Study On The Regulatory Role Of P2X₇ Receptor In Mediating Immune Inflammatory Responses Of Monocyte-macrophage In Type 2 Diabetes Mellitus

Background: Diabetes mellitus（DM） has become the world’s third largest disease in terms of incidence and risk to damage human health, only behind the cancers and cardiovascular diseases. Abnormal lipid metabolism（such as free fatty acids） plays an important role in the pathogenesis of type 2 diabetes mellitus,（T2DM）. Meanwhile, T2 DM also involves low grade inflammation. The inflammatory reactions during insulin resistance often cause the body to produce abnormal inflammatory factors, increase acute phase proteins and activate inflammatory pathways. As an important participant in both innate immunity and adaptive immunity, the monocytes / macrophages can influence and regulate the immune inflammatory response by secreting cytokines and antigen-presenting process. During inflammatory processes, ATP（Adenosine triphosphate） will be passively released into the extracellular space from stressed cells or dead cells and induce various physiological effects by binding to different purinergic receptors（such as P2X₇ receptor） on the cell membrane. P2X₇ receptor is a kind of non-selective ATP-gated cation channel ligands, whose upregulated activity in a variety of inflammatory pathologies will alter the expression and release of inflammatory mediators, particiapte in immune inflammatory responses, and induce cell damage or apoptosis. Long non-coding RNAs（Lnc RNAs） generally are RNA transcripts longer than 200 nucleotides. Lnc RNAs located in the nucleus or cytoplasm are lack of the ability to encode proteins. However lnc RNAs may be involved in regulation of cell apoptosis, proliferation, and differentiation at the RNA epigenetic level, gene transcription and post-transcriptional modifications. Uc.48+（Ultraconserved.48） is298 bp long and belongs to the lnc RNAs.Prospective studies have demonstrated that C reactive protein（CRP） levels can predict the onset of T2 DM in the non-diabetic population, independent of obesity, fat distribution, insulin resistance and other factors. In addition, CRP may be directly involved in inflammation, artery atherosclerosis and other cardiovascular diseases and also is the most powerful predictor and risk factor for coronary heart disease inT2 DM. Abnormal lipid metabolism may cause lipid deposition or toxicity which can cause β-cell dysfunction. Although the fatty acids（such as phospholipids） play the role in maintaining the integrity of cell membranes, abnormal increase in the concentrations of free fatty acids（FFAs） can activate a variety of signaling pathways（endoplasmic reticulum stress, reactive oxygen species（ROS）, apoptosis,inflammation pathway, etc.） and induce lipotoxicity. Under inflammation status,peripheral blood monocytes enter into tissues and differentiate to macrophages which will contribute to the improvement or deterioration related to tissue function by releasing a series of growth factors and cytokines to summon other cells（such as fibroblasts, endothelial cells, etc.）. The macrophages in T2 DM patients can be induced to produce inflammatory cytokines which activate complement and induce endothelial cells to produce adhesion molecule, leading to endothelial dysfunction to accelerate inflammatory response. P2X₇ receptor is widely expressed in various immune cells（e.g., monocytes, macrophages, Nature Kill cells, etc.）, whose activation can facilitate the inflammatory response via increasing inflammatory cytokines secreted by immune cells and regulate immune response through adjustment of B cell or T cell function. The first part of this study explored the role and mechanism of the inflammatory response of THP-1 macrophages mediated by P2X₇ receptor after high glucose and lipid treatment.In the development of T2 DM, macrophage inflammatory reaction and lipid metabolism display mutually synergistic interaction. It means that inflammation can lead to lipid metabolism disorder and abnomal lipid metabolism may also induce inflammation. Studies have shown that macrophages may play important role to decide the ultimate fate of the injured area by releasing a series of growth factors and cytokines. Lnc RNAs have the tissue-specific expression feature and can be induced in various tissues. Thus lnc RNAs can be considered as biomarkers or therapeutic targets because lnc RNAs secreted from cells may show the specific expression for the disease types, stages, and tissues. Abnormal lnc RNAs expression is related with the pathological changes of some diseases. We have observed that macrophages treated with high glucose and high lipid can lead to uc.48+ expression upregulated,suggesting uc.48+ may be involved in the pathological changes of T2 DM. Theresearch related uc.48+ in monocyte/macrophages has not been reported yet.Therefore, the second part of this study explored the uc.48+ role and its possible mechanism in diabetes due to inflammatory pathological damage of RAW264.7macrophages mediated by P2X₇ receptor after high glucose and lipid treatment.Under T2 DM status, the macrophage infiltration may cause the adipose tissue,liver, muscle and pancreas to the inflammation state. Studies have shown that the status of tissue macrophages play a key regulatory role in the occurrence and progression of T2 DM. On the other hand, unusual lnc RNAs expression in specific tissues can cause human body dysfunction and induce various diseases; for example lnc RNAs may be involved in the insulin metabolic effects and insulin resistance. The third part of this study explored the uc.48+ role in pathological changes in type 2diabetic mice through assessment of the effect of uc.48+ siRNA on blood glucose,blood pressure, neuropathological pathological changes and peritoneal macrophages’ pathological changes mediated by P2X₇ receptor in these mice.Objectives:1. To explore the role and underlying mechanism of P2X₇ receptor in the immune inflammation responses in T2 DM through assessments of clinical parameters and the P2X₇ receptor in peripheral blood monocytes among three groups of subjects（T2DM patients with normal CRP levels, T2 DM patients with high CRP levels and healthy control subjects） and in cultured THP-1 macrophages following treatment of high glucose and high lipids.2. To study the uc.48+ role and possible mechanism in P2X₇ receptor- mediated inflammatory pathological damage due to diabetes via detecting uc.48+ and cytokines in serum among three groups of populations（T2DM patients with normal CRP levels,T2 DM patients with high CRP levels and healthy control subjects） and in cultured RAW264.7 macrophages after high glucose and lipid treatment.3. To explore the role of uc.48+ in pathological changes in type 2 diabetic mice through observing the effect of uc.48+ siRNA on type 2 diabetic mice and peritoneal macrophages whose P2X₇ receptor were upregulated.Methods:1. After selected individulas were sorted to three groups（T2DM patients withnormal CRP levels, T2 DM patients with high CRP levels and healthy control subjects）, clinical parameters（including fasting plasma glucose, postprandial glucose,total cholesterol, triglyceride, low-density lipoprotein, high density lipoprotein,glycosylated serum protein, fasting insulin） were first determined. Then P2X₇ receptor expression in peripheral blood monocytes was detected by flow cytometry.The data were analysed to compare the potential difference in three groups. After the best concentrations of high glucose and FFAs to be used in this study were determined by MTS assay, P2X₇ receptor expression in THP-1 macrophages were knocked down by small interfering RNA（P2X₇ siRNA） at high glucose and FFAs treatment. The levels of P2X₇ receptor m RNA and protein were detected by RT-PCR and Western blotting respectively. Internal ROS and calcium concentration, the cytokine content in culture supernatant（including TNF-α, IL-10, IL-1β） were detected by fluorescent probes and enzyme-linked immunosorbent assay（ELISA）respectively. The ERK signaling pathway was assessed by Western blotting.2. The content of cytokines（including TNF-α, IL-10, IL-1β） and the expression of uc.48+ in collected serum from three groups of people（T2DM patients with normal CRP levels, T2 DM patients with high CRP levels and healthy control subjects）were measured by ELISA and RT-PCR respectively. After RAW264.7 cells were transfected with uc.48+ siRNA at high glucose and FFAs treatment, the m RNA levels of uc.48+ and P2X₇ receptor were detected by RT-PCR. The protein mass of P2X₇ receptor and ERK signaling pathway were assessed by Western blotting. ROS and calcium concentration, culture supernatant cytokine content（including TNF-α, IL-10,IL-1β） were detected by fluorescent probes and ELISA respectively. The cell viability and apoptosis were determined by MTS test and flow cytometry respectively.3. Type 2 diabetic mice were induced by high-sugar and high-fat diet combined with intraperitoneal injection of low-dose streptozotocin（STZ）. After validation of successful generation of type 2 diabetic mice with hyperglycemia by blood glucose test, uc.48+ siRNA was intraperitoneally injected to these mice. Lipids and cytokines in mouse serum, blood pressure and heart rate, mechanical pain threshold and thermal pain threshold were examined. After collection and identification of peritoneal macrophages in the mice, the formation of P2X₇ receptor channel, m RNA levels ofuc.48+ and P2X₇ receptor, the protein mass of P2X₇ receptor and changes of ERK signaling pathway were evaluated by calcein-AM and YO-PRO dyes, RT-PCR and Western blotting respectively.Results:1. Significant increases in fasting plasma glucose, postprandial plasma glucose,glycosylated serum proteins and homeostasis model assessment for insulin resistance were observed in T2 DM patients compared to healthy subjects. In addition,triglyceride, total cholesterol and low-density lipoprotein contents in T2 DM patients were higher than that in healthy people. T2 DM patients with high CRP levels displayed higher fasting plasma insulin（FPI）, glycosylated serum proteins and homeostasis model assessment for insulin resistance than healthy control subjects and T2 DM patients with normal CRP levels. The mean fluorescence intensity（MFI）values of P2X₇ receptor in monocytes from T2 DM patients with high CRP levels were significantly increased compared with those obtained from healthy control subjects and T2 DM patients with normal CRP levels. The dosages of high glucose and lipid were set at 44.4 m M glucose combined with 1.0 m M free fatty acids（FFAs）as determined by MTS test. m RNA and protein levels of P2X₇ receptor were significantly elevated compared to the control group, whereas they were significantly knocked down by P2X₇ siRNA transfection. P2X₇ siRNA transfection significantly attenuated the up-regulation of ROS and calcium concentrations and p-ERK1/2activation in THP-1 macrophage due to high glucose and high FFAs treatment. TNF-αand IL-1β levels were up- regulated while IL-10 level was down-regulated by high glucose and high FFAs treatment of THP-1 macrophages; P2X₇ siRNA transfection caused significant diminishment of these effects.2. TNF-α and IL-1β levels in T2 DM patients were significantly higher than healthy control subjects, while their levels in T2 DM patients with high CRP levels were significantly higher than T2 DM patients with normal CRP levels. IL-10 level in T2 DM patients with high CRP levels was significantly lower than the other two groups. The uc.48+ expression in T2 DM patients was significantly higher than healthy control subjects, meanwhile its expression in T2 DM patients with high CRP levels was significantly higher compared to T2 DM patients with normal CRP levels.Uc.48+ level in RAW264.7 cells could be significantly knocked down by uc.48+siRNA transfection. The up-regulated expression of m RNA and protein of P2X₇ receptor of RAW264.7 cells due to high glucose and high FFAs treatment were significantly downgraded after uc.48+ siRNA transfection. The down-regulated proliferation of RAW264.7 cells due to high glucose and high FFAs treatment was significantly upgraded after uc.48+ siRNA transfection. In addition, the up-regulated apoptosis, elevated ROS and calcium concentrations in RAW264.7 cells due to high glucose and high FFAs treatment were significantly inhibited by uc.48+ siRNA transfection. The high glucose and high FFAs treatment-induced increase of TNF-αand IL-1β levels and decrease of IL-10 level in RAW264.7 cells were also significantly attenuated by transfection of uc.48+ siRNA. Up-regulated p-ERK1/2activation by high glucose and high FFAs treatment of RAW264.7 cells was also significantly mitigated after uc.48+ siRNA transfection.3. Feeding high-sugar and fat diet in combination with intraperitoneal injection of low-dose STZ in mice resulted in significant increase of the fasting plasma glucose and postprandial glucose compared to the control group, indicating successful establishment of type 2 diabetic animal models. The fasting plasma glucose and postprandial glucose were significantly reduced after intraperitoneal injection of uc.48+ siRNA into type 2 diabetic mice. The lipids of the diabetic mice group were significantly increased compared with the control group, however there was no significant difference of lipids between the diabetic mice group, DM+NC siRNA group and DM+uc.48+ siRNA group after uc.48+ siRNA injection.MWT（Mechanical withdrawal threshold） and TWL（Thermal withdrawal latency） in diabetic mice group were significantly decreased compared with the control group,but such effects were significantly relieved after uc.48+ siRNA injection. The heart rates of the diabetic mice group were significantly increased compared with the control group. However, there was no significant difference of heart rates between the diabetic mice group, DM+NC siRNA group and DM+uc.48+ siRNA group after uc.48+ siRNA injection. Both systolic and diastolic blood pressure in diabetic mice group were significantly increased compared with the control group, but these changes were significantly diminished after uc.48+ siRNA injection. Diabetic micegroup displayed significantly higher levels of TNF-α and IL-1β compared to the control group, and these alterations were significantly alleviated after uc.48+ siRNA injection. IL-10 concentration in diabetic mice group was significantly decreased compared with the control group, whereas uc.48+ siRNA injection could retore its concentration. In addition, the level of ion channels formation, the m RNA and protein expression of P2X₇ receptor, and the p-ERK1/2 activity of peritoneal macrophages were significantly increased in diabetic mice group compared with the control group;however all these changes could be significantly attenuated after uc.48+ siRNA transfection in vivo.Conclusions:1. The elevated expression of P2X₇ receptor in monocytes in T2 DM patients with high CRP levels may be associated with increased formation of insulin resistance. Treatment of THP-1 macrophages with high glucose and FFAs up-regulated P2X₇ receptors, inflammatory cytokines and p-ERK signaling pathway,whereas P2X₇ receptor knockdown could reduce or improve these effects. This suggested that P2X₇ receptor may regulate the inflammatory response in THP-1macrophages.2. The serum uc.48+ expression and levels of inflammatory cytokines in T2 DM patients with high CRP levels were significantly increased, suggesting T2 DM patients were at the active immune and inflammatory status. Treatment of RAW264.7 cells with high glucose and FFAs whose uc.48+ expression increased evoked P2X₇receptor-mediated immune and inflammatory response through a variety of ways（such as cytokines secretion, ROS formation, activation of ERK signaling pathway,etc.）; uc.48+ siRNA could regulate these scenarios and thus influence the course and outcome of immune and inflammatory responses mediated by P2X₇ receptor.3. The uc.48+ expression of peritoneal macrophages was significantly increased in diabetic mice group compared with the control group. On the one hand uc.48+ siRNA could improve blood glucose, blood pressure, neuropathological changes in type 2 diabetic mice; on the other hand it could downregulate the expression of P2X₇ receptor and could regulate inflammatory response, oxidative stress and ERK signaling pathway in the peritoneal macrophage mediated by P2X₇ receptor from type 2 diabetic mice. This suggested uc.48+ may play important role through mediation P2X₇ receptor in the pathological changes of blood glucose, blood pressure, neuropathological changes and peritoneal macrophages in T2 DM.