Effects Of Astragalus Membranaceus On The Production Of Inflammatory Cytokines In BMDM Cells

Objective: diabetic nephropathy(Diabetic Nephropathy,DN)is a serious complication of diabetes mellitus(Diabetes Mellitus,DM),which is serious and harmful.The pathogenesis of the disease is related to chronic inflammation.In the animal experiment and the clinical research of DN patients,it was found that the inflammatory cells in the kidney tissue were infiltrated by macrophages.There was a positive correlation between the degree of invasion and the degree of renal tissue damage.Therefore,the inflammatory reaction mediated by macrophages is one of the important reasons for accelerating the development of DN,TLR4 is the pattern recognition receptors on the macrophage surface,and its ligand activation promotes macrophage proinflammatory cytokines(TNF-α,IL-1β)to accelerate the inflammation,promote the occurrence and development of DN.Our previous studies also found that TLR4 on the surface of macrophages induced by high glucose and increase the expression of many clinical and experimental results show that the expression of TLR4 in high glucose conditions increased,and its endogenous ligand of HMGB1 or HSP60 in the serum of patients with diabetes were significantly higher in both cell surface binding promotes macrophage activation,the activation occurs repeatedly,promote proinflammatory cytokine macrophage(TNF-α,IL-1β)generation,and anti-inflammatory cytokines(IL-10)have reduced or not enough to adjust the DM state balance,long-term effects lead to the occurrence of DN.Therefore,it may be an effective means to delay the onset of DN by selecting an immunomodulator to regulate TLR4 mediated inflammation.Astragalus is a traditional Chinese herbal medicine,the main role of immune regulation,Astragalus has been used in clinical treatment of DM and DN,but the specific mechanism is not fully understood.Therefore,this study intends to obtain the bone marrow cells of Balb/C mice,in vitro stimulation with the neutral granulocyte colony stimulating factor(GM-CSF),induced BMDM,TLR4 endogenous ligand HMGB1 and exogenous ligand LPS were stimulated,and then intervened with different concentrations of Astragalus membranaceus,To investigate whether Astragalus can regulate the inflammatory reaction mediated by BMDM TLR4,and further clarify its regulatory mechanism.Method:1 BMDM induction Select the Balb/C rats,bone marrow cells obtained from thigh bone,the cells with 1640 medium washing again,and then the cells spread to the 6 well plates,no holes in the mixed solution of 1ml 1640 cells and cultured with 50ng/ml,GM-CSF induced 72 hours,remove the 6 hole plate in the electron microscope under the observation of cell morphology,each hole in 1ml 1640 medium,adding 50ng/ml GM-CSF 72 hours of induction.2 LPS,HMGB1 and Astragalus stimulation of BMDM cells after the production of inflammatory factors The successful induction of BMDM to 2×105/ hole concentration seeded in 12 well plates,cultured 2 hour respectively,after adding different concentrations of LPS,HMGB1,astragalus,after 24 hours,the supernatant was collected to detect the expression of ELISA,IL-1β、TNF-α、IL-6、IL-10.3 Effects of Astragalus membranaceus on inflammatory cytokines produced by LPS and HMGB1 stimulated BMDM cells BMDM the same treatment,first add LPS(1μg/ml)or HMGB1(200ng/ml),and then add different concentrations of Astragalus,respectively,in the role of 4,12,24 hours after collecting supernatant,ELISA detection of IL-1β、TNF-α、 IL-6、IL-10 expression.Result:1.After LPS stimulation of BMDM cells for 24 hours,there was no significant change in the expression of IL-6,IL-1β,TNF-α and IL-10 were increased with the increase of LPS concentration.2.The expression of IL-1β and TNF-α in supernatant was not changed after HMGB1 stimulated BMDM cells for 24 hours.The expression of IL-6 increased with the increase of HMGB1 concentration,and the IL-10 decreased with the increase of HMGB1 concentration.3.Astragalus stimulation of BMDM cells after 24 hours,no significant changes in the supernatant of IL-1βexpression in each group;but all were lower than the control group;the expression of TNF-α was lower than the control group,1mg/ml was the most obvious,with the increasing concentration of Astragalus has increased,but no significant differences among the groups;the expression of IL-6 is with the increase of the concentration of Astragalus decreased,decreased significantly in a concentration dependent manner in 8mg/ml,the most significant;IL-10 expression in each group was higher than that of control group,with increasing concentration of Astragalus is increased,there are significant differences only in the concentration of 8mg/ml.4.Astragalus on HMGB1(200ng/ml)stimulated the expression of inflammatory cytokines in BMDM: IL-1β: HMGB1 stimulation alone,no significant change compared with the different concentration of Astragalus group,Astragalus was significantly higher in 2mg/ml;TNF-α: HMGB1 alone resulted in a downward trend,but did not change significantly,decreased significantly in Astragalus 2mg/ml,value of each group were lower than the control group;IL-6:HMGB1 alone stimulated,increased performance in Astragalus 2,4 and 8mg/ml significantly increased in a concentration dependent manner,and each group was higher than that of control group;IL-10: HMGB1 alone stimulated expression decreased,1mg/ml decreased significantly in Astragalus,but each the value increased with stimulus concentration increased,and the value of each group were lower than the control group.5.Astragalus on LPS(1μg/ml)to stimulate the expression of inflammatory cytokines in BMDM cells: IL-1β: At the time of individual stimulation,the expression of 24 hours was significantly higher,and the concentration of Astragalus in the concentration of 2,4,8mg/ml was significantly increased,which was concentration dependent.TNF-α: LPS stimulation alone,in the 12 h,24h concentrations in each group were increased to 12 hours,after the Astragalus,the concentration of 12 h in each group showed no significant change in 24 h,24h,8mg/ml and LPS group decreased.IL-6:LPS alone resulted in the highest in 4h,decreased with time,Astragalus 1、8mg/ml decreased its expression at this time,reduce the 1mg/ml concentration in 12 h is still,but the other was not significantly increased,24 h decreased the concentration of Astragalus,but there was no significant difference between.IL-10:LPS alone stimulated,the highest expression level at 12 h,24h decreased significantly,After 2,4 and 8mg/ml stimulation in Astragalus,the expression of 12 h was increased,the concentration of 4mg/ml was the most significant,and the concentrations of 24 h were significantly lower than those of group LPS.Conclusion:1.LPS stimulated BMDM cells to produce TNF-α,IL-1β,IL-10,and concentration dependent.2.HMGB1 stimulated BMDM production of IL-6,but inhibited the expression of IL-10.3.Astragalus can inhibit the expression of TNF-α and IL-6 in BMDM cells and promote the expression of IL-10.4.Astragalus in the low concentration,HMGB1 inhibited the expression of TNF-α and IL-10,and promoted the expression of IL-6 and IL-1β in BMDM cells.5.Astragalus inhibited LPS stimulated BMDM cells to produce TNF-α,IL-6,promote the expression of IL-10.6.Astragalus is an important immunomodulator,there were different levels of inhibitory effects on TNF-α,IL-1β and IL-6,but it can promote the anti-inflammatory factor IL-10.Moreover,it can inhibit the inflammatory response of LPS and HMGB1,thus slowing the development of macrophage mediated inflammatory response and providing a new treatment basis for the treatment of DN.