Ganoderma total sterol (GS) and GS1 protect rat cerebral cortical neurons from hypoxia/reoxygenation injury

doi:10.1016/j.lfs.2004.08.013

Life Sciences

Volume 76, Issue 9, 14 January 2005, Pages 1027-1037

https://doi.org/10.1016/j.lfs.2004.08.013 Get rights and content

Abstract

The effect of Ganoderma total sterol (GS) and its main components(GS₁) on rat cortical neuronal cultures exposed to hypoxia/reoxygenation (H/R) was studied in vitro. GS (0.01,0.1,1 μg/ml) increased neuron viability following H/R. GS also significantly reduced malondialdehyde content and reactive oxygen species production and increased manganese superoxide dismutase (Mn-SOD) activity; furthermore, the translocation of nuclear factor-kappa B and the production of interleukin-1β and tumor necrosis factor α induced by H/R were also blocked. These findings suggest that GS might be useful in treating H/R-induced oxidative stress and inflammatory response. We also hypothesized that Mn-SOD might play a critical role in the neuroprotective effect of GS against H/R injury. In addition, pretreatment with GS₁ (0.01, 0.1, 1 μg/ml) significantly attenuated the decline of neuron viability and the formation of reactive oxygen species. Furthermore GS₁ possessed more potent protective effect on neurons compared with GS at the same dose. These findings demonstrated that GS₁ is the main component in GS; and play a critical role in the neuroprotective effect of GS against H/R.

Introduction

The molecular basis for the development of delayed neuronal death induced by brain ischemia/reperfusion is not well understood. Among various speculations, oxidative stress and systemic inflammatory response are considered to be two major factors (Chan, 1996) Oxidative stress can be defined as the pathogenic outcome of the overproduction of oxidants that overwhelm the antioxidant capacities of free radical quenching enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Changes induced by ischemia-reperfusion can include an increase in oxidant factors, impairment of endogenous antioxidant systems, or both. The inflammatory response following cerebral ischemia involves the infiltration of peripheral leukocytes from the circulation into the brain as well as the upregulation of pro-inflammatory cytokine tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6).

Ganoderma lucidum, historically a very popular medicinal fungi in oriental cultures, was listed in the first folk pharmacopoeia in the second century B.C. In modern clinical trials, the preparations of Ganoderma lucidum show therapeutic efficacy in treating cancer, diabetes, neurasthenia, and insomnia, and so forth (Lin, 2002). Sterol is one of the main components purified from Ganoderma lucidum. Many studies have reported that plant sterols possess anti-oxidant bioactivity (Shi et al., 1998, Van Rensburg et al., 2000) in part due to an increase the activities of anti-oxidant enzymes such as cytosolic SOD, catalase and GSH-px(Cai et al., 2002). Other investigations of plant sterol have shown it to have direct immunomodulatory activity: the secretion of the monokines IL-6 and TNF-α by endotoxin activated human monocytes was significantly inhibited (Lee et al., 2003).

Our previous study had provided the in vivo evidence that GS protects rats against cerebral ischemia reperfusion injury. GS decreased rat infarct volume and brain edema, improved cerebral histological damage and rat behavioral outcomes. And the protective effect of GS may be associated with its antioxidant activity (Zhao et al., in press).

In the present study, we examined the effects of GS and GS₁ on hypoxia/ reoxygenation injury in cortical neurons in vitro and characterized the mechanisms involved in these activities. In addition, we compared the effect of GS with GS₁.

Section snippets

Cell culture

Dissociated cortical cells from Sprague-Dawley 1-day-old rats were grown in DMEM (Gibco/BRL, Geithersburg, MD, USA). Cells were plated with a density of 1 × 10⁶ cells/ml in plates coated with 12.5 mg/l of poly-L-lysine (Sigma, St. Louis, MO, USA). Cells were cultured for 7 days in an atmosphere of 5% CO₂/95% air at 37 °C. Cytosine arabinoside (10 μmol/l, Sigma, USA) was added 4 days after plating to inhibit the replication of nonneuronal cells. Our neuronal cultures consisted of approximately

Cell viability

H/R caused neuronal death, as shown by a decrease in MTT reduction; Pretreatment with GS and GS₁ (1 μg/ml) significantly attenuated the decline of MTT reduction. And GS₁ played more marked protective effect on neurons (Table 1).

Oxidative stress and SODs’ activities

We performed confocal analysis with a ROS-specific fluorogen, DCF-DA, to examine the changes in ROS production in rat cortical neurons. The effect of GS and GS₁ on H/R-induced stimulation of ROS is shown in Fig. 1.A. Exposing cortical neurons to H/R significantly

Discussion

In this study, we confirmed that H/R exerts neurotoxic effects on rat cultured cortical neurons. These effects were accompanied by free radical generation, lipid peroxidation production, NF-κB activation, and the production of inflammatory cytokines IL-1β and TNFα. Our results show that in cortical neurons, GS lends remarkable protection against H/R through mechanisms including a decrease in free radical production and an inhibition of cytokine production. And GS₁ have more great effect on

Conclusion

In summary, this is the first report showing that GS and GS1 exhibits remarkable protection against hypoxia/reoxygenation injury in rat cortical neurons. As a natural (plant) drug, Ganoderma lucidum sterol has fewer undesirable side effects than other agents. Hence, our findings could provide novel therapeutic targets to prevent neuronal death following stroke.

Acknowledgements

Sterol extract from Ganoderma lucidum was kindly provided by Professors LIN Shu-Qian of the Fuzhou Institute of Green Valley Bio-Pharm Technology. This research was supported by the Research Fund of Shanghai Green Valley Holding Co., Ltd.

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Anti-inflammatory effects of Ganoderma lucidum sterols via attenuation of the p38 MAPK and NF-κB pathways in LPS-induced RAW 264.7 macrophages
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The anti-inflammatory activity of Ganoderma triterpenes is also associated with the MAPK signaling pathways, some triterpenes significantly suppress LPS-induced phosphorylation of ERK1/2 and JNK but not p38 (Dudhgaonkar et al., 2009). However, few studies have looked into anti-inflammatory activities and underlying molecular mechanism of Ganoderma sterols, which are another important kind of active ingredients in G. lucidum with various biological and pharmacological activities such as anti-tumor (Gan et al., 1998; Chen et al., 2017), antiviral (el-Mekkawy et al., 1998), cholesterol lowering (Hajjaj et al., 2005), neuroprotection (Zhao et al., 2005), antifungal (Smania et al., 2003), and antimicrobial (Vazirian et al., 2014) etc. So far, it remains unclear whether sterols contribute to the anti-inflammatory activity of G. lucidum.
Ganoderma lucidum exhibits pronounced anti-inflammatory effects, polysaccharides and triterpenoids are regarded as major constituents displaying the anti-inflammatory activities, whether sterols contribute to this activity remains unclear. Herein Ganoderma lucidum sterols (GLS) were innovatively isolated by a single-step procedure, the profile of GLS was characterized by HPLC-ELSD and shown similar to that of sterols separated by a traditional method, but much higher in content. Furthermore, GLS inhibited inflammation in macrophages by significantly attenuating LPS-induced cell polarization as well as releases and mRNA expressions of pro-inflammatory mediators NO, TNF-α, IL-1β and IL-6. Moreover, the anti-inflammatory activity of GLS was mediated by MAPK and NF-κB pathways, GLS suppressed MAPK pathways by blocking phosphorylation of p38 but not ERK and JNK, which is complementary with inhibitory effects of Ganoderma polysaccharides and triterpenes on JNK and ERK, indicating Ganoderma sterols may exert synergistic anti-inflammatory effect with polysaccharides and triterpenes. GLS also inhibited NF-κB pathways by restraining phosphorylation and degradation of IκB-α and blocking phosphorylation of NF-κB p65. Molecular docking confirmed that sterols of GLS were directly bound to active sites of p38 and p65 to suppress their activation. Therefore, our findings suggest GLS as natural and safe anti-inflammatory agents to prevent and treat inflammatory diseases.
Protective effects of curcumin and Ganoderma lucidum on hippocampal damage caused by the organophosphate insecticide chlorpyrifos in the developing rat brain: Stereological, histopathological and immunohistochemical study
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Recent studies have demonstrated benefits to the brain; for example, the water extract of G. lucidum (WGL) induces neuronal differentiation and neurite growth of PC12 cells and has hypnotic and antidepressant effects as in vivo (Chu et al., 2007; Matsuzaki et al., 2013). G. lucidum polysaccharides (GLP), one of the main active components of G. lucidum, also protect neurons from hypoxia/reoxygenation damage as in vitro (Zhao et al., 2005). In addition, fat obtained from G. lucidum spores protects dopaminergic neurons and improves behavioral deficits in a Parkinson's disease rat model (Ding et al., 2010).
The aim of this study is to draw attention to the possible effects of chlorpyrifos exposure on the developing rat hippocampus in the prenatal period and to determine whether these effects can be reduced with various antioxidant substances.
Pregnant rats were divided into 7 groups.; Chlorpyrifos (CPF), Curcumin (CUR), Ganoderma lucidum (GNL), Chlorpyrifos + Curcumin (CPF + CUR), Chlorpyrifos + Ganoderma lucidum, (CPF + GNL), SHAM and Control (C). After the experiments, brain tissues were evaluated by stereological and immunohistochemical methods.
As a result of the stereological analyzes, it was determined that the number of pyramidal neurons in the hippocampus of the CPF group decreased significantly from all other groups. In contrast, the number of neurons in the CPF + CUR and CPF + GNL groups was significantly higher than the CPF group. In addition, immunohistochemical analyzes showed that the density of cells stained with glial fibrillar acidic protein (GFAP) positive in all areas in the hippocampus of the rats in the CPF group was significantly higher compared to the control group, whereas in the CPF + CUR and CPF + GNL groups were less than the CPF group. As a result, the exposure of CPF in the prenatal period caused neurotoxicity in the brain hippocampus, whereas CUR and GNL reduced this toxicity caused by CPF.
Preventive and therapeutic effect of Ganoderma lucidum on kidney injuries and diseases
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Ganoderma lucidum (G. lucidum, Lingzhi) is a well-known Chinese traditional medicine to improve health and to treat numerous diseases for over 2000 years in Asian countries. G. lucidum has the abundant chemical components such as triterpenes and polysaccharides, which have various biological activities including anti-oxidation, anti-inflammation, anti-liver disorders, anti-tumor growth and metastasis, etc. Recently, many lines of studies have elucidated the therapeutic effects of G. lucidum and its extractions on various acute kidney injury (AKI) and chronic kidney disease (CKD) pathogenesis, including autosomal dominant polycystic kidney disease, diabetic nephropathy, renal proximal tubular cell oxidative damage and fibrotic process, renal ischemia reperfusion injury, cisplatin-induced renal injury, adriamycin-induced nephropathy, chronic proteinuric renal diseases, etc. Clinical researches also showed potent anti-renal disease bioactivities of G. lucidum. In this chapter, we review experimental and clinical researches and provide comprehensive insights into the renoprotective effects of G. lucidum. In recent years, renal diseases have gradually aroused attention on account of their booming prevalence worldwide and lack of effective therapies. Although the complicated pathogenesis of kidney diseases, such as acute kidney injury (AKI) and chronic kidney diseases (CKD) have been intensively studied. The morbidity and mortality of AKI and CKD still rise continuously. Thanks to the conventional experience and the multi-target characteristics, natural products have been increasingly recognized as an alternative source for treating renal diseases.
Ganoderic acid -loaded solid lipid nanoparticles ameliorate D-galactosamine induced hepatotoxicity in Wistar rats
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G. lucidum, basidiomycetes fungi is a medicinal mushroom; sterols, triterpenes and polysaccharides are the vital pharmacologically active constituents of G. lucidum [1]. Earlier studies have revealed that the ganoderic acid (GA) (triterpenes) from G. lucidum have hepatoprotective [2], and neuroprotective activity [3]. In addition, GA also exhibited antihepatitis [4], hypocholesterolemic, antihistaminic, antitumor [5] and anti-HIV effects [6].
Ganoderic acid has been known as a key bioactive constituent in Ganoderma lucidum, commonly used for preventing and treating hepatopathy of various etiologies. This study was designed to investigate the hepatoprotective and antioxidant activity of ganoderic acid loaded solid lipid nanoparticles in d-galactosamine induced hepatotoxicity in male Wistar rats. Ganoderic acid loaded solid lipid nanoparticles was prepared by solvent injection technique and prepared formulation was characterized for various parameters and final formulation evaluated for its hepatoprotective study in Wistar rats. The prepared optimized formulation presented particle size of 122.7 ± 8.6 nm with polydispersity index of 0.27 ± 0.03 and zeta potential of −18 ± 1.4 mV. The drug loading, entrapment efficiency and nanoparticle yield were found to be 21.6 ± 1.23%, 70.16 ± 3.21% and 60.5 ± 2.75%, respectively. In vitro release study of ganoderic acid from nanoparticles at pH 7.4 exhibited biphasic release pattern with an initial burst release followed by a sustained release. Treatment of rats with prepared formulation significantly restored the levels of serum hepatic markers and antioxidant enzymes as compared to free ganoderic acid in d-galactosamine induced hepatotoxicity in rats. Histopathological examination also supported a marked hepatoprotective effect of prepared formulation. In conclusion, nano-formulation of ganoderic acid has significantly attenuated markers of hepatic damage compared to free ganoderic acid and may represent a potential hepatoprotective nano-formulation for amelioration of d-galactosamine induced liver injury.
Pharmacological effects of natural Ganoderma and its extracts on neurological diseases: A comprehensive review
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Citation Excerpt :
These findings suggested that GS might help treat H/R-induced oxidative stress and inflammatory responses. Furthermore, since pretreatment with GS1 significantly attenuated the decline of neuronal activity and the formation of ROS, and that GS1 had a stronger protective effect on neurons than GS at the same dose in the neuroprotective effect of GS on H/R [187]. In the model of I/R, results showed that pretreatment with GLA for 3 days and 7 days reduced the loss of neurons in the hippocampus, decreased the contents of malondialdehyde in the hippocampus and serum, and decreased the levels of TNF-α and IL-8 levels.
Ganoderma, has been used for clinical applications for thousands of years as a highly-nutritious and significantly-effective medicinal herb. The active components and efficacy of Ganoderma are constantly being explored and supplemented every year. In recent years, more and more literature has reported the pharmacological effects of Ganoderma on anti-tumor, liver protection and immunity enhancement, especially on neuroprotection. Numerous research works on the neuroprotective effects of Ganoderma have been documented (e.g., modulation of neurogenesis, amelioration of Alzheimer's disease, therapeutic effect on epilepsy, the protective effect on neural cells in stroke injury, etc.) thus it has drawn increasing attention. However, an integrated and comprehensive review of recent research findings has not been detailed in any great depth. Therefore, the purpose of this review is to summarize and elucidate recent progress of neuroprotective effects of natural Ganoderma and its extracts.
Neuroprotective effects of water-soluble Ganoderma lucidum polysaccharides on cerebral ischemic injury in rats
2010, Journal of Ethnopharmacology
To investigate the neuroprotective effects of water-soluble Ganoderma lucidum polysaccharides (GLPS) on cerebral ischemic injury in rats, and to explore the involved mechanisms.
Two models [middle cerebral artery occlusion (MCAO) in Sprague–Dawley (SD) rats and oxygen and glucose deprivation (OGD) in primary cultured rat cortical neurons] were employed to mimic ischemia–reperfusion (I/R) damage, in vivo and in vitro, respectively. Cerebral infarct area was measured by tetrazolium staining, and neurological functional deficits were assessed at 24 h after I/R. Neuronal apoptosis was studied by Nissl staining and DNA fragmentation assay. Neuronal injury was assessed by morphological examination using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) leakage, cell viability was measured by sodium 3′-1- (phenylaminocarbonyl)-3, 4-tetrazolium-bis (4-methoxy-6-nitro) benzene sulfonic acid (XTT) reduction. Neuronal apoptosis was determined by flow cytometry, and electron microscopy was used to study morphological changes of neurons. Caspase-3, -8 and -9 activation and Bcl-2, Bax protein expression were determined by western blot analysis.
Oral administration of GLPS (100, 200 and 400 mg/kg) significantly reduced cerebral infarct area, attenuated neurological functional deficits, and reduced neuronal apoptosis in ischemic cortex. In OGD model, GLSP (0.1, 1 and 10 μg/ml) effectively reduced neuronal cell death and relieved cell injury. Moreover, GLPS decreased the percentage of apoptotic neurons, relieved neuronal morphological damage, suppressed overexpression of active caspases-3, -8 and -9 and Bax, and inhibited the reduction of Bcl-2 expression.
Our findings indicate that GLPS protects against cerebral ischemic injury by inhibiting apoptosis by downregulating caspase-3 activation and modulating the Bcl-2/Bax ratio.

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Ganoderma total sterol (GS) and GS1 protect rat cerebral cortical neurons from hypoxia/reoxygenation injury

Abstract

Introduction

Section snippets

Cell culture

Cell viability

Oxidative stress and SODs’ activities

Discussion

Conclusion

Acknowledgements

Chemistry & Biology

Journal of Molecular and Cellular Cardiology

Life Sciences

Oxidative metabolism modulates signal transduction and micronucleus formation in bystander cells from alpha-particle-irradiated normal human fibroblast cultures

Little JB Cancer Research

Antioxidative and free radical scavenging effects of ecdysteroids from Serratula strangulate

Canadian Journal of Physiology and Pharmacology

Role of oxidants in ischemic brain damage

Stroke

Transcriptional regulation of endothelial cell adhesion molecules: NF-k B and cytokine-inducible enhancers

FASEB Journal

Superoxide dismutases

Annual Review of Biochemistry

Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome c and subsequent DNA fragmentation after permanent focal cerebral ischemia in mice

Journal of Neuroscience

Rapid poteolysis of IκB-alpha is necessary for activation of transcription factor NF-κB

Nature

Ganoderma total sterol (GS) and GS₁ protect rat cerebral cortical neurons from hypoxia/reoxygenation injury