Therapeutic principle of ganoderma lucidum capsules against experimental autoimmune encephalomyelitis pathogenesis and its bioactivity-directed quality control study

Abstract

Abstract: Multiple Sclerosis (MS) is an immune-mediated, neurological disease in central nervous system (CNS). It affects over 2.22 million people globally and usually is diagnosed in young adults at working age. With the poor therapeutic outcome, most patients eventually develop into handicap. Therefore, seeking effective therapeutic strategy for MS treatment is timely important. In the present study, I proposed a potential therapeutic Traditional Chinese Medicine processed product Ganoderma lucidum Capsule (GLC), exhibited efficacy in active experimental autoimmune encephalomyelitis (EAE) with amelioration of clinical symptoms, alleviation of disease progression, and pathological improvements. On the other hand, aiming at the deficiency of quality control of Ganoderma lucidum, I established an efficacy-fingerprint analysis method for quality control of Ganoderma lucidum capsule. In the therapeutic study, our results revealed that GLC extract alleviated EAE progression via reducing inflammation and demyelination in spinal cords, inhibiting microglia activation and suppressing the immune response in the peripheral system. Consistent with that in vitro study, GLC extract inhibited p65 phosphorylation and down-regulated the expression of IL-1β, IL-6, iNOS and 3NT in the microglia under LPS stimulation. Then, I reported that GLC extract has neuroprotective effect against inflammatory infiltration- derived neuronal apoptosis, through mediating the protein expression levels of capcase-3, Bax and Bcl-2. Meanwhile, GLC decreased nitrative stress via reducing the peroxynitrite (ONOO¯) production with downregulated iNOS. I also found that GLC extracts has neuroprotective effects on SIN-1 challenged PC12 via upregulating the expression level of bcl-2 and reducing ONOO¯-induced nitrative stress by decreasing ONOO¯ production. Overall, our study sheds novel light on the therapeutic effect of GLC on MS and its underlying mechanisms are associated with anti-inflammation effect and neuroprotective effect under microglia-derived nitrative damage. For chemical studies, I firstly investigated the potential bioactive compounds from GLC extract. Six Ganoderic acids from GLC extract have been screened out with strong ONOO¯ scavenging capability, including Ganoderic acid C2, G, B, H, D and F. Moreover, Ganoderic acid A and H were found to cross the blood-brain-barrier (BBB) in active EAE. Based on the bioactive compounds, a reliable quantitative analysis methods and fingerprint profile of GLC for quality control was successfully established by HPLC-VWD system. In summary, to my knowledge, this is the first report of the application of Ganoderma lucidum in the treatment of EAE/MS, and the first application of the combination of bioactive properties and fingerprint analysis for the quality control of Ganoderma lucidum. These results serve to establish new therapeutic strategy and develop drug candidates for the treatment of EAE/ MS. Meanwhile, it lays the foundation for the quality control of Ganoderma lucidum and its processed products. (430 words)