Mechanism Of Ligustilide In Delaying Thymic Immune Senescence By Regulating The Spatial Distribution Of Thymic Epithelial Cells

Objective The thymus is a crucial organ involved in the processes of aging and immunity.This study aimed to investigate the abnormalities in the spatial distribution of thymic epithelial cells(TECs)in the acute immunosenescent thymus.To achieve this,Adriamycin(ADM)-induced acute thymic degeneration mice and their organ models were constructed.The aim of the study was to investigate the effect and mechanism of Ligustilide(LIG),an active component of traditional Chinese medicine Angelica sinensis,in delaying thymic immune senescence.Methods Section One The acute thymic degeneration in mice was induced by intraperitoneal injection of ADM,while Ligustilide was administered either subcutaneously or via acupoint injection at the Tiantu point.The experiment was divided into the following groups:normal group,model group,group receiving acupoint/subcutaneous injection of PBS,groups receiving acupoint/subcutaneous injection of Ligustilide at low,medium,and high doses(0.2,1,5 mg/kg),rapamycin group(2 mg/kg i.p.),and metformin hydrochloride group(200 mg/kg i.p.).The experiment monitored age-related characteristics such as the spontaneous activity,grip strength,and microcirculation blood flow at the thymus.Additionally,thymic output function was evaluated using the TRECs method.The mRNA expressions of Sirt6,P53,P21,PTMα,Tβ4,Tβ15,Tβ10 in thymus were analyzed by qPCR.Thymus structure and the spatial distribution of TECs were examined using H&E staining and immunofluorescence.The thymus cell subsets was analyzed by flow cytometry.Lastly,Western Blot method was used to detect the expressions of CYCLIN D1,α-TUBULIN,ECADHERIN,PROFILIN 2,and P53 proteins in thymus.An ADM-induced senescent thymic epithelial cell model was prepared.0.01,0.1,1 μM Ligustilide were used to interfere with aging thymic epithelial cells.The MTT assay was used to assess the cell survival rate;the β-galactosidase-specific staining method was used to detect cell aging.qPCR was used to measure the mRNA expressions of Sirt6,Sirt3,P53,P21,senescence-associated secretory phenotype(SASP)Il-1α,Il-1β,Il-6,Il-8,Hgf,Cxcl 9,Cxcl 2,as well as PTMα,Tβ4,Tβ15,Tβ10,Tα1,and Tslp in cells.Fluorescently labeled F-actin and G-actin immunofluorescence were used to observe cell skeleton,while Western blot analysis was used to assess the expressions of CYCLIN D1,α-TUBULIN,VIMENTIN,ECADHERIN,PROFILIN 2,and P53 proteins in cells.ADM was utilized to induce thymic senescence in vitro,and the thymic structure,the spatial distribution of TECs,and the T cell subsets were observed by the above methods.Actin polymerization inhibitor,Tβ15 and Tβ4 overexpressing adenovirus were utilized to verify the potential mechanism of action.Section Two Tβ15 knockdown and overexpression techniques were used to intervene the thymus to observe the changes of thymus structure and the distribution of TECs.The expressions of CD3,CD4,CD8,and TCRβ in the thymus were detected using immunohistochemistry.The mRNA expressions of Aire,Fezf2,Llcam,β2M,H2-K1,Gimap3,Gimap4,S1p1,CD69,Spl,Icam-1,and Vcam-1 in the thymus were detected using qPCR.The change of thymocyte subsets in the thymus was analyzed using flow cytometry.Artificial thymus organoids were constructed and transplanted into the axillary region of nude mice.The structural characteristics of thymic organoids,distribution of TECs,and thymic output function were assessed using the aforementioned methods.Knockdown and overexpression techniques targeting Tβ15 in iTECs were employed to elucidate the potential mechanisms underlying Tβ15’s regulation of TEC spatial distribution.Results Section One Compared to the normal group,the model group of mice showed disordered thymus structure,accelerated thymus output(P<0.01),and signed of aging,as indicated by increased P53 and P21 levels(P<0.05),decreased Tβ4 and Tβ15 levels(P<0.01).Compared to the model group,the thymus output function was decreased in the acupoint/subcutaneous injection of PBS group(P<0.01).However,microcirculation blood flow in the thymus was increased in the acupoint injection of PBS group(P<0.05),along with an increased proportion of CD3+TCR β+and CD4SP T cells(P<0.01).Meanwhile,the number of spontaneous activities of mice in the subcutaneous injection PBS group increased(P<0.01).Compared to the control group,the thymic corticomedullary boundary of the acupoint injection Ligustilide group was clear,the number of medullary reticular epithelial cells increased,P53 and P21 levels decreased,Tβ4 and Tβ15 levels increased,and positive selection process of thymic T cells was restored.Compared to the control group,the subcutaneous injection Ligustilide group could significantly increase mouse weight,grip strength and microcirculation blood flow volume,reduce P53 and P21 levels,increase the number of thymic nurturing cells and the ratio of DP T cell.Ligustilide treatment of adriamycin-induced iTECs resulted in a decrease in senescent cells.Furthermore,there was an increase in the structure of microfilaments.The expressions of aging-related factors,such as P53 and P21,were decreased,while the expressions of PROFILIN 2,α-TUBULIN,and CYCLIN D1 were increased.In vitro,adriamycin induced thymus structure disorder,resulting in a decrease in the number of TECs and a blockage in DN T cell development.Ligustilide reversed the process,increaseed the number of TECs,promoted DN to DP T cells,and increased the proportion of CD3+TCR β+and CD4SP T cells.Mechanism studies have found that adriamycin reduced the microfilaments in iTECs and reduced the expressions of PROFILIN 2,CYCLIN D1,and α-TUBULIN proteins,while increasing the levels of Tβ4 and Tβ15.Ligustilide achieved a balance in the microfilament assembly of TECs by regulating the expression of Tβ4 and Tβ15,with particular emphasis on Tβ15.Section Two The overexpression of Tβ15 inhibited thymus shrinkage in vitro,reduced the number of TECs and the proportion of CD4SP T cells,and lowered the mRNA levels of Aire,L1cam,S1p1,and other factors.In contrast,knockdown of Tβ15 promoted thymus atrophy(P<0.05),expanded the medulla,and reversed all changes.In the experiments on artificial thymus organoids,the volume of thymic organoids decreased significantly(P<0.01),and the proportion of DN T cells increased when thymocytes were co-cultured with iTECs overexpressing Tβ15.On the contrary,the organoid exhibited enlargement,and the proportion of DN T cells decreased when Tβ15 was knocked down.After transplantation of thymic organoids with Tβ15 OX into the armpit of nude mice,it was observed that the thymic output was reduced,and the differentiation rate of T cells was accelerated.In contrast,after transplantation of thymic organoids with Tβ15 knockdown,the TECs network structure within the organoids became denser,thereby accelerating the output of immature T cells.Overexpression of Tβ15 did not affect cell migration ability but decreased the expression of FGFR2Ⅲb and Leptin while increasing the levels of TNF-α and IL-1β.Conversely,knockdown of Tβ15 enhanced the proliferation and migration of iTECs,increased the expressions of and and decreased the levels of TNF-α and IL-1β.The co-culture of iTECs with high expression of Tβ15 and thymocytes inhibited the proliferation and increased the apoptosis rate of thymocytes,while the knockdown of Tβ15 promoted the proliferation and reduced the apoptosis rate of thymocytes.Conclusion Ligustilide possessed the ability to reverse adriamycin-induced acute thymic degeneration and might exhibit an anti-thymic immunosenescence effect.This effect was attributed to the inhibitory impact of ligustilide on Tβ15 expression,resulting in a deceleration of actin depolymerization and the maintenance of dynamic equilibrium in actin cycling behavior.Tβ15,known as the most potent actin-binding protein,plays a critical role in regulating the assembly rate of TECs,thereby preserving their normal morphology and function and ensuring the stability of thymic structure and output.