与肠道氧化应激及肠道屏障功能障碍之间存在显著相关性.综上表明,内质网应激在桔青霉素诱导的空肠屏障功能障碍中具有重要调控作用.

Zearalenone (ZEA) is a mycotoxin commonly found in cereals and feedstuffs, which can induce oxidative stress and inflammation to cause liver damage in humans and animals. Betulinic acid (BA) is extracted from pentacyclic triterpenoids of many natural plants and has anti-inflammatory, and anti-oxidation biological activities in many studies. However, the protective effect of BA on liver injury induced by ZEA has not been reported. Therefore, this study aims to explore the protective effect of BA on ZEA-induced liver injury and its possible mechanism. In the mice experiment, ZEA exposure increased the liver index and caused histopathological impairment, oxidative damage, hepatic inflammatory responses, and increased hepatocyte apoptosis. However, when combined with BA, it could inhibit the production of ROS, up-regulate the proteins expression of Nrf2 and HO-1 and down-regulate the expression of Keap1, and alleviate oxidative damage and inflammation in the liver of mice. In addition, BA could alleviate ZEA-induced apoptosis and liver injury in mice by inhibiting the endoplasmic reticulum stress (ERS) and MAPK signaling pathways. In conclusion, this study revealed the protective effect of BA on the hepatotoxicity of ZEA for the first time, providing a new perspective for the development of ZEA antidote and the application of BA.

People of all ages could suffer from sleep disorders, which are increasingly recognized as common manifestations of neurologic disease. Acorus tatarinowii is a herb that has been used in traditional medicine to promote sleep. β-asarone, as the main component of volatile oil obtained from Acorus tatarinowii, may be the main contributor to the sleeping-promoting efficacy of Acorus tatarinowii. In the study, adult male C57BL/6 mice were administered β-asarone at 12.5 mg/kg, 25 mg/kg, and 50 mg/kg. Behavioral experiments showed that β-asarone at 25 mg/kg could significantly improve sleep duration. It was also observed that the proportion of NREM (Non-Rapid Eye Movement) sleep increased considerably after administration of β-asarone. In the PVN (paraventricular nucleus of hypothalamus) region of the hypothalamus, it was observed that the glutamate content decreased after β-asarone treatment. At the same time, the expression of VGLUT2 (vesicular glutamate transporters 2) decreased while the expression of GAD65 (glutamic acid decarboxylase 65) and GABARAP (GABA Type A Receptor-Associated Protein) increased in the hypothalamus, suggesting that β-asarone may suppress arousal by reducing glutamate and promoting transformation of glutamate to the inhibitory neurotransmitter GABA (γ-aminobutyric acid). This study is the first to focus on the association between β-asarone and sleep, shedding perspectives for pharmacological applications of β-asarone and providing a new direction for future research.

Damage to the reproductive system is the key factor leading to male infertility. Citrinin (CTN) is produced by Penicillium and Aspergillus in nature, and is definitely found in food and animal feed. Studies have revealed that CTN can cause damage to male reproductive organs and reduce fertility, but the mechanism of toxicity has not been revealed. In the present study, male Kunming mice were given different doses of CTN (0, 1.25, 5 or 20 mg/kg BW) by intragastric administration. The results demonstrated that CTN exposure caused disorder of androgen, a decline in sperm quality, and histopathological damage of testis. The inhibition of the expression of ZO-1, claudin-1 and occludin suggests that the blood-testis barrier (BTB) was damaged. Simultaneously, CTN inhibited the activity of antioxidant enzymes such as CAT and SOD, and promoted the production of MDA and ROS, resulting in oxidative damage of testis. Additionally, apoptotic cells were detected and the ratio of Bax/Bcl-2 was increased. Not only that, CTN activated the expression of endoplasmic reticulum stress (ERS)-related proteins IRE1, ATF6, CHOP, and GRP78. Interestingly, 4-Phenylbutyric Acid (4-PBA, an ERS inhibitor) treatment blocked the adverse effects of CTN exposure on male reproduction. In short, the findings suggested that CTN exposure can cause damage to mouse testis tissue, in which ERS exhibited an important regulatory role.

Citrinin, a secondary metabolite, can pose serious risks to the environment and organisms, but its hepatotoxic mechanisms are still unclear. Histopathological and ultrastructural results showed that citrinin-induced liver injury in Kunming mice, and the mechanism of citrinin-induced hepatotoxicity was studied in L02 cells. Firstly, citrinin mades L02 cell cycle arrest in G2/M phase by inhibition of cyclin B1, cyclin D1, cyclin-dependent kinases 2 (CDK2), and CDK4 expression. Secondly, citrinin inhibits proliferation and promotes apoptosis of L02 cells via disruption of mitochondria membrane potential, increase Bax/Bcl-2 ration, activation of caspase-3, 9, and enhance lactate dehydrogenase (LDH) release. Then, citrinin inhibits superoxide dismutase (SOD) activity and increases the accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS), resulting oxidative damage in L02 cells; upregulates the protein expression of binding immunoglobulin protein (Bip), C/EBP homologous protein (CHOP), PKR-like ER kinase (PERK) and activating transcription factor6 (ATF6), inducing ER stress in L02 cells; increases the phosphorylation of AMP-activated protein kinase (AMPK) and decreases the content of adenosine-triphosphate (ATP), activating AMPK pathway in L02 cells. Eventually, pretreatment with NAC, an ROS inhibitor, alleviates citrinin-induced cell cycle G2/M arrest and apoptosis by inhibiting ROS-mediated ER stress; pretreatment with 4-PBA, an ER stress inhibitor, reversed ER stress and p-AMPK; pretreatment with dorsomorphin, an AMPK inhibitor, decreases citrinin-induced cell cycle G2/M arrest and apoptosis. In summary, citrinin induces cell cycle arrest and apoptosis to aggravate liver injury by activating ROS-ER stress-AMPK signaling pathway.

Zearalenone (ZEA) is a mycotoxin with estrogen-like biological activity, which widely present in feed and raw materials, with strong reproductive system toxicity and a major threat to animal reproduction. Betulinic acid (BA) is a natural plant compound with antioxidant, anti-inflammatory and other pharmacological activities. However, the mechanism of ZEA-induced uterine injury and the protective effect of BA have not been reported. Our results show that ZEA could cause uterine histopathological damage and cellular ultrastructural damage, affecting the secretion of sex hormones, such as estradiol (E2) and progesterone (P4), and increase the mRNA and protein expression of estrogen receptor α (ERα). ZEA could inhibit the activities of catalase (CAT) and superoxide dismutase (SOD), increase the production of malondialdehyde (MDA) and reactive oxygen species (ROS), and cause uterine oxidative stress. Furthermore, ZEA affected the homeostasis of uterine cell proliferation and death by regulating the expression of proliferating cell nuclear antigen (PCNA) and activating the mitochondrial apoptotic pathway. ZEA-induced uterine injury might be related to the activation of p38/ERK MAPK signaling pathway. However, the regulatory effect of ZEA on the uterus was reversed after BA treatment. In conclusion, the uterus is an important target organ attacked by ZEA, and BA showed a good therapeutic effect.