| ObjectiveTo explore the effects and possible mechanism of PNS on hematopoietic and immune functions on AA by establishing a suitable immune-mediated AA mice model and inducing DC2.4 maturation in vitro.To provide experimental support for the clinical treatment of AA and expanded application of PNS.MethodsC57BL/6 mice were randomly divided into control group,total body irradiation group,model group,cyclosporine A treatment group(CsA)(25 mg·kg-1),PNS lowdose(50 mg·kg-1),medium-dose(100 mg·kg-1)and high-dose(200 mg·kg-1)group.The immune-mediated AA mice model was established by X-ray total body irradiation and mixed lymphocyte infusion from allogeneic mice.All mice were sacrificed after 15 days of drug intervention,peripheral blood trilineage cells and bone marrow nucleated cells(BMCs)were counted,bone marrow pathology was detected by H&E staining,organs indexes were calculated,the levels of pro-inflammatory and antiinflammatory factors in the serum were detected by ELISA kits to comprehensively evaluate the effects of PNS on AA mice.The expression of CD11 c and proteins related to the TLR4/TLR2-NF-κB pathway in bone marrow were detected by Western blot to explore the possible mechanism.DC2.4 maturation model was induced by lipopolysaccharide(LPS)in vitro.DC2.4 was divided into normal control group,LPS group(1 μg·mL-1),PNS low-dose(50 μg·mL-1),medium-dose(100 μg·mL-1),high-dose(200 μg·mL-1)group.The cells and supernatant of each group were collected after 24h of drug intervention.To evaluate the effects of PNS on DC2.4 maturation and its specific mechanism,the expression of CD86,CD40 and MHC-II in DC2.4 surface were detected by Flow Cytometry,the levels of TNF-α,IL-6 and IL-10 in the supernatant were detected by ELISA.The proteins expression related to TLR4/TLR2-NF-κB pathway were detected by Western blot.Results1.Compared to mice in control group,the bone marrow hematopoietic structure of mice in model group was significantly damaged,the levels of peripheral blood trilineage and BMCs were significantly decreased,indicating that the immunemediated AA model mice was successfully established.At the same time,spleen index and the levels of TNF-α,IFN-y and IL-2 in the serum were significantly increased,thymus index and the level of IL-10 were decreased.Besides,the expression of CD11c,TLR4,TLR2,MyD88,NF-κB and Akt proteins in bone marrow were significantly up regulated.After PNS treatment,the hematopoietic structure and functions of mice bone marrow were improved,the levels of BMCs,RBC and PLT were significantly increased.The levels of IFN-γ,IL-2 and TNF-α in the serum and spleen index were down regulated,the levels of IL-10 and thymus index were up regulated.And the overexpression of CD11c,TLR4,TLR2,NF-κB and Akt proteins in bone marrow were inhibited by PNS,while there was no significant change in the expression of MyD88 and MAPK proteins.2.LPS could promote the phenotype and functional maturation of DC2.4.Compared to DC2.4 in control group,the expression of CD40,CD86 and MHC-Ⅱ in the cell surface and levels of TNF-α,IL-6 in the supernatant were significantly increased,IL-10 was decreased.Besides,the expression of TLR4,TLR2,MyD88 and Akt proteins were significantly increased,but NF-κB and MAPK had no significant change.PNS could inhibit the maturation of LPS-treated DC2.4 by down regulating the expression of CD86,MHC-Ⅱ,inhibiting the secretion of TNF-α,IL-6,and promoting the secretion of IL-10,while PNS had no significant effect on the expression of CD40.High dose of PNS can significantly reduce the expressions of TLR4,TLR2,MyD88 and Akt proteins in DC2.4 cells.ConclusionPNS can ameliorate AA bone marrow injury and immune disorders,regulate cytokine levels and promote hematopoiesis,and its mechanism may be related to the inhibition of the maturation of DCs and the expression of some proteins in the TLR4/TLR2-NF-κB pathway. |
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