Studies On The Biosynthesis Of Gastrodin By Armillaria Mellea And Its Anti-UVB Damage Effect

The long-term radiation of ultraviolet B（UVB）can cause skin damage,resulting in skin loss of elasticity,wrinkles,redness,swelling,skin and other photoaging phenomena.Therefore,the search for anti-ultraviolet drugs has become a research hotspot.Gastrodia elata,as a food and medicinal herb in China,has many effects such as antidepressant and anti-inflammatory.Gastrodin,as the main active component in the tuber of Gastrodia elata Bl.Gastrodin is mainly obtained through raw drug extraction and chemical synthesis in the market.However,gastrodiae raw material has long cultivation period and heterogeneous quality,and chemical synthesis is highly toxic,costly and environmentally unfriendly.However,biosynthesis has the advantages of mild reaction conditions,high catalytic efficiency,low environmental pollution,and can catalyze some reactions that are difficult to be completed by chemical synthesis.Armillaria species are diverse,which provide rich germplasm resources for screening strains that can produce gastrodin.Previous studies have found that Armillaria luteo-virens Sacc can produce gastrodin,but the biosynthesis of gastrodin by Armillaria sinapina has not been reported.The effect of gastrodin on UVB-induced skin damage and the differential expression of related genes need to be studied.It is of great significance for the further development and utilization of drugs against ultraviolet radiation damage.In this study,the ability of different Armillaria strains to synthesize gastrodin was evaluated by HPLC method,and the optimal strain was selected.Secondly,ABTS⁺,DPPH and FRAP methods were used to evaluate the antioxidant effect of gastrodin in vitro.Thirdly,a mouse model of UVB damage was established,and histopathological staining was used to observe the damage of gastrodin on UVB-damaged skin and the content of collagen fibers and elastic fibers.At the same time,the degree of keratinization of mice skin tissue and malondialdehyde（MDA）,superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）,catalase（CAT）The activity of matrix metalloproteinase-1（TIMP-1）,matrix metalloproteinase-9（MMP-9）and inflammatory cytokines were evaluated.Finally,through the combined analysis of transcriptome and network pharmacology of mouse skin tissue,the protective pathway of gastrodin against UVB damage was explored.Findings were as follows:1.Effects of three Armillaria millaria strains on gastrodin biosynthesis:The three Armillaria millaria strains had different gastrodin biosynthesis abilities.Among them,strain A had the strongest transformation effect,and its gastrodin content was up to 154.57 ug/ml（P<0.05）,which were the ideal transformants.2.Gastrodin has a certain scavenging effect on DPPH and ABTS+free radicals in vitro.The 50%inhibitory concentration（IC50）of gastrodin for scavenging DPPH and ABTS+was0.81 mg/ml and 0.89 mg/ml,respectively.The reduction ability to Fe²⁺was enhanced with increasing concentration in a concentration-dependent manner,with an IC50 of 0.60 mg/ml.3.The effect of gastrodin on UVB-damaged skin of mice Gastrodin can improve the appearance of UVB-damaged skin and significantly improve the keratinization of UVB-damaged skin.Masson staining and Victoria blue staining showed that the content of collagen and collagen fibers in the back skin of mice decreased and the arrangement of collagen fibers was disordered after UVB irradiation.The positive drug and（low,medium and high）gastrodin groups could inhibit the reduction of collagen and elastic fibers,and the improvement effect of gastrodin high dose group was close to that of the blank control group.Gastrodin increased the activities of antioxidant enzymes（SOD,GSH-px,CAT）in UVB-damaged skin,inhibited the increase of MDA and the increase of pro-inflammatory cytokines IL-6 and TNF-αcaused by skin photoaging.4.Transcriptome analysis of the mechanism of gastrodin against UVB damage in mouse skin showed that there were 614 genes changed in the gastrodin group compared with the model group（log2>1,P-value≤0.05）,of which 219 genes were up-regulated and 395 genes were down-regulated.GO and KEGG enrichment analysis of these differential genes showed that the differential genes were mainly involved in hydrolase activity,keratinizing envelope,response to external biological stimuli,immune response,response to cytokines,and response to external stimuli.The metabolic pathways involved included cytokine-cytokine receptor interaction,NOD-like receptor signaling pathway,cell adhesion molecules,TNF signaling pathway,IL-17 signaling pathway,light transduction,arachidonic acid metabolism,C-type lectin receptor signaling pathway,etc.According to transcriptome analysis,gastrodin may regulate inflammatory response and immune response through NOD/IL-17/TNF signaling pathway,and Il1b,Ccl20,Cxcl10,Nod2,Ccl2,Cxcl1,Irf1 and other genes.Network pharmacology analysis showed that the signaling pathways of gastrodin against skin photoaging mainly included IL-17 signaling pathway,TNF signaling pathway,apoptosis,etc.The key core targets included TNF,VEGFA,CASP3,EGFR,IL6,MMP9,TLR4,NFKB1,MYD88,etc.Co-analysis of transcriptome and network pharmacology showed that TLR4 was enriched in both network pharmacology prediction and transcriptome sequencing results.TLR4 could promote the secretion of pro-inflammatory factors such as IL-6 and TNF-αthrough TLR4/My D88/NF-κB pathway.In the transcriptome analysis,gastrodin down-regulated the UVB-induced TLR4gene expression.In Chapter II,gastrodin suppressed the secretion of pro-inflammatory cytokines IL-6 and TNF-αin the mouse skin after photodamage.The combined analysis of transcriptome and network pharmacology indicated that gastrodin might play a protective role against UVB-induced photoaging by regulating TLR4 gene,IL-17 signaling pathway,TNF signaling pathway,Toll-like receptor signaling pathway and C-type lectin receptor signaling pathway.