Effect Of Ganoderma Lucidum Polysaccharides From Changbai Mountain On The Expression Of Inflammatory Mediators In Macrophages Stimulated By Pg-LPS

Background:With plaque biofilm as the initiating factor,periodontitis is a multifactorial inflammatory disease mediated by host immunity and characterized by destruction of periodontal soft and hard tissues.During the occurrence and development of periodontitis,the destruction of periodontal tissue is mostly caused indirectly by the host immune inflammatory response to resist stimulation rather than directly caused by infectious microorganisms.Therefore,inhibiting excessive immune inflammatory response in the process of periodontitis has become one of the entry points for treatment.Anti-inflammatory therapy assisted by traditional Chinese medicine has attracted much attention due to its obvious therapeutic effect,low side effects and low drug resistance.Ganoderma lucidum polysaccharides have therapeutic effects on inflammatory reactions in heart,liver,intestine,pancreas and other organs and tissues.In the previous experiments,our research group found that the crude extract of Ganoderma lucidum polysaccharides from Changbai Mountain could inhibit periodontitis inflammation in mice and slow down alveolar bone absorption.On this basis,this experiment further purified the crude polysaccharide of Ganoderma lucidum from Changbai Mountain,analyzed its physical and chemical properties,and explored its influence and action pathway in the model of periodontitis in vitro,hoping to lay the foundation for the prevention and treatment of periodontitis with Ganoderma lucidum polysaccharides and provide new strategies for the treatment of periodontitis with traditional Chinese medicine.Methods :1.Purified Ganoderma lucidum polysaccharide was obtained by DEAE-52 cellulose column chromatography and molecular sieve purification after the extracting crude polysaccharide by the water extraction-ethanol precipitation technology.High performance size exclusion chromatography-multiangle laser light scattering,high performance anion exchange chromatography and Fourier transform infrared spectroscopy were used to detect the molecular weight,monosaccharide composition and glycosidic bond of polysaccharides.2.CCK8 assay was used to detect the effect of Ganoderma lucidum polysaccharides on the proliferation of RAW264.7 macrophages under normal and inflammatory conditions.Transwell migration assay was used to detect the effect of Ganoderma lucidum polysaccharides on the migration of RAW264.7 macrophages under inflammatory conditions.3.The effect of Ganoderma lucidum polysaccharides from Changbai Mountain on the m RNA expression levels of TNF-α,IL-1β,IL-10 and i NOS in RAW264.7macrophages under inflammatory conditions was detected by quantitative Real-time PCR.4.The effect of Ganoderma lucidum polysaccharides on the expression of inflammatory mediators TNF-α,IL-1β,IL-10 in the supernatant of RAW264.7macrophages under inflammatory state was detected by ELISA.5.Nitrate reductase method was used to detect the effect of Ganoderma lucidum polysaccharides on NO content in the supernatant of RAW264.7 macrophages under inflammatory state.Results :1.On the basis of previous work,this study further isolated and purified the relatively purified Ganoderma lucidum polysaccharide from Changbai Mountain,with a weight-average molecular weight of 6.465 k Da.It contains a variety of monosaccharide components mainly composed of glucose,galactose,mannose,xylose,rhamnose and glucuronic acid（composition ratio > 5%）,including O-H,C-H,C=O,C-H,C-OC,C-O-H,α-pyranosyl and β-D-pyranosyl linkages.2.After stimulation with 10 μg/m L Pg-LPS for 24 h,the proliferation activity and migration activity of RAW264.7 macrophages were enhanced,and the supernatant content and expression of related m RNA of the inflammatory mediators TNF-α,IL-1β,IL-10 and NO were increased.3.25 μg/m L,50 μg/m L,100 μg/m L concentration of Ganoderma lucidum polysaccharides can promote the proliferation of RAW264.7 macrophages in normal and inflammatory state stimulated by Pg-LPS,inhibit the migration of RAW264.7macrophages in inflammatory state.4.25 μg/m L,50 μg/m L,100 μg/m L concentration of Ganoderma lucidum polysaccharides reduce the m RNA and protein expression of pro-inflammatory cytokines TNF-α,IL-1β of RAW264.7 macrophage under inflammatory state,and improve anti-inflammatory cytokines IL-10 m RNA expression and secretion level.5.25 μg/m L,50 μg/m L,100 μg/m L Ganoderma lucidum polysaccharides can reduce the i NOS m RNA expression and the NO content of RAW264.7 macrophage under inflammatory state.Conclusions :1.In this study,Ganoderma lucidum polysaccharide from Changbai Mountain was successfully isolated and purified.The analysis of physicochemical properties showed that it was a compound Ganoderma lucidum polysaccharide containing multiple monosaccharide components and glycosidic bonds.2.Ganoderma lucidum polysaccharides at different concentrations（25 μg/m L,50μg/m L,100 μg/m L）can change the biological behavior of RAW264.7 macrophages,including promoting the proliferation of RAW264.7 macrophages under normal and inflammatory conditions,and inhibiting their migration under inflammatory conditions.3.Ganoderma lucidum polysaccharides at different working concentrations（25μg/m L,50 μg/m L,100 μg/m L）could regulate the contents of inflammatory mediators TNF-α,IL-1β,IL-10,and NO in the supernatant of RAW264.7 macrophages and the expression of intracellular related m RNA,and inhibit the inflammatory response of macrophages in periodontal inflammatory microenvironment in vitro.