Study On The Antagonistic Protective Effects And Mechanisms Of P2X7 Receptor-mediated Neuropathic Pain And Depression Comorbidity In Animals

Background:The International Association for the Study of Pain（IASP）redefined pain in 2020 as unpleasant feelings or emotional experiences related to or similar to actual or potential tissue damage,including pain sensations and the resulting emotional changes.As a type of pain,chronic pain is often associated with mood disorders,such as anxiety,depression,and sleep disorders.Studies have shown that people with chronic pain are more likely to suffer from depression,a phenomenon that is often overlooked.At the same time,depression enhances the perception and duration of chronic pain.Chronic pain and depression share a common pathophysiological process.Neuroinflammation is involved in the pathogenesis of chronic pain and depression comorbidity.Studies have shown that microglia maintain the homeostasis of the internal environment in the central nervous system and that microglia play an important role in pain and depression.Inflammation stimulates microglia activation,inducing the opening of gap junction hemichannels and the release of ATP and neurotoxic substances,which can trigger the release of various cytokines and the production of various pathological responses,ultimately leading to brain damage such as chronic pain,neuronal damage and depression.The International Diabetes Federation（IDF）Diabetes Atlas X provides that 537million adults worldwide have diabetes in 2021 and that diabetes is one of the leading causes of premature death.There are an estimated 388 million people with pre-diabetes in China,and the prevalence of pre-diabetes among Chinese adults is as high as 35.7%.Twelve percent of global health expenditure,or US$727 billion,is spent on diabetes and its complications;the cost of diabetes-related healthcare in China was US$110billion in 2017.Chronic low-grade inflammation is a key factor in the pathogenesis of diabetes.The global epidemic of pre-diabetes and diabetes has led to a corresponding epidemic of its complications,for which there is no effective prevention or treatment,the most common complication being diabetic neuropathy.Diabetic neuropathic pain（DNP）neuropathy is a clinically common peripheral neuropathy.Typical patients with diabetic neuropathic pain may experience burning pain,pins and needles,electric shock pain,etc.The pain may be noticeable at night and can seriously affect sleep,quality of life and work efficiency.Diabetic neuropathic pain neuropathy has a different pathogenic basis to other pain neuropathies and is characterized by spontaneous pain,nociceptive hyperalgesia and abnormal pain,making it a major challenge in the field of pain management.Depression is the most common mental disorder,with hundreds of millions of people having suffered from it and a suicide risk rate of around 15%,severely affecting human physical and mental health.Comorbidity refers to the co-existence of two or more chronic illnesses in the same patient,and patients with chronic pain often have symptoms associated with depression.The prevalence of diabetes mellitus complicated by depression is on the rise every year.The exact mechanisms underlying the co-morbidity of diabetic neuropathic pain and depression are unknown,and effective treatments are not known.Diabetic neuropathic pain is often complicated by depression.It is important to investigate the pathogenesis and prevention of type 2 diabetic neuropathic pain and depression comorbidity through a comprehensive study.Ferroptosis is a form of programmed cell death driven by iron-dependent lipid peroxidation.Ferroptosis is triggered by an imbalance in intracellular iron homeostasis,resulting in an increase in the production of iron-mediated toxic reactive oxygen species（ROS）.The absence or reduced expression of glutathione peroxidase 4（GPX4）leads to reduced cellular antioxidant capacity,accumulation of ROS,increased production of the lipid peroxidation product malonaldehyde（MDA）,mitochondrial shrinkage and ultimately oxidative cell death（ferroptosis）.When intracellular iron homeostasis is disrupted,excess iron is converted to ROS from hydrogen peroxide and lipid peroxides by the Fenton reaction,leading to ferroptosis.The process of ferroptosis often accompanied by inflammation,and when the inflammatory response exceeds a certain limit,a large amount of pro-inflammatory cytokines released,leading to damage to the body.Glucose metabolism is closely related to iron metabolism and high glucose levels can lead to iron overload and ultimately to ferroptosis,which is involved in the pathological changes of diabetes mellitus.Purinergic receptors include P1 and P2 receptors,which can be subdivided into P2X（1-7）receptors and P2Y(_{1,2,4,6,11-14})receptors.ATP can be released into the inflammatory microenvironment in response to organ or tissue injury.ATP acts as a signaling molecule through paracrine or autocrine activation of P2 receptor,affecting the homeostasis of the internal environment and the development of the inflammatory response.Among the P2 receptors,P2X7 has an important role in activating lymphocyte,macrophage and glial cell responses.Even though clinical studies are inconclusive as of today,P2X7 receptor offers new directions for targeting anti-inflammatory effects.P2X7 receptor mediates the pathological changes in chronic pain,diabetic co-morbid neuropathic pain,and depression.Antagonizing P2X7 receptor ameliorates pathological changes in animal models of diabetic neuropathic pain complicated by depression.Objective:1.To investigate the effect and mechanism of gallic acid on the comorbidity of pain and depression by establishing a chronic constriction injury（CCI）and chronic unpredictable stimulation（CUMS）comorbidity rat model.2.To determine whether long non-coding RNA（lnc RNA）UC.471+sh RNA can inhibit hippocampal P2X7 receptor expression and attenuate neuronal cell injury caused by increased ROS production,thereby improving the pathological changes of neuropathic pain and depression comorbidity in type 2 diabetic rats.3.To investigate whether Cp G ODN 2088 can be involved in the treatment of diabetic neuropathic pain through P2X7 receptor and its related mechanisms in a rat model of diabetic neuropathic pain.Method:1.A rat model of chronic constrictive injury（CCI）and chronic unpredictable stimulation（CUMS）co-morbidity was established,and the success of the model was established by detecting changes in mechanical and thermal pain sensitivity,and detecting depression-like behaviors such as sugar-water preference,forced swimming and the open-field test.The rats were randomly divided into five groups after one week of adaptation:sham-operated group（Sham）,Sham+Gallic acid drug control group（Sham+Gallic acid）,CCI+CUMS model group（CCI+CUMS）,CCI+CUMS+Gallic acid treatment group（CCI+CUMS+Gallic acid）and CCI+CUMS+Saline solvent control group（CCI+CUMS+NS）.Molecular docking predicted the interaction of gallic acid with P2X7 receptor.Real-time quantitative PCR（q RT-PCR）and protein blotting were used to detect the expression levels of P2X7receptor and TNF-αin rat spinal cord.Immunofluorescence assay detected co-localization of P2X7 with microglia marker OX42,and protein blotting detected expression of TNF-αconverting enzyme（TACE）,nuclear factor kappa-B（NF-κB）,p-STAT3,signal transduction and transcriptional activator 3（STA3）and glutathione（GSH）,GPX4,ROS,MDA and tissue iron content were detected by kit.Mitochondrial damage was detected by transmission electron microscopy.2.A rat model of diabetic neuropathic pain combined with depression was established,and the success of the model was established by examining the pain and depression behavior of the rats.Experimental groups:normal control group（Ctrl）,diabetic neuropathic pain combined with depression model group（DNP+DP）,diabetic neuropathic pain combined with depression plus lnc RNA UC.471+short hairpin RNA（sh RNA）group（DNP+DP+lnc RNA UC.471+sh RNA）,diabetic neuropathic pain combined with depression plus sh RNA negative control treatment group（DNP+DP+NC sh RNA）.Behavioral changes in mechanical and thermal nociceptive sensitization were measured in rats.lnc RNA UC.471+interaction with the P2X7 receptor promoter SP1 was measured by RNA Binding Protein Immunoprecipitation Assay（RIP）.The direct interaction of SP1 with the P2X7 receptor was detected by a dual luciferase assay.The expression levels of UC.471+RNA,P2X7 receptor,transcription factor nuclear factor erythroid 2-related factor 2（Nrf2）,transferrin receptor 1（TFR-1）,ferritin heavy chain 1（FTH-1）and GPX4 m RNA in the hippocampus of each group of rats was detected by q RT-PCR.The expression of P2X7 receptors and inflammatory markers TNF-αand interleukin-1beta（IL-1β）in the hippocampus of rats was measured by western blotting,and the protein expression of ferroptosis-related markers Nrf2,TFR-1,FTH-1 and GPX4 was measured.The alterations in hippocampal neurons were detected by Nissl staining for Nissl vesicles.Hippocampal iron deposition was measured by Prussian blue staining.GSH,GPX4,ROS,MDA and tissue iron content were measured by kits.Mitochondrial structural and morphological changes were detected by transmission electron microscopy.3.A rat model of diabetic neuropathic pain was established,and the success of the model was established by measuring the blood glucose and pain behavior of the rats.Experimental groups:normal control group（Ctrl）,diabetic neuropathic pain model group（DNP）,diabetic neuropathic pain plus Cp G ODN 2088 treatment group（DNP+Cp G ODN 2088）,diabetic neuropathic pain plus Cp G ODN Control group（DNP+Cp G ODN Control）,diabetic neuropathic pain plus Cp G ODN Control group（DNP+Cp G ODN Control）,diabetic neuropathic pain plus Cp G ODN 2088 treatment group（DNP+Cp G ODN 2088）.P2X7 small interfering RNA group（DNP+P2X7sh RNA）.The m RNA expression of P2X7 receptor,Nrf2,SLC7A11 and GPX4 in the DRG of the rats was measured by q RT-PCR.The protein expression of macrophage colony-stimulating factor（MCSF）,macrophage colony-stimulating factor receptor（CSF1R）,IL-1β,interleukin-6（IL-6）,inflammation-related TLR9-NF-κB pathway and P2X7 receptor,and the protein expression of ferroptosis-related indicators Nrf2,recombinant Solute Carrier Family 7,member 11（SLC7A11）,heme oxygenase 1（HO-1）and GPX4 were detected by western blotting in each group of rat DRG.Fluorescence expression of MCSF,toll-like receptor 9（TLR9）and P2X7 by immunofluorescence technique in DRG.GSH,GPX4,ROS and tissue iron levels were measured by kits.Mitochondrial structural and morphological changes were detected by transmission electron microscopy.Establishment of a neuron and satellite glial cell co-culture cell model.Neurons treated with high glucose and high lipid were used to act on satellite glial cells,and neuronal inflammation-related factors and inflammatory signalling pathway as well as ferroptosis-related indicators in satellite glial cells were detected in the cell model by q RT-PCR,western blotting and kits.Result:1.The results of our experiments showed that in the CCI plus CUMS group,pain behavior and depression-like behavior were significantly worse compared to the sham-operated group,the expression levels of P2X7,TACE,TNF-α,NF-κB and p-STAT3 were up-regulated and GPX4 expression was down-regulated.the release of GSH and GPX4 was reduced,the release of ROS,MDA was increased and tissue iron content was increased.After gallic acid treatment,pain and depression-like behaviors were relieved,the P2X7-ROS signaling pathway was inhibited,GSH and GPX4 release was increased,and MDA release and tissue iron content were decreased.2.The results of the study revealed a direct interaction between lnc RNA UC.471+and the P2X7 receptor promoter by an immunoprecipitation assay.Dual luciferase assays revealed that SP1 overexpression promoted the expression of the P2X7 receptor.In situ hybridization and Real-time PCR showed that the expression of hippocampal UC.471+was significantly higher in the DNP+DP model group than in the control group.Pain and depressive behaviors were significantly increased in the DNP+DP model group,and P2X7 receptor m RNA and protein expression levels were significantly higher than in the control group.The co-expression of P2X7 receptor and microglia marker OX42 was observed in fluorescent double-labeling experiments.At the same time,the upregulated expression of TNF-αand IL-1βin the hippocampus and the protein expression of ferroptosis-related markers Nrf2,TFR,FTH and GPX4 were abnormally changed.After treatment with lnc RNA UC.471+sh RNA,pain and depression-like behaviors were alleviated,inflammatory and ferroptosis signaling pathways were inhibited,GSH and GPX4 release was increased,MDA and ROS release was reduced and tissue iron content decreased.3.The expression of P2X7 receptor,inflammation-related cytokines macrophage colony-stimulating factor（MCSF）,macrophage colony-stimulating factor receptor（CSF1R）,and inflammatory factors IL-1βand IL-6 were upregulated in the DRG of diabetic rats with reduced mechanical and thermal pain sensitivity thresholds.The expression of TLR9,p-NF-κB and NF-κB was also increased.In addition,the expression of Nrf2,SLC7A11,HO-1 and GPX4 were down-regulated.After treatment with Cp G ODN 2088,the abnormal expression of these markers was reversed.The results showed that the expression of P2X7 receptor in satellite glial cells was increased,the expression of Nrf2,SLC7A11,HO-1 and GPX4 was down-regulated,and the expression of TLR9,p-NF-κB,NF-κB,IL-1β,IL-6,MCSF and CSF1R was up-regulated in neurons.After application of Cp G ODN 2088 treatment,the improvement results were consistent with in vivo experiments.Conclusion:1.Chronic constrictive injury and chronic unpredictable stimuli stimulate painful and depressive-like behaviors in rats.Gallic acid alleviates cellular ferroptosis by inhibiting P2X7 receptor and its downstream TNF-αand NF-κB release and STAT3 phosphorylation,thereby increasing GSH and GPX4 expression,reducing ROS accumulation,MDA production and tissue iron deposition,and ultimately alleviating pain and depression-like behaviors in rats with pain and depression comorbidity.2.Pain and depressive behaviors were evident in rats with diabetic neuropathic pain combined with depression.The expression of hippocampal lnc RNA UC.471+was significantly increased in the model rats,while the expression of P2X7 receptor m RNA and protein was upregulated in the hippocampus.lnc RNA UC.471+sh RNA decreased the expression of hippocampal P2X7 receptor and lnc RNA UC.471+in the diabetic neuropathic pain combined with depression model rats.It also inhibited the release of inflammatory factors and ferroptosis in hippocampal microglia,ultimately alleviating pain and depression symptoms.3.Combining in vivo and in vitro results,we suggest that Cp G ODN 2088 affects neuronal MCSF,CSF1R,IL-1β,IL-6 and TLR9/NF-κB signaling pathways thereby inhibiting the release of inflammatory factors and suppressing P2X7 receptor-mediated ferroptosis in satellite glial cells,ultimately alleviating pain behaviors in diabetic neuropathic pain rats.