| Diabetes is a kind of metabolic disease caused by multifactor such as genetics, environments, and immunity. It has been well known as a serious threat to human health and together with tumors, cardiovascular and cerebrovascular diseases it has been called as "three killers" by the World Health Organization. According to the data of the New England Journal of Medicine in2010, the number of diabetes patients in China has reached90million, and the pre-diabetes patients have been reached about150million. With the development of aging over the world, Westernized diet and sedentary, unhealthy living habits, the prevalence of diabetes has gradually increased. Therefore, it has important clinical significance to study of the diabetes pathogenesis and to find effective therapeutic targets.Obesity related low grade inflammatory response has been widely accepted as an important reason to cause insulin resistance and diabetes. Its basic pathophysiological mechanisms can be characterized as following. In obese patients, adipose tissue increasing, and they increase the catabolism of fat cells to produce the free fatty acids which can reverse stimulate the fat cells and macrophages in the adipose tissue to occur inflammatory reaction, and with the occurrence of inflammatory reaction,macrophages can adapt to secrete pro-inflammatory cytokines such as IL-1? and TNF-?. These pro-inflammatory cytokines can step to promote some effectors cells such as skeletal muscle cells insulin resistance, and the course of above is the main reason for obese patients prone to form insulin resistance and diabetes. However, the published data show that macrophages are treated with the free fatty acids for approximately3hours to produce increased expression of inflammatory cell molecule, after the removal of free fatty acids.12h hours later, the macrophage inflammatory protein such as JNK expression reduce back to the value of the base level. Additionally, the anti-inflammatory cytokines such as IL-10can help to improve insulin resistance in mice skeletal muscle cells and has a potential role in the treatment of diabetes. These evidences show that the free fatty acid-induced inflammatory response of macrophages is reversible and can be regulated, and the regulation of macrophage inflammatory response may become an important research direction for the treatment of diabetes and other metabolic diseases.Astragalus polysaccharide (APS) is a kind of Chinese traditional medicine, which is widely used in developing countries, and mainly used against microbial infection or as immuno-stimulaters. APS is the extract of Astragalus including the main active ingredient. The use of astragals polysaccharide is still limited in the experimental stage and has not been approved for large-scale use in clinical practice. APS injection has been available and adjuvant therapy for cancer patients in clinical. Many studies had shown that the astragals polysaccharides regulate blood sugar and improve insulin resistance. The mechanism APS up-regulate insulin resistance is still unclear. The previous data showed that APS could increase AMPK activity in skeletal muscle. AMPK has been widely recognized as an important target of the regulation of macrophage inflammatory response. The inflammatory response has been significantly demonstrated it is closely associated with the occurrence of diabetes. Whether APS can activate macrophage AMPK and play the roles in the anti-inflammatory is a key issue to be resolved in this study. Palmitate, a kind of free fatty acid, produce inflammatory reaction in macrophages and it is a vitro inflammatory model in this experimental. APS was added before the palmitate treatment as an intervention factors and to analysis the levels of secretion of various cytokines and inflammatory proteins to comprehensive understand the roles of APS on macrophage inflammatory reaction. At the same time, after inhibiting AMPK activity by transfecting AMPK negatively regulate plasmid (DN-AMPK) or using an AMPK inhibitor Compound C, the APS and palmitate were treated as above on the regulation of the inflammatory response to further explore whether there is the AMPK activity involved in the mechanism. The whole studies are divided into three parts of the experiment as followning.Part I APS exerts anti-inflammatory effects in macrophage RAW264.7cellsAims:APS were demonstrated that could improve insulin resistance and regulate blood glucose concentration, but its effect on macrophage inflammatory reaction is still need to investigate. Therefore in this vitro experiment APS was used as a treatment to culture macrophage RAW264.7for several hours, or APS was used as an intervenion factor, was added before Palmitate treatment in macrophage, cytokines genes or protein levels including various pro-inflammatory or anti-inflammatory were analysized to comprehensive understand the roles of APS on inflammatory reaction of macrophage RAW264.7cells.Methods:APS (400?g/ml) is used as the treatment factor to treat the macrophages for24hours, the cells were harvest and the total mRNA was extract, Real time PCR method was used to analysize the expression of cytokines genes, including anti-inflammatory genes IL-10, Arginase, MMR, YM-1, YM-2, Dectin-1and pro-inflammatory genes TNF-a, IL-1?, iNOS, IL-6, MCP-1, CD11c and so on; In addition, a kind of free fatty acid,Palmitate was used to copy the model of macrophage pro-inflammatory reaction in vitro, on the basis of this pro-inflammatory model, APS was used as an intervention factor for24hours, the supernatant of culture medium was collect to detect the protein levels of IL-1? and IL-10on the ELISA method; At the same time, the cells were harvest to detect the genes levels of various cytokines,including the anti-inflammatory genes: IL-10, Arginase, MMR, YM-1, YM-2, Dectin-1and the pro-inflammatory genes:TNF-?. IL-1?. iNOS. IL-6, MCP-1, CD11c.Results:(1) APS significantly increase the expression levels of anti-inflammatory cytokines (IL-10:3.4±0.3fold. P?0.001; MMR:1.3±0.1fold, p<0.01; Arginase:1.5±0.2 fold. p<0.01; Dectin-1:5.6±0.4fold. p<0.001; YM-1:4.9±0.6fold. p<0.001; YM-2:4.4±0.3fold. p<0.001). among those pro-inflammatory cytokines. only TNF-a and iNOS is slightly increased (TNF-a:1.0±0.0vs1.8±0.2, iNOS:1.0±0.0vs1.7±0.6, p<0.05), and there were no significant changes in levels of other cytokines (IL-1(3:1.0±0.0vs1.1±0.2, IL-6:1.0±0.0vs1.2±0.1, CD11c:1.0±0.0vs1.1±0.1, p?0.05). even the expression of MCP-1decreased after the treatment of APS (MCP-1:1.0±0.0vs0.8±0.1, p<0.05).(2) The Palmitate acid can promote macrophage RAW264.7forming a differentiation toward the pro-inflammatory direction, but when APS was used as the intervention factor, the pro-inflammatory effects of palmitate has been inhibited. Many anti-inflammatory cytokines in the roles of APS has been significantly increased, including IL-10, Arginase, MMR, YM-1, YM-2, Dectin-1(IL-10:2.8±0.4fold. p?0.01; MMR:3.4±0.6fold, p<0.01; Arginase:4.5±0.5fold, p<0.001; Dectin-1:2.6±0.2fold. p?0.001; YM-1:2.0±0.3fold, p?0.01; YM-2:23.1±1.6fold, p<0.001); while many pro-inflammatory cytokines in the treatment of APS has significantly inhibit, such as iNOS, IL-1?IL-6, MCP-1, CDllc (IL-1?:14.9±0.8vs6.3±0.5,p<0.001; iNOS:19.8±1.6vs10.3±0.7, p<0.001; MCP-1:18.4±2.3vs5.0±0.5, p<0.001; CD11c:3.1±0.4vs2.1±0.3, P?0.01; IL-6:10.0±0.6vs2.3±0.2, p?0.001). At the same time, APS inhibited Palmitate induced RAW264.7cell secretion of IL-1? (1071.7±15.0pg/ml vs515.0±117.7pg/ml, p<0.05); conversely, APS can improve the secretion levels of IL-10inhibited by Palmitate (20.3±9.9pg/ml vs78.6±9.1pg/ml, p?0.001).Conclusions:(1) APS can alter macrophage polarity towards the direction of anti-inflammatory macrophages.(2) APS can inhibit the Palmitate-induced inflammatory reaction in macrophage RAW264.7cells.Part II APS activate AMPK activity in the macrophages.Aims:According to the the previous study, the APS were confirmed that could stimulate AMPK activity in rat skeletal muscle cells and L6cells. Many studies have demonstrated that AMPK has become a key regulation target of inflammatory reaction in macrophages. However, whether the APS can activate macrophage RAW264.7cells AMPK is still unclear, so in this present experiment the APS treat the macrophage RAW264.7cells for some time or under some special concentrations; additionally, the APS was used as an intervention factor, added before the treatment of Palmitate in macrophages RAW264.7cells, the cells were harvest and the changes of AMPK activity was analysized to clarify whether APS can activate AMPK and has the potential to become an agonists of inflammation or a treatment drug for diabetic mellitus.Methods:APS in the concentration of400?g/ml treat macrophages RAW264.7cells for several different time (0,15,30,60,120and240minutes), or the cells were treat with APS under different concentrations (0?10,100,200,400,800?g/ml) for3hours. In addition, APS was added before Palmitate as an intervention factor for about3hours, and the time of Palmitate treatment is2hours. The RAW264.7cells were harvest and the total protein was extract, Phos-AMPK, AMPK was analysized by the method of Western blotting against the primary mouse antibody come from cell signaling.Results:(1) APS (400?g/ml) can activate macrophage AMPK in15minutes, the ratio of p-AMPK and AMPK increase2.3±0.2fold (p?0.01), while the ratio of P-AMPK and AMPK increased slowly and maintained at a certain levels after that time (30min:2.6±0.3fold,60min:2.5±0.3fold,120min:2.8±0.2fold,240min:2.9±0.2fold), and there is no statistically difference among them, APS can increase the ratio of AMPK phosphorylation with its escalating concentration (0?g/ml:1.0±0.0;10?g/ml:1.4±0.3, p<0.05;100?g/ml:1.7±0.3,p<0.001;200?g/ml:2.2±0.2, p?0.001;400?g/ml:2.7±0.2?p?0.001;800?g/ml:4.7±0.7. p?0.001).(2) APS (400?g/ml) can reverse the Palmitate (0.25,0.5mM) induced-inhibition of AMPK phosphorylation in macrophage RAW264.7cells. Compared the APS+Palmitate (0.25mM) group with the Palmitate (0.25mM) group, the ratio of p-AMPK and AMPK increase1.8±0.2fold (p?0.01). At the same time. Compared the APS+Palmitate (0.5mM) group with the Palmitate (0.5mM) group, APS (400?g/ml) increase the ratio of p-AMPK and AMPK2.1±0.3fold(p<0.01). Conclusions:(1) APS can quickly activate macrophage RAW264.7cells AMPK, and it is aconcentration dependent.(2) APS can reverse the Palmitate induced-inhibition of AMPK phosphorylation inmacrophage RAW264.7cells.Part ? APS regulate inflammatory reaction through the AMPK pathwayAims:The results from part one have confirmed that APS can modulate macrophage inflammatory reaction; however, the regulation mechanism of APS in macrophages is still unclear. AMPK has been widely accepted as a key target of inflammatory reaction in macrophages, and we confirmed in the part two experiments that APS could stimulate AMPK activity in macrophage. However, there is no evidence that could prove the inhibition inflammation reaction role of APS is related to the AMPK activity caused by APS in macrophage RAW264.7cells. In present study, AMPK negative plasmid (D"N-AMPK) or an AMPK inhibitor Compound C was used to inhibit AMPK activity, in order to further explore whether the regulation role of APS is AMPK activity dependent.Methods:Macrophage RAW264.7cells in six plate wells were transfected with plasmid DN-AMPK, and pcDNA-Zeo plasmid was used as a control. After24hours, the DN-AMPK transfected groups would treated with APS (0?g/ml,200?g/ml,400?g/ml) for3hours to analysize the phosphorylation level of AMPK and after24hours to analysize the IL-10gene and protein. pcDNA-Zeo groups were also used as control groups. In addition, DN-AMPK groups were pre-treated with APS (400?g/ml) for about3hours, remove the Astragalus polysaccharides, and with Palmitate (0.5mM) for next2hours, the cells were collected to detect the levels of AMPK phosphorylation.the groups without APS pretreatment were used as control groups. Furthermore, DN-AMPK macrophage RAW264.7cells were treated with APS (400?g/ml) for24hours, remove the APS medium, and Palmitate (0.5mM) was treated for another12hours, the supernatants and the cells were all collected to detect IL-10, IL-1? protein and their genes expression levels. Furthermore, besides IL-10and IL-1?. many inflammation related cytokines were detected including anti-inflammatory genes such as Arginase, MMR, YM-1, YM-2, Dectin-1, and pro-inflammatory genes containing iNOS, TNF-?,1L-1?, IL-6, MCP-1, and CD11c. Finally, Compound C was used as an AMPK inhibitor to pretreat the macrophages for1hours, then removed it, APS (400?g/ml) treat macrophages for next24hours, and then the palmitate (0.5mM) was used for12hours, the supernatants and the cells were all collected to analysize IL-10, IL-1? protein and their genes expression levels, respectively.Results:(1) After AMPK impairment, the increase phosphorylation of AMPK induced by APS (0,200,400?g/ml) in RAW264.7cells was abrogated.The AMPK impairment abrogated the increase of IL-10gene and protein induced by APS (0,200,400?g/ml) in RAW264.7cells. In DN-AMPK group, the expression of IL-10mRNA is0.9±0.2,1.3±0.2and1.5±0.2(p<0.05) respectively. Additionally, APS (0?g/ml,200?g/ml,400?g/ml) stimulate RAW264.7cells secretion of IL-10protein:25.7±8.2pg/ml,53.5±4.9pg/ml,62.8±7.1pg/ml (p<0.01). And there is no statistically difference among them.(2) The AMPK impairment abrogated the recovery of phosphorylation AMPK caused by APS (400?g/ml) in macrophage RAW264.7cells. The AMPK impairment abrogated the inhibition of IL-1? and the recovery of IL-10induced by the pretreatment of APS (400?g/ml) before Palmitate treatment. The AMPK impairment abrogated the inhibition of pro-inflammatory genes and the recovery of anti-inflammatory genes induced by the pretreatment of APS (400?g/ml) before Palmitate treatment.Conclusions:APS can regulate macrophage inflammatory reaction dependent on the AMPK activity. |
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