Exploratory Study On The Anti-Inflammatory Effect Of Dendrobium Officinale Neutral Polysaccharides On Human Oral Mucosal Epithelial Keratinocytes

Objective:The active polysaccharide component was extracted from Dendrobium officinale to study the effect of Dendrobium officinale polysaccharide（DOP）on the proliferation and differentiation of human normal oral mucosal epithelial keratinocytes（Keratinocytes,KCs）.And the effect on the expression level of inflammatory cytokines（IL-6,IL-8,TNF-α）and activation level of Pg-LPS/TLR2 signaling pathway in KCs in vitro inflammation model.To clarify the effect of Dendrobium officinale polysaccharide on the proliferation,differentiation and anti-inflammatory protection of human oral keratinocytes,and provide a reference for the development of Dendrobium officinale for oral diseases.Methods:1.Extract and isolate the most active polysaccharide component from Dendrobium officinale and further purify it:The crude polysaccharide is deproteinized by the Sevage method,and then subjected to DEAE-52 cellulose column chromatography,and decolorized and preliminary fractionated by gradient elution using deionized water,0.05,0.1,0.2,0.3,0.5M Na Cl solution,and separated in sequence.Six polysaccharides are numbered DOP-1,DOP-2,DOP-3,DOP-4,DOP-5,and DOP-6 in the order of elution.Among them,DOP-1 was eluted from deionized water and was a neutral polysaccharide;the remaining five were eluted from a group of 0.05-0.5M gradient Na Cl solution and were acidic polysaccharides.Purification by GFC method:The DOP-1 with the highest content was further purified through a Sephadex-G100 gel chromatography column to obtain a single DOP-1.2.In vitro culture,passage and expansion of human normal oral mucosal epithelial keratinocytes,and freeze storage in time,so that the cells used in the experiment are in the 4th to6th generation.3.CCK-8 reagent was used to detect the effect of 6.25-400μg/ml DOP-1 on KCs cell viability.4.CCK-8 reagent was used to detect the effect of DOP-1 on the proliferation ability of KCs.5.Serum-free,high Ca²⁺induced KCs differentiation,RT-PCR was used to detect the effects of DOP-1 on the expression of KCs differentiation marker proteins:loricrin（LOR）,involucrin（IVL）gene expression.6.KCs were stimulated with Pg-LPS to establish an in vitro inflammation model.RT-PCR was used to detect the expression levels of IL-6,IL-8 and TNF-α,and ELISA was used to detect the levels of IL-6,IL-8 and TNF-αin the culture supernatant.Western Blot was used to detect the activation level of TLR2 activated by Pg-LPS.Result:1.The extraction rate of Dendrobium officinale polysaccha-rides were 19.70%,and the total polysaccharide content was 63.76%.2.DOP-1concentration≥200μg/ml had significant effect on inhibiting cell viability of KCs（P<0.0001）,while concentration≤100μg/ml had no effect on inhibiting cell viability.In the concentration range of 12.5-100μg/ml,DOP-1 has a negative correlation between KCs proliferation and its concentration.3.High concentration of DOP-1（50,100μg/ml）significantly inhibited KCs expression of loricrin（P<0.01）.There is no significant effect on the expression of involucrin,indicating that DOP-1 at this concentration has a certain inhibitory effect on KCs keratinization.4.DOP-1 can inhibit KCs expression,secrete IL-6,IL-8,and TNF-αinduced by Pg-LPS in a concentration range of 12.5～100μg/ml,and it has a positive effect on the concentration of TLR2 receptor protein.The relationship indicates that DOP-1 has anti-inflammatory and anti-apoptotic effects in a concentration-dependent manner.Conclusion:1.The most active polysaccharide component in Dendrobium officinale is neutral polysaccharide（DOP-1）.2.Low concentration of DOP-1（≤100μg/ml）promotes the proliferation of KCs,higher concentration of DOP-1will delay the differentiation of KCs,and high concentration（≥200μg/ml）will obviously inhibit the cell activity of KCs.3.DOP-1 can improve the anti-inflammatory ability of keratinocytes and has a positive correlation with its drug concentration.