Lignosus rhinocerus TM02® Extract (xLr®) Alleviates Hyperglycaemia-induced Endothelial Dysfunction By Modulating Oxidative Stress

Hyperglycaemia-induced oxidative stress, common in diabetes, often results in endothelial dysfunction, is a major contributor to cardiovascular disorders. Lignosus rhinocerus (Tiger Milk mushroom), used as folk medicine by native people in Southeast Asia and China, has been discovered to have various bioactivities including anti-inflammatory, antioxidant and anti-cancer. However, its effect in mitigating endothelial dysfunction is not fully understood. This study explored the protective effects and underlying mechanism of the patented cold-water extract from cultivated L. rhinocerus TM02 ® (xLr ® ) on isolated mouse aortas and human umbilical vein endothelial cells (HUVECs). HUVECs and aortas isolated from C57BL/6J mice were incubated with normal glucose (NG, 5.5 mM), high glucose (HG, 30 mM), xLr ®, aminoguanidine (AG) and apocynin (Apo) for 48 hours, respectively. Wire myograph was used to evaluate aortic vascular reactivity. The effect of xLr ® on the reactive oxygen species (ROS) formation, nitric oxide (NO) bioavailability and expression of oxidative stress markers and glycation proteins were assessed via dihydroethidium fluorescence (DHE), 4-amino-5-methylamino-2’,7’-difluorofluorescein (DAF-FM DA) and Western blot, respectively. Ex vivo treatment with xLr ® significantly restored endothelium-dependent relaxations in isolated aortas following incubation in HG. Elevated oxidative stress, increased RAGE protein expression and decreased NO bioavailability in HUVECs exposed to HG were normalised by xLr ® treatment. This demonstrates that xLr ® reversed impaired endothelial function and shields endothelial cell from hyperglycaemia-induced destruction by regulating oxidative stress, hence enhancing bioavailability of NO partly via inhibition of AGE. This study delineates the potential of xLr ® in maintaining endothelial health and preventing cardiovascular complications associated with diabetes. Lignosus rhinocerus TM02 ® Extract ( xLr ® ) Alleviates Hyperglycaemia-induced Endothelial Dysfunction By Modulating Oxidative Stress Sun Wern, Tan a , Siti Sarah, M. Sofiullah f, Sharifah Zamiah Syed Abdul Kadir a, Ker Woon, Choy c,d, Muhammad Fazril, Mohamad Razif b, Szu Ting, Ng e, Chon Seng, Tan e, Shin-Yee, Fung b*, Dharmani Devi, Murugan a* a Department of Pharmacology, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia b Medicinal Mushroom Research Group (MMRG), Department of Molecular Medicine, Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia c Department of Anatomy, Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sungai Buloh Campus, Jalan Hospital, 47000, Sungai Buloh, Malaysia d Institute of Pathology, Laboratory and Forensic Medicine (I-PPerForM), Universiti Teknologi MARA (UiTM), Sungai Buloh Campus, Jalan Hospital, 47000, Sungai Buloh, Malaysia e LiGNO Research Initiative, LiGNO Biotech Sdn.Bhd, Jalan Perindustrian Balakong Jaya 2/2, Taman Perindustrian Balakong Jaya 2, 43300 Balakong Jaya, Selangor, Malaysia f Xiamen University Malaysia, Jalan Sunsuria, Bandar Sunsuria, 43900 Sepang, Selangor Darul Ehsan *Corresponding author: Dharmani Devi Murugan Department of Pharmacology, Faculty of Medicine, Universiti Malaya, 50603 Kuala Lumpur, Malaysia; email: [email protected]; Tel: +603-7967 7566; Fax: +603-7967 4971 Fung Shin Yee Medicinal Mushroom Research Group (MMRG), Department of Molecular Medicine, Faculty of Medicine, Universiti Malaya, 50603 Kuala Lumpur, Malaysia email: [email protected] Tel: +603-7967 5745

Hyperglycaemia-induced oxidative stress, common in diabetes, often results in endothelial dysfunction, is a major contributor to cardiovascular disorders. Lignosus rhinocerus (Tiger Milk mushroom), used as folk medicine by native people in Southeast Asia and China, has been discovered to have various bioactivities including anti-inflammatory, antioxidant and anti-cancer. However, its effect in mitigating endothelial dysfunction is not fully understood. This study explored the protective effects and underlying mechanism of the patented cold-water extract from cultivated L. rhinocerus TM02 ® (xLr ® ) on isolated mouse aortas and human umbilical vein endothelial cells (HUVECs). HUVECs and aortas isolated from C57BL/6J mice were incubated with normal glucose (NG, 5.5 mM), high glucose (HG, 30 mM), xLr ®, aminoguanidine (AG) and apocynin (Apo) for 48 hours, respectively. Wire myograph was used to evaluate aortic vascular reactivity. The effect of xLr ® on the reactive oxygen species (ROS) formation, nitric oxide (NO) bioavailability and expression of oxidative stress markers and glycation proteins were assessed via dihydroethidium fluorescence (DHE), 4-amino-5-methylamino-2’,7’-difluorofluorescein (DAF-FM DA) and Western blot, respectively. Ex vivo treatment with xLr ® significantly restored endothelium-dependent relaxations in isolated aortas following incubation in HG. Elevated oxidative stress, increased RAGE protein expression and decreased NO bioavailability in HUVECs exposed to HG were normalised by xLr ® treatment. This demonstrates that xLr ® reversed impaired endothelial function and shields endothelial cell from hyperglycaemia-induced destruction by regulating oxidative stress, hence enhancing bioavailability of NO partly via inhibition of AGE. This study delineates the potential of xLr ® in maintaining endothelial health and preventing cardiovascular complications associated with diabetes.

Endothelial dysfunction; hyperglycemia; Lignosus rhinocerus TM02 ® cold-water extract xLr ® ; oxidative stress Abbreviations : ACh, acetylcholine; AG, aminoguanidine; AGE, advanced glycation end products; Apo, apocynin; DAF-FM, 4-amino-5-methylamino-2’,7’-difluorofluorescein; DHE, dihydroethidium; EDR, endothelium-dependent relaxation; EIR, endothelium-independent relaxation; eNOS, endothelial nitric oxide synthase, Glo1, glyoxalase-1; HUVECs, human umbilical vein endothelial cells; MG, methylglyoxal; NADPH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; NOX, NADPH oxidase; O 2- , superoxide anions; ONOO - , peroxynitrite; RAGE, receptor of AGEs; ROS, reactive oxygen species; SNP, sodium nitroprusside; xLr ® , cold water extract of Lignosus rhinocerus cultivar TM02 ®

The vascular endothelium is made up of an endothelial cell monolayer that lines the tunica intima of blood vessels. It serves as a selective permeable barrier between blood and tissues, regulating vasomotor function, modulating inflammatory processes, and maintaining vascular homeostasis [1]. Vascular health is maintained by the balance between vasoconstriction and vasodilation factors released by the endothelium. Endothelial dysfunction is considered as an early indication of blood vessel destruction that disrupts the permeability of the endothelial barrier and causes an inflammatory response in the development of cardiovascular disorders. The characteristics of endothelial dysfunction include elevated reactive oxygen species (ROS) levels, decreased nitric oxide (NO) bioavailability and proinflammatory factors, leading to impairment of endothelium-dependent vasodilation [2]. Endothelial dysfunction is considered a characteristic of various human pan-vascular disorders, such as atherosclerosis, hypertension, and diabetes [3]. Hyperglycaemia is known to cause metabolic derangements in endothelial cells [4]. Hyperglycaemia leads to elevated ROS production, including superoxide anions (O 2- ), which are produced when vasculature nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) partially reduces molecular oxygen to O 2- [5]. ROS production, which is mediated by NOXs, can lead to endothelial nitric oxide synthase (eNOS) uncoupling. When O 2- reacts readily with NO, forming toxic peroxynitrite (ONOO - ), this uncouples eNOS and accelerates free radicals production that further exacerbate oxidative stress and decrease NO bioavailability, eventually resulting in endothelial dysfunction [6]. Another primary mechanism of diabetes-associated endothelial cell dysfunction involves the formation of hyperglycaemia-associated advanced glycation end products (AGEs). These end-products bind to cellular receptors of AGEs (RAGE), leading to the subsequent activation of NOXs, which initiates oxidative stress/inflammation cascade [7]. Lignosus rhinocerus, commonly known as Tiger Milk mushroom, has a history of traditional medicinal use among native communities of Southeast Asia. Its sclerotium has been used as a folk medicine to relieve fever, asthma, cough, cancer, and food poisoning [8]. According to a chronic toxicity study, the consumption of L. rhinocerus sclerotia up to 1000 mg/kg did not cause any observed adverse effects in Sprague Dawley rats [9]. These results indicate that L. rhinocerus could potentially serve as a complementary medicine, offering various health benefits with a good safety profile. Additionally, its bioactive properties including anti-oxidative, anti-inflammatory, and anti-proliferative activities have been reported in various scientific studies [10-14] . In 2022, Yap et al. [14] showed that consuming L. rhinocerus sclerotia (TM02 ® cultivar) can reduce oxidative stress in a chronic hyperglycaemia-induced diabetic model. Their results showed improved glycaemic control, lowered plasma AGE concentration, and increased mitochondrial ATP levels in both the cardiac and skeletal muscles of rats treated with TM02 ® . Although the anti-oxidative effects of L. rhinocerus have been shown, there is still a lack of studies on the effect of L. rhinocerus on vascular function and NO bioavailability under hyperglycaemic conditions. Therefore, this study explored the effects of patented cold-water extract from cultivated L. rhinocerus TM02 ® (xLr ® ) in alleviating oxidative stress and restoring impaired endothelial function induced by hyperglycaemia in diabetic models, both in vitro and ex vivo .

The aortic rings from mice treated with high glucose (HG, 30 mM) for 48 h significantly reduced the maximal relaxation and sensitivity to ACh (R max 66.20%, pEC 50 - 6.81 ± 0.08), compared to the control (NG, 5.5 mM) group (R max 89.40%, pEC 50 -7.47 ± 0.16). Co-treatment with xLr ® (10, 30, and 50 μg/mL) showed significant improvements in the maximal relaxation and sensitivity to ACh-induced relaxation compared to those treated in HG (Figures 1A and Table 1) . Moreover, co-incubation with aminoguanidine (AG, 100 μM) restored the impaired relaxation, which has a similar vascular protective effect with xLr ® (Figures 1B and Table 1) . The SNP-induced EIR showed no significant differences among the groups, indicating that the treatment with xLr ® did not affect the dilatory function of vascular smooth muscle (Figures 1C-D and Table 1) . 2.2 Effect of xLr ® on ROS production and NOX2 protein expression in HUVECs HUVECs incubated in HG produced a significantly elevated level of vascular ROS, compared to NG, as detected by dihydroethidium (DHE) fluorescence. Compared to HG, the co-treatment with xLr ® (30 and 50 μg/mL) significantly decreased intracellular ROS production in HUVEC. Similarly, co-treatment with aminoguanidine (AG, 100 μM), which is an inhibitor for advanced glycation end products (AGEs), and apocynin (Apo, 100 μM), which is a ROS scavenger, also significantly normalised the increased ROS in HUVECs incubated in HG, comparable to the highest dose of xLr ® (50 μg/mL). Moreover, the level of intracellular ROS in HUVECs in NG, co-treated with xLr ® (50 μg/mL) was comparable to the NG-treated group ( Figure 2A ). In line with that, the measurement of NOX2 protein in HG-treated HUVECs showed a greater amount of NOX2 protein than in the NG-treated group (Figure 2B ). Co-treatment with xLr ® (50 μg/mL), aminoguanidine (AG, 100 μM), and apocynin (Apo, 100 μM) significantly decreased the expression of NOX2 protein. No significant differences in NOX2 protein expression were observed between the co-treatment of xLr ® (50 μg/mL) in NG and the NG-treated group. The NO bioavailability in HUVECs incubated in HG was significantly lower, indicating that incubation with HG attenuated NO production in HUVECs. The co-treatment with xLr ® (50 μg/mL), aminoguanidine (AG, 100 μM), and apocynin (Apo, 100 μM) significantly restored the decreased NO levels in HUVECs challenged with HG. Meanwhile, co-treatment of xLr ® (50 μg/mL) did not alter the NO level in HUVECs incubated in NG. The effects of xLr ® on glycation-associated proteins, receptor of AGEs (RAGE) and glyoxalase 1 (Glo1) in HUVECs were then explored via Western Blot. Although not significant, both RAGE and Glo1 showed up-regulated trends in HG-induced HUVECs. Although not significant, there is a decreasing trend observed in the groups co-treated with xLr ® (50 μg/mL), aminoguanidine (AG, 100 μM), and apocynin (Apo, 100 μM).

This study demonstrated that xLr ® treatment effectively alleviate endothelial dysfunction mice aortas exposed to hyperglycaemic conditions. The treatment with xLr ® normalised the harmful effects of high glucose, as showed by significant improvements in endothelium-dependent relaxations in the aorta and enhanced NO production in HUVECs. The treatment with xLr ® also reduced superoxide production and expression of NOX2, as well as reduced RAGE and normalised Glo1 expression in HUVECs. These findings suggest that xLr ® can protect the endothelium from hyperglycaemia-induced vasorelaxant dysfunction by minimising oxidative stress and enhancing the NO bioavailability. Tiger Milk mushroom, scientifically known as L. rhinocerus, has been reported to have a rich nutrient composition that exhibits antioxidant capacity, especially in the sclerotial water extracts, as demonstrated in previous in vitro studies [15,16] . Parallel to this, proteomics analysis identified two potential isoforms of manganese superoxide dismutase (Mn-SOD) in L. rhinocerus, which plays a significant role in antioxidant defence against superoxide radicals, suggesting that the TM02 ® sclerotium can be a promising source to improve antioxidant status [17]. However, the effect of L. rhinocerus on endothelial dysfunction related to oxidative stress and NO bioavailability is not well known. The current study demonstrated the therapeutic potential of L. rhinocerus in preserving endothelial function by inhibiting ROS overproduction and enhancing NO bioavailability. Excessive ROS generation, elevated oxidative stress, enhanced production of inflammatory factors, and reduced nitric oxide (NO) bioavailability are key factors that lead to endothelial cell dysfunction, a hallmark of cardiovascular complications [18]. Long-term exposure of arteries to elevated glucose levels (HG, 30 mM) significantly decreased ACh-induced endothelium-dependent relaxation, but did not affect SNP-induced endothelium-independent relaxation, which is in agreement with previous studies [19-21]. In addition, the dysfunction was significantly reversed by treatment with xLr ® suggesting that it has a vascular protective effect against hyperglycaemia-induced endothelial dysfunction. The improvement in endothelium function shown by xLr ® is comparable to aminoguanidine (AG). The positive benefits of xLr ® are more likely attributable to increased endogenous NO bioavailability than improved vascular smooth muscle sensitivity, as evidenced by the fact that all experimental groups showed no changes in relaxation to SNP, an exogenous NO donor. To further explore the mechanisms behind the vascular protective effects of xLr ®, HUVECs were exposed to oxidative stress induced by high glucose and co-incubated with different concentrations of xLr ® . It has been shown that ROS is induced in endothelial cells under hyperglycaemic conditions, which may ultimately contribute to endothelial dysfunction [22-24]. Membrane-bound NADPH oxidase subunits, including NOX2, are a primary source of ROS in the vasculature, where high glucose levels stimulate the activation of NOX2, leading to increased ROS production [25]. In the present study, HUVECs exposed to high glucose showed elevated ROS levels associated with increased NOX2 expression, both of which were then reduced by treatment with xLr ® . The antioxidant effect of xLr ® was comparable to that of apocynin (Apo), an antioxidant, and aminoguanidine (AG), an AGE inhibitor. This aligns with previous research indicating that compounds in the L. rhinocerus extract can mitigate oxidative stress by inhibiting ROS generation in both HT22 cells and PC12 cells exposed to apoptotic environment [26,27] . Studies on the chemical constituents of L. rhinocerus TM02 ® cultivar found that its aqueous extracts contained a high proportion of polysaccharides, β-glucan, phenolic and water-soluble compounds that have antioxidant properties [15,16,28]. Therefore, this study showed that xLr ® effectively improved hyperglycaemia-induced endothelial dysfunction by reducing oxidative stress. Elevated levels of ROS contribute to the development of endothelial cell dysfunction, which is accompanied by the inactivation of endothelial nitric oxide synthase (eNOS) and a reduction in NO levels. The eNOS uncoupling generates superoxide anion (O 2 − ) instead of NO, thus becoming a source of free radicals that further worsens oxidative stress and decreases the NO bioavailability via the production of peroxynitrite radicals, which further impairs vascular function [6]. However, following co-treatment with a higher concentration of xLr ® (50 μg/mL), NO production was significantly increased in HUVECs. This also aligned with the improved vascular function of HG-incubated aorta co-treated with xLr ® . Therefore, it is possible that hemostatic imbalance was restored by the ROS-inhibiting effect of xLr ®, which increased NO levels in high-glucose-stimulated endothelial cells, thereby improving endothelial function. Protein glycation is a random, nonenzymatic reaction between sugars and proteins triggered by diabetes and ageing, leading to the formation of advanced glycation end products (AGEs). AGEs bind to their receptors (RAGE) and promote ROS production and oxidative stress that is responsible for endothelial dysfunction [7]. In this study, the expression of RAGE in HUVECs exposed to high glucose levels increased, which is parallel to the result of another study that exposed Schwann cells to high levels of glucose [29]. Similarly, a study found that the medium-molecular-weight (MMW) fraction from the cold-water extract of L. rhinocerus sclerotia powder possessed strong anti-glycation activity, effectively inhibiting the formation of N𝜀-(carboxymethyl)lysine and other advanced glycation end products (AGEs) in a human serum albumin-glucose model [16]. In 2022, another study showed that treatment with L. rhinocerus TM02 ® cultivar sclerotia decreased AGE levels in high-fat diet-induced diabetic rats [14] demonstrating the anti-glycation potential of TM02 ® . Although not significant, a decrease in RAGE expression was observed following treatment with xLr ®, supporting the antiglycation potential of TM02 ® . Similarly, studies have found that polysaccharides isolated from Ganoderma lucidum (Lingzhi) attenuated myocardial collagen cross-linking in rats with diabetes, following decreased levels of AGEs and enhanced antioxidant enzyme activities [30]. According to Lau et al [31], the significant constituents of L. rhinocerus are carbohydrates, with β-glucans being the predominant glucans in the aqueous extracts. Thus, we speculate that the abundant bioactive components in the xLr ® , such as β-glucans, may potentially contribute to down-regulating RAGE expression, thus alleviating oxidative stress induced by hyperglycaemia. Glyoxalase 1 (Glo1) is essential in detoxifying reactive dicarbonyl compounds, like methylglyoxal (MG), which are major precursors to AGEs [32]. This detoxifying mechanism is downregulated due to elevated MG formation and reduced glyoxalase 1 (Glo1) activity in endothelial cells cultured under prolonged high glucose conditions [33] and in the brain or kidney tissue of diabetic rodent models. Besides, overexpression of Glo1 reduces the levels of AGEs and oxidative stress induced by hyperglycaemia [34], lessens endothelial dysfunction, and alleviates the onset of renal damage in diabetic rats [35]. However, a contrasting result was observed in the present study whereby 48 hours of high glucose exposure upregulated Glo1 protein expression; however, the expression of Glo1 in all treatment groups was lower compared to the group exposed to high glucose levels. This phenomenon can be explained by enhanced glucose metabolism, MG production, and decline in Glo1 activity on a time- and glucose concentration-dependent basis. This is corroborated by previous research that demonstrated increased MG production in HAEC cells under high glucose conditions after incubation for 1-2 days without a decrease in Glo1 activity [36]. This may represent an adaptive survival mechanism to counteract the accumulation of MG induced by high glucose levels. Elevated levels of MG may trigger the upregulation of Glo1, facilitating the detoxification of MG accumulation during the early stages of hyperglycaemia. According to a study investigating the effects of pyridoxamine on methylglyoxal-induced macrophage dysfunction and healing in diabetic wounds, pyridoxamine treatment resulted in a reduced expression of Glo1 in both THP-1 M1-like macrophages and wound tissue of diabetic mice. It is hypothesised that pyridoxamine directly quenches MG, thereby alleviating MG-induced cellular stress more rapidly, and reducing the need for increased Glo1 synthesis during MG detoxification [37]. It is possible that xLr ® may also neutralise MG directly, thus reducing the necessity for increased Glo1 expression. A study has identified genes that code for Glo1, catalase-peroxidases, and superoxide dismutase (SOD) in the genome-transcriptome data of L. rhinocerus, which plays an important role in its anti-glycation mechanism [16]. However, further studies are required to validate this effect. While this study highlights the antioxidant properties of L. rhinocerus, certain limitations and future directions should be considered. Further research should explore the mechanistic pathways underlying ROS attenuation, such as receptor-mediated NADPH oxidase signalling and AGE-RAGE axis modulation, particularly through in vivo studies. Expanding investigations to obese or diabetic models could elucidate its metabolic benefits, including effects on adipose tissue inflammation, insulin sensitivity, and vascular function. Additionally, identifying and characterizing the bioactive compounds responsible for these effects would support drug development and standardization. Limitations of the current work include reliance on in vitro and unresolved specific responses. Addressing these gaps would strengthen the foundation of L. rhinocerus as a therapeutic agent for oxidative stress-related diseases.

LiGNO Biotech Sdn Bhd (Balakong Jaya, Selangor, Malaysia) supplied the crude sclerotial powder of TM02® cultivar, which was verified through DNA fingerprinting by sequencing the internal transcribed spacer (ITS) regions of ribosomal RNA using specific primers: F-TM and ITS4 [8]. TM02 ® is a cultivated mushroom grown using solid-state fermentation technology, specifically cultivated for its sclerotium to maximise its medicinal potency. The method used to prepare the xLr ® was adapted from Yap et al. [15]. Briefly, cold-water extraction was carried out by stirring the mixture at 4°C for 24h with a 1:20 (g mL -1 ) mass-to-volume ratio. The solution was then filtered using Whatman No.1 filter paper, and the extract was collected and freeze-dried. Male C57BL/6J mice (12 weeks old) were ordered from the Animal Experimental Unit (AEU), Universiti Malaya. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC), Universiti Malaya (Ethics reference no: 2022-240210/PHAR/R/DDM). The animals were housed in a well-ventilated room at 24 ± 1°C with 12h light/dark cycles and had free access to standard chow and tap water. Mice’s aorta was carefully excised and submerged in ice-cold phosphate buffer saline (PBS) (pH 7.4) after the mice were sacrificed via carbon dioxide (CO 2 ) inhalation. Under a microscope, fat and connective tissues were carefully removed from the isolated mouse aortae, which were then cut into 2-4 mm long segments. The aortae were cultured in either normal glucose (NG, 5.5 mM) or high glucose (HG, 30 mM), either alone or in combination with xLr ® (10, 30 and 50 μg/mL), or aminoguanidine (AG, 100 μM) 37°C for 48 hours in Dulbecco’s Modified Eagle’s Media (DMEM; Nacalai Tesque, Japan) with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 U/mL penicillin (ScienCell, USA). After incubation, the aortae from each group were carefully placed in oxygenated Krebs solution for vascular function analysis using a wire myograph. The rings were mounted on a wire myograph (Danish Myo Technology, Aarhus, Denmark), filled with Krebs solution at 37°C, and continuously aerated with carbogen (95% O 2 and 5% CO 2 ). Isometric tension changes in the aorta in response to different drugs were recorded using the PowerLab LabChart 6.0 recording system (AD Instruments, Australia). After equilibrating for about 45 minutes at their optimal resting tension (5 mN), the rings were pre-contracted with 60 mM KCl solution to assess viability, then rinsed three times with Krebs solution. To induce a steady contraction, phenylephrine (3 μM) was added when the tension stabilised and returned to baseline. Acetylcholine (ACh) was added cumulatively from 3 nM to 10 μM to generate endothelium-dependent relaxation, while sodium nitroprusside (SNP) was added cumulatively from 1 nM to 10 μM to produce endothelium-independent relaxation. The concentration-response curves were represented as the percentage reduction in contraction induced by phenylephrine prior adding ACh or SNP. The cumulative concentration-response curves were used to determine concentration required to produce 50% of R max (pEC 50 ) and maximum effect (R max ). Human umbilical vein endothelial cells (HUVECs; Lonza, Basel, Switzerland; No.CC-2517) were cultivated in endothelial cell growth medium (ECM) containing endothelial cell growth supplement, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum (FBS), (ScienCell, USA). The cells were cultured at 37°C in a humidified 5% CO 2 incubator. Experiments were conducted when the cells from passages 5-7 achieved 90% confluency. The cells were incubated in either normal glucose (NG, 5.5 mM) or high glucose (HG, 30 mM) co-incubated with xLr ® (10, 30, and 50 μg/mL), apocynin (Apo, 100 μM) and aminoguanidine (AG, 100 μM) for 48 hours. Treated HUVECs were exposed to dihydroethidium dye (DHE, 5 μM, Invitrogen, Carlsbad, CA, USA) in normal physiological saline solution (NPSS) for 15 min, then rinsed three times with NPSS. The fluorescence intensity was recorded using the Leica TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany) with an excitation of 515 nm and emission of 585 nm. Using Image J, the intensity of DHE fluorescence was analyzed, and the fold change in fluorescence intensity compared to the control group was indicated. 4-amino-5-methylamino-2’,7’-difluorofluprescein (DAF-FM DA; Molecular Probes) dye was used to measure the amount of nitric oxide (NO) produced. Briefly, treated HUVECs were incubated with DAF-FM diacetate dye (5 μM) for 20 min at 37°C. After being washed with NPSS, the cells were stimulated for 10 mins with 5 μM calcium ionophore, A23187 (SigmaAldrich, StLouis, MO, USA). The absorbance was measured using a microplate reader at fluorescence excitation and emission of 495/515 nm. The intracellular NO level differences were assessed by calculating the relative fluorescence intensity (F1/F0), where F0 denotes the average fluorescence signals before adding A23187, and F1 indicates fluorescence signals recorded at specific time intervals after A23187 addition. Treated HUVECs were homogenised in ice-cold 1X RIPA buffer, and the lysates were centrifuged at 15,000 xg for 30 min. The supernatant was collected, and a BCA protein assay kit (Thermo, USA) was used to determine the protein concentrations, following the manufacturer protocols. The total protein concentration of 25 μg was separated in 10% sodium dodecyl sulphate (SDS)-polyacrylamide gel and then transferred to an immobilon-P polyvinylidene difluoride (PVDF) membrane at 110 V for 75 min to 120 min. After blocking with 5% bovine serum albumin (BSA) for one hour at room temperature, the membranes were incubated overnight at 4°C with either primary rabbit polyclonal or primary mouse antibody against receptor of AGE (RAGE; 1: 500, Santa Cruz), glyoxalase 1 (Glo1; 1:1000, Santa Cruz), NADPH oxidase 2 (NOX2; 1:500, Santa Cruz), and β-actin (1: 10,000, Santa Cruz). The blots were rinsed with Tris buffered saline containing 0.2% Tween-20 (TBST) on the next day, followed by incubation with respective horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature. After developed with Amersham TM ECL plus Western Blotting detection system, the blots were exposed on X-ray films, which were automatically processed by SRX-101 (Konica, Wayne, NJ). Quantity One 1-D analysis software (Bio-Rad) was used to perform densitometric analysis. Protein expression levels of RAGE, Glo1, and NOX2 were normalised to the housekeeping protein β-actin and then compared to the control.

are presented as means ± standard error of the mean (SEM) from n experiments. Non-linear regression was used to analyse the concentration-response curves. Statistical analysis of the data obtained was performed using the statistical software GraphPad Prism version 10.0.0 for Windows (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com). The two-tailed Student’s t-test was employed to determine statistical significance for the comparison of two groups. One-way ANOVA, followed by Bonferroni’s post-hoc multiple comparison test, was employed for comparisons involving more than two treatments. It is considered significant when the p-value is less than 0.05.

In conclusion, this study demonstrated that both ex vivo and in vitro treatments with xLr ® significantly prevent endothelial dysfunction induced by high glucose by reducing oxidative stress and enhancing the bioavailability of NO partly via inhibition of AGE. These findings further support the use of L. rhinocerus TM02 ® and its extract, the xLr ® as a functional food to prevent cardiometabolic disorders by reducing oxidative stress and safeguarding endothelial function in hyperglycaemic environment. However, clinical studies would be encouraged to investigate whether these effects benefit patients with cardiovascular diseases. Author’s Contribution T.S.W and S.S.S conducted the experiments and analysed the data. T.S.W wrote the manuscript. N.S.T and T.C.S authenticated TM02 ® . S.K.Z and D.D.M provided the funding. C.K.W, S.K.Z, S.Y.F., M.F.M.R and D.D.M designed the study, supervised the work and revised the manuscript. All authors read and approved the final manuscript. Ethics Approval and Consent to Participate The care and use of animals were approved by the Institutional Animal Care and Use Committee (IACUC), Universiti Malaya (Ethics reference no: 2022-240210/PHAR/R/DDM). Declaration of competing interest All authors declare no conflict of interest.

This work is funded by the Ministry of Higher Education (MOHE) Malaysia via the Fundamental Research Grant Scheme (FRGS/1/2021/SKK0/UM/02/8)

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Results are expressed as mean ± SEM (n = 3). #p < 0.05 compared with NG; *p < 0.05 compared with HG | pEC 50 (log M) | R max (%) | pEC 50 (log M) | R max (%) | | | NG (5.5 mM) | -7.47 ± 0.16 | 89.39 ± 1.87 | -7.86 ± 0.14 | 100.6 ± 1.96 | | HG (30 mM) | -6.81 ± 0.08 # | 66.20 ± 3.66 # | -7.79 ± 0.12 | 96.76 ± 1.45 | | HG + xLr ® (10 μg/mL) | -7.07 ± 0.14 | 85.81 ± 3.74 * | -7.86 ± 0.16 | 94.60 ± 0.86 | | HG + xLr ® (30 μg/mL) | -7.16 ± 0.16 | 87.58 ± 2.84 * | -8.10 ± 0.14 | 96.28 ± 1.21 | | HG + xLr ® (50 μg/mL) | -7.29 ± 0.23 | 92.02 ± 0.50 * | -8.12 ± 0.37 | 96.17 ± 1.93 | | HG + AG (100 μM) | -7.12 ± 0.18 | 84.91 ± 5.93 * | -7.74 ± 0.06 | 97.98 ± 2.05 | Figure legends FIGURE 1 : Assessment of isolated aortas from C57BL/6J mice incubated in normal glucose (NG, 5.5 mM), high glucose (HG, 30 mM), xLr ® (10, 30, and 50μg/mL) and aminoguanidine (AG, 100 μM) for 48 h in ACh-induced relaxation (A, B) and SNP-induced relaxation (C, D) . Results are presented as mean ± SEM (n = 3). #p < 0.05 compared with NG; *p < 0.05 compared with HG FIGURE 2 : Reactive oxygen species (ROS) production (A) and the presence of NOX-2 protein (B) measured in HUVECs after treatment with normal glucose (NG, 5.5 mM), high glucose (HG, 30 mM), xLr ® (10, 30 and 50 μg/mL), apocynin (Apo, 100 μM), and aminoguanidine (AG, 100 μM) for 48 h. The upper panel shows representative Western Blots and the bottom panel shows the quantitative data of NOX2 protein expression (B) . Results are presented as mean ± SEM (n = 4). # p < 0.05 compared with NG; *p < 0.05 compared with HG FIGURE 3 : Nitric oxide (NO) production before and after the addition of calcium ionophore A23187 (5 μM) for 10 min in HUVECs incubated with normal glucose (NG, 5.5 mM), high glucose (HG, 30 mM), xLr ® (10, 30 and 50 μg/mL), apocynin (100 μM), and aminoguanidine (100 μM) for 48 h as measured by fluorescence imaging of 4-amino-5-methylamino-2’-7’-digluorofluorescein (DAF-FM DA). Results are presented as mean ± SEM (n = 4). #p < 0.05 compared with NG; *p < 0.05 compared with HG. FIGURE 4 : Western blot and quantitative data showing (A) RAGE, and (B) Glo1 proteins in HUVECs incubated with normal glucose (NG, 5.5 mM), high glucose (HG, 30 mM), xLr ® (50 μg/mL), aminoguanidine (AG, 100 μM) and apocynin (Apo, 100 μM) for 48 h. Results are presented as mean ± SEM (n = 3). #p < 0.05 compared with NG; *p < 0.05 compared with HG. Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Metrics & Citations Metrics Article Usage 142views 83downloads Citations Download citation Tan Sun Wern, M. Sofiullah Siti Sarah, Sharifah Zamiah Syed Abdul Kadir, et al. Lignosus rhinocerus TM02® Extract (xLr®) Alleviates Hyperglycaemia-induced Endothelial Dysfunction By Modulating Oxidative Stress. Authorea. 30 September 2025. 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