The Effect Of 25-hydroxycholesterol On IL-6 Secretion And Senescence Of Thymic Epithelial Cells In Thymic Atrophy

Objective:The thymus begins to degenerate early,leading to a decreased adaptive immune response to tumors and emerging infectious diseases.But the mechanism is not fully clarified.Accumulating evidence has uncovered that the microenvironment dysfunction driven by the secretion of inflammatory factors from thymic stromal cells plays an important role in thymic atrophy.Emerging studies have found the imbalance of cholesterol homeostasis in thymic epithelial cells of aged mice,accompanied by an inflammatory response.We find that the expression of cholesterol 25 hydroxylase(Ch25h)is significantly upregulated in thymic epithelial cells after LPS stimulation,25-hydroxycholesterol(25-HC)may regulate the expression of inflammatory factors.The study aims to elucidate the expression changes of cholesterol 25 hydroxylase(Ch25h)in thymic stromal cells during sepsis.To explore the role and molecular mechanism of cholesterol metabolite 25-HC in thymic atrophy and thymic epithelial cells senescence.To investigate the internal relationship between the cholesterol metabolism pathway and inflammatory cytokine production in the thymic epithelial cell,which provides a theoretical potential therapeutic target for rejuvenation of the immune function.Methods1)The expression of Ch25 h and inflammatory factors are verified in the cortical thymus epithelial cells(c TECs)after being treated with LPS.The c TEC-sh RNACh25 h cell lines are constructed.Western blot is used to determine the Ch25 h gene silencing level.The inflammatory factor IL-6 is measured in the sh RNA-Ch25 h cell lines after being treated with LPS or 25-HC respectively.2)The changes of the phosphorylation IκB are detected with or without 25-HC in the sh RNA-Ch25 h cell lines.The secretion of IL-6 is detected in the culture supernatant after 25-HC treated with or without the IκB phosphorylation inhibitor Bay 11-7082.3)The expression of liver X receptor(LXR)is detected with or without 25-HC treated.The IL-6 secretion and the NF-κB signal-related protein expression are analyzed with or without LXR agonist-stimulated in the sh RNA-Ch25 h cells culture supernatant.4)Acute thymic atrophy is induced by constructing the sepsis model in mice,and the expression differences of Ch25 h and inflammatory factors are verified in vivo.The IL-6 is detected in the thymic stromal cells through tail vein injection 25-HC in mice.5)The expression of Ch25 h is detected in mice thymic stromal cells of different ages,and the serum is also collected to analyze the expression changes of IL-6.6)The cell senescence is induced by DOX in vitro.Cell viability is detected after treatment with different concentrations of 25-HC.The number changes of the senescent cells are detected by cell senescence staining.And cells apoptosis is detected by flow cytometry.Results1)The expression of Ch25 h and inflammatory factors are significantly increased in the c TECs after being treated with LPS.And the cholesterol metabolite 25-HC,catalyzed by enzyme Ch25 h,can promote the secretion of inflammatory factor IL-6,but not the IL-1β and TNF-a.2)Compared with the control group,the secretion of IL-6 is decreased with LPS treated in the sh RNA-Ch25 h cell culture supernatant.And the secretion of IL-6 is increased with the 25-HC treatment in the sh RNA-Ch25 h culture supernatant.3)25-HC can promote the secretion of IL-6 by phosphorylation of IκB,which is blocked by the IκB phosphorylation inhibitors Bay 11-7082.4)The activity of LXR is increased after 25-HC treatment,T0901317 used as the LXR agonist can block the IL-6 secretion by inhibiting the activation of the NF-κB signal pathway.5)Thymus volume decreased significantly in the mice sepsis models.The expression of Ch25 h is significantly increased in the thymus stromal cells.The IL-6 secretion is promoted after being treated with 25-HC in vivo.6)Compared with newborn and young mice,the expression of Ch25 h is slowly decreased with age in the thymic stromal cells,while the IL-6 increases in the serum.7)Doxorubicin(DOX)can induce the senescence of medullary thymic epithelial cells in vitro.8)Compared with the non-senescent cells(without DOX treatment)group,25-HC can significantly reduce the viability of the senescent cells at a particular concentration(10 mM),while not on non-senescent cells.Accordingly,we find that 25-HC can increase the proportion of apoptotic cells and LDH release in senescent cells and ameliorate the accumulation of the senescent cells.These results suggest that 25-HC can clear the senescent m TECs by inhibiting phosphorylation of IκB and decreasing the expression of anti-apoptotic protein Bcl-2.ConclusionThe Ch25 h expression is elevated in thymic stromal cells during acute thymic atrophy,which catalyzes cholesterol to synthesize the metabolite 25-HC.It induces the inflammatory IL-6 secretion in the c TECs by promoting the phosphorylation of IκB.Meanwhile,LXRa plays a negative feedback role in regulating the IL-6 secretion,which can be activated by 25-HC.We also find that the expression of Ch25 h is decreased with age in the age-related thymic degeneration,25-HC can clear the Doxorubicin-induced m TECs senescent cells,which provides a research basis for rejuvenation of the thymic function.