Inonotus Sanghuang Polyphenols Modulate The Crosstalk Between Macrophages And Adipocytes

BackgroundObesity is a kind of chronic metabolic disease caused by multiple factors,which is characterized condition in which adipose tissue loses its normal proportion to other tissues due to excessive accumulation and/or abnormal distribution of adipose in the body.However,the etiological mechanism is unknown.Epidemiological surveies show the incidence of obesity is on the rise in the world.What’s more,obesity leads to a series of problems of health,social and psychological.From a health perspective,obese people are not a single group.Since the 1980 s,studies on the correlation between obesity and metabolic diseases have been increasing.The metabolic status of obese people is divided into "normal" and "high-risk",according to whether obesity is associated with insulin resistance,body fat distribution,lipid and adipokine spectrum,inflammatory factor spectrum,genetic polymorphism and so on.And the "high-risk" obese patients with energy metabolism disorder are more likely to suffer from atherosclerosis,type 2 diabetes mellitus(T2DM),hypertension,cardiovascular diseases,stroke,malignant tumors and other diseases,leading to early death,disability and the decline in the quality of life.Obesity results from the metabolic imbalance of energy in the body,which is manifested on the level of cells as excessive hypertrophy and appreciation of adipocytes,intracellular triglyceride accumulation and intracellular endocrine dysfunction.Adipose tissue is not only an energy storage organ,but also an important endocrine tissue,which can secrete a variety of adipocytokines to regulate the energy metabolism balance of the body,and play an important role in obesity-related inflammation and metabolic disorders.These adipocytokines are produced by adipocytes or macrophages in adipose tissue,which are called adipose tissue macrophages(ATMs).There is substantial evidence that ATMs exhibit the phenotypic change from M2 macrophages to M1 macrophages polarization during the course of obesity,thereby accelerating adipose tissue inflammation.M1 macrophages secrete proinflammatory cytokines and promote the decomposition of adipocytes and the continuous production of free fatty acids(FFA).Dysregulation of adipocytokines and overproduction of FFA will not only result in ectopic lipid accumulation and lipotoxicity(this is directly related to the occurrence of obesity-related complications such as atherosclerosis,hypertension,metabolic syndrome,insulin resistance and so on),but also activate pro-inflammatory signaling pathways such as Toll-like receptor 4(TLR4)signaling in macrophages and promote the release of inflammatory factors,promoting the development of chronic inflammation in adipose tissue.The vicious cycle augments chronic inflammatory changes.What we need to do is to curb the vicious cycle and arrest or reduce obesity-related complications.Sanghuang mushroom(sanghuang)is a popular medicinal polypore used throughout East Asia and plays a physiological function in resistive effects of oxidize and germ and tumor,as well as reducing the blood sugar and lipemia and improving immunity.Inonotus sanghuang(I.sanghuang)is one species of sanghuang mushroom and a white-rot fungus in the family of hymenochaetaceae.Ethanol extract of I.sanghuang mycelia produced from liquid fermentation scavenged DPPH and hydroxyl radicals has been shown to have antioxidant activity due to the existing phytochemicals(polyphenolics)such as rutin,eriodictyol,naringenin,and sakuranetin.Our recent in vitro study has shown that the anti-oxidant,anti-proliferative,and anti-microbial activities have been found in I.sanghuang exact(polyphenols)including from another sanghuang species,wild I.sanghuang from the Aershan Region of Inner Mongolia(Inner Mongolia,China).However,there are no studies exploring the anti-inflammatory and immunomodulating properties of wild I.sanghuang from the Aershan Region of Inner Mongolia and its use for the prevention/treatment of inflammation-related diseases.ObjectiveThis study was to explore the effect of Inonotus sanghuang extract(ISE)on macrophages in adipose tissue and the interaction between macrophages and adipocytes using the coculture system between murine RAW264.7 macrophages and 3T3-L1 adipocytes that could induce the inflammatory response of adipose tissue.Methods1.Murine RAW264.7 macrophages were divided into five groups including normal control group,the LPS group,LPS + ISE(0.5,1.0 and 2.0μg/mL)group.In the presence of different doses of ISE,RAW264.7 macrophages were stimulated with LPS for 24 hours and then the cell-free suprenantant were collected to measure the levels of the inflammatory cytokines including NO,TNF-α,IL-6 and MCP-1 by ELISA.In addition,the cell viability of ISE on RAW264.7 macrophages was determined by CCK8 method.2.Murine RAW264.7 macrophages were divided into five groups including normal control group,the L1 CM group,L1 CM + ISE(0.5,1.0 and 2.0μg/mL)group.In the presence of different doses of ISE,RAW264.7 macrophages were stimulated with conditioned medium of hypertrophied 3T3-L1 adipocytes(L1CM)for 24 hours and then the cell-free suprenantant were collected to measure the levels of the inflammatory cytokines including NO,TNF-α,IL-6 and MCP-1 by ELISA.3.In the presence of different doses of ISE,RAW264.7 macrophages were cocultured with differentiated 3T3-L1 adipocytes.RAW264.7 macrophages and 3T3-L1 adipocytes were cultured separately under the same cultured conditions for control groups.Different concentrations of ISE were administrated meanwhile as that of coculture.After cocultured for 24 hours,the cell-free suprenantant were collected to measure the levels of the inflammatory cytokines including NO,TNF-α,IL-6 and MCP-1 by ELISA.4.RAW264.7 macrophages were stimulated with LPS or L1 CM in the presence ISE for 24 h.The MAPK(JNK,p38 and ERK),NF-κB and STAT3 signals were determined by western blotting.5.After RAW264.7 macrophages were treated with different doses of ISE for 1h,they were put into the upper chamber of transwell and then put into 24-well plate that was added the L1 CM in 24-well plate.The migrated cells were counted under microscope after incubation for 4 hours.Results1.ISE inhibited inflammatory mediators including NO,TNF-α,IL-6 and MCP-1 released by LPS-or L1CM-induced RAW264.7 macrophages.2.ISE administration decreased the secretion of proinflammatory cytokines including NO,TNF-α and IL-6 released by the cocultures of RAW264.7 macrophages and 3T3-L1 adipocytes.3.ISE blocked the L1CM-stimulated migration of RAW264.7 macrophages.4.The anti-inflammatory effects of ISE were caused by inhibition of p38 MAPK,ERK MAPK and STAT3 signals activity in LPS-or L1CM-induced RAW264.7 macrophages.ConclusionCurrent study indicated that ISE suppressed the interaction between macrophages and adipocytes which might contribute to attenuating chronic inflammation in adipose tissue and improving obesity-related complication.Data suggest that ISE might be a valuable medicinal food effective in improving obesity-related complications.