Early smoking lead to worse prognosis of COPD patients: a real world study.

BACKGROUND: Smoking remains a major risk factor for the development and progression of chronic obstructive pulmonary disease (COPD). Due to the adolescent smoking associated with worse health state, the age, at which an individual started smoking, might play a key role in shaping the trajectory of COPD development and the severity. METHODS: We conducted an observational study from September 2016 through January 2023 of eligible patients hospitalized with COPD. Patients who started smoking during the alveolar development stage (ADS, smoking initiation ≤ 24 years old) were defined as early smoking patients, and patients who started smoking after ADS (smoking initiation > 24 years old) were defined as late smoking patients. We collected demographic and clinical data characterizing the patients and documented their condition from hospital discharge to follow-up. The primary endpoints were short-term (within one year), 3-year, and long-term (beyond 3 years) all-cause mortality after discharge. RESULTS: Among 697 COPD patients, early smoking patients had a lower smoking cessation rate (P < 0.001) and a higher smoking index (P < 0.001) than late smoking patients. Although adjusted smoking index, early smoking patients still had poorer lung function (P = 0.023), thicker left ventricular diameters (P = 0.003), higher frequency of triple therapy use during stable stage (P = 0.049), and more acute exacerbations in the past year before enrollment (P < 0.05). Survival analysis showed that they had a higher risk of death after discharge within three years (P = 0.004) and beyond three years (P < 0.001). Furthermore, even in early smoking COPD patients who quit smoking after adjusting the smoking index had poorer lung function (P < 0.05) and thicker left ventricular diameters (P = 0.003), and survival analysis also showed that they had a higher long-term mortality rate (P = 0.010) and shorter survival time (P = 0.0128). CONCLUSION: Early smoking COPD patients exhibited multiple adverse clinical outcomes, including heavy cigarette addiction, compromised pulmonary function, augmented left ventricular diameter, and elevated mortality risk. Additional, smoking cessation could not bring enough improvement of health state in early smoking COPD patients as late smoking COPD patients. Consequently, early intervention and specialized cessation approaches for younger smokers are of paramount importance in this context.

ESCRT-I protein UBAP1 controls ventricular expansion and cortical neurogenesis via modulating adherens junctions of radial glial cells.

Intricate cerebral cortex formation is orchestrated by the precise behavior and division dynamics of radial glial cells (RGCs). Endocytosis functions in the recycling and remodeling of adherens junctions (AJs) in response to changes in RGC activity and function. Here, we show that conditional disruption of ubiquitin-associated protein 1 (UBAP1), a component of endosomal sorting complex required for transport (ESCRT), causes severe brain dysplasia and prenatal ventriculomegaly. UBAP1 depletion disrupts the AJs and polarity of RGCs, leading to failure of apically directed interkinetic nuclear migration. Accordingly, UBAP1 knockout or knockdown results in reduced proliferation and precocious differentiation of neural progenitor cells. Mechanistically, UBAP1 regulates the expression and surface localization of cell adhesion molecules, and ß-catenin over-expression significantly rescues the phenotypes of Ubap1 knockdown in vivo. Our study reveals a critical physiological role of the ESCRT machinery in cortical neurogenesis by regulating AJs of RGCs.

DNA dioxygenases TET2 deficiency promotes cigarette smoke induced chronic obstructive pulmonary disease by inducing ferroptosis of lung epithelial cell.

Chronic obstructive pulmonary disease (COPD) is a significant global cause of morbidity and mortality currently. Long-term exposure of cigarette smoke (CS) inducing persistent inflammation, small airway remodeling and emphysematous lung are the distinguishing features of COPD. Ferroptosis, occurred in lung epithelial cells has recently been reported to be associated with COPD pathogenesis. DNA dioxygenase ten-eleven translocation 2 (TET2) is an important demethylase and its genetic mutation is associated with low forced expiratory volume in 1 s (FEV1) of lung function. However, its role in COPD remains elusive. Here, we found that TET2 regulates CS induced lipid peroxidation through demethylating glutathione peroxidase 4 (GPx4), thus alleviating airway epithelial cell ferroptosis in COPD. TET2 protein levels were mainly reduced in the airway epithelia of COPD patients, mouse models, and CS extract-treated bronchial epithelial cells. The deletion of TET2 triggered ferroptosis and further exaggerated CS-induced airway remodeling, inflammation, and emphysema in vivo. Moreover, we demonstrated that TET2 silencing intensified ferroptosis, while TET2 overexpression inhibited ferroptosis in airway epithelial cell treated with CSE. Mechanically, TET2 protected airway epithelial cells from CS-induced lipid peroxidation and ferroptosis through demethylating the promoter of glutathione peroxidase 4 (GPx4). Finally, co-administration of methylation inhibitor 5'-aza-2'-deoxycytidine (5-AZA) and the antioxidant N-acetyl-cysteine (NAC) have more protective effects on CS-induced COPD than either administration alone. Overall, our study reveals that TET2 is an essential modulator in the lipid peroxidation and ferroptosis of airway epithelial cell, and could act as a potential therapeutic target for CS-induced COPD.

Myricetin relieves the symptoms of type 2 diabetes mice and regulates intestinal microflora.

To verify the role of myricetin in alleviating the symptoms of type 2 diabetes and regulating the intestinal flora, we established a type 2 diabetes mouse model. After being fed a high-fat and high-sugar diet for six weeks, mice were intraperitoneally injected with streptozotocin (80 mg/kg body weight [BW]) 2-3 times. Type 2 diabetes mice were randomly divided into type 2 diabetes control (T2DM) and myricetin intervention groups. Water and food intake, fasting blood glucose (FBG), and BW were monitored weekly. After six weeks of myricetin administration, superoxide dismutase (SOD) levels and blood lipid content were measured. Furthermore, 16S rRNA sequencing was used to detect the gut microbiota composition. FBG and blood lipid levels of T2DM mice were significantly reduced upon myricetin treatment, while SOD levels were increased. Myricetin improved polydipsia, polyphagia, polyuria, and weight loss in T2DM mice. In addition, the signature type 2 diabetes microflora was established by analyzing the microflora structure of healthy mice, type 2 diabetes mice, and mice treated with myricetin. Results showed that type 2 diabetes disrupted the mice intestinal flora, and myricetin intervention normalized the intestinal flora. In conclusion, our results indicate that myricetin alleviates type 2 diabetes in mice and regulates the intestinal microflora.

Effects of Asphaltene Concentration and Test Temperature on the Stability of Water-in-Model Waxy Crude Oil Emulsions.

In oil fields, the formation of water-in-waxy crude oil emulsion is inevitable. The dissolved/crystallized state wax can interact with asphaltenes and then greatly affect the emulsion stability. However, studies on this aspect are still insufficient. In this work, the effects of the test temperature (30 °C well above the wax appearance temperature (WAT) and 15 °C well below the WAT) and asphaltene concentration (0∼1.5 wt %) on the stability of the water-in-model waxy crude oil emulsions containing 10 wt % wax were systematically investigated. When the model crude oils contain no wax, the flowability of the oils is good and the asphaltene concentration has little influence on the oil rheology. Increasing the asphaltene concentration facilitates the adsorption of asphaltenes to the oil-water interface, thus reducing the interfacial tension and water droplet size while enhancing the interfacial dilatational modulus. The stability of the emulsions improves with the increase in the asphaltene concentration, but the emulsions are still unstable. When the model crude oils contain 10 wt % wax, the WAT slightly decreases from the initial 25 to 24 °C after the addition of asphaltenes. The oil rheology is greatly improved by the addition of 0.05 wt % asphaltenes. With the further increase of the asphaltene concentration, the improved rheological ability of the asphaltenes deteriorates rapidly. At the asphaltene concentration of 1.5 wt %, the oil rheology is dramatically aggravated. The stability of the emulsion containing 10 wt % wax is mainly controlled by two aspects: on the one hand, the dissolved-state wax (30 °C) could facilitate the adsorption of asphaltenes to the interface, further reduce the interfacial tension and the water droplet size, and enhance the interfacial dilatational modulus; on the other hand, the wax crystals precipitated in the oil phase (15 °C) can form a stronger network structure at relatively high asphaltene concentrations (0.5∼1.5 wt %) and then immobilize the water droplets. The above two aspects greatly improve the sedimentation and coalescence stabilities of the emulsions at 15 °C. In addition, we did not find persuasive evidence showing that the wax could crystallize around the water droplets and strengthen the oil-water interfacial films.

Asthma Patients Benefit More Than Chronic Obstructive Pulmonary Disease Patients in the Coronavirus Disease 2019 Pandemic.

Background: Coronavirus disease 2019 (COVID-19) has raised many questions about the role of underlying chronic diseases on disease outcomes. However, there is limited information about the effects of COVID-19 on chronic airway diseases. Therefore, we conducted the present study to investigate the impact of COVID-19 on patients with asthma or chronic obstructive pulmonary disease (COPD) and ascertain risk factors for acute exacerbations (AEs). Methods: This single-center observational study was conducted at the Second Xiangya Hospital of Central South University, involving asthma or COPD patients who had been treated with inhaled combination corticosteroids (ICSs), such as budesonide, and one long-acting beta-2-agonist (LABA), such as formoterol, for at least a year before the COVID-19 pandemic. We conducted telephone interviews to collect demographic information and clinical data between January 1, 2019, and December 31, 2020, focusing on respiratory and systemic symptoms, as well as times of exacerbations. Data for asthma and COPD were then compared, and the risk factors for AEs were identified using logistic regression analysis. Results: A total of 251 patients were enrolled, comprising 162 (64.5%) who had asthma and 89 who had COPD, with none having COPD/asthma overlap. Frequency of AEs among asthma patients was significantly lower in 2020 than in 2019 (0.82 ± 3.33 vs. 1.00 ± 3.16; P < 0.05). Moreover, these patients visited the clinic less (0.37 ± 0.93 vs. 0.49 ± 0.94; P < 0.05) and used emergency drugs less (0.01 ± 0.11 vs. 007 ± 0.38; P < 0.05) during the COVID-19 pandemic. In contrast, among COPD patients, there were no significant differences in AE frequency, clinic visits, or emergency drug use. Furthermore, asthma patients visited clinics less frequently during the pandemic than those with COPD. Logistic regression analysis also showed that a history of at least one AE within the last 12 months was associated with increased AE odds for both asthma and COPD during the COVID-19 pandemic (odds ratio: 13.73, 95% CI: 7.04-26.77; P < 0.01). Conclusion: During the COVID-19 pandemic, patients with asthma showed better disease control than before, whereas patients with COPD may not have benefited from the pandemic. For both diseases, at least one AE within the previous 12 months was a risk factor for AEs during the pandemic. Particularly, among asthma patients, the risk factors for AE during the COVID-19 pandemic were urban environment, smoking, and lower asthma control test scores.

Spinal P2X7R contributes to streptozotocin-induced mechanical allodynia in mice.

Painful diabetic neuropathy (PDN) is a diabetes mellitus complication. Unfortunately, the mechanisms underlying PDN are still poorly understood. Adenosine triphosphate (ATP)-gated P2X7 receptor (P2X7R) plays a pivotal role in non-diabetic neuropathic pain, but little is known about its effects on streptozotocin (STZ)-induced peripheral neuropathy. Here, we explored whether spinal cord P2X7R was correlated with the generation of mechanical allodynia (MA) in STZ-induced type 1 diabetic neuropathy in mice. MA was assessed by measuring paw withdrawal thresholds and western blotting. Immunohistochemistry was applied to analyze the protein expression levels and localization of P2X7R. STZ-induced mice expressed increased P2X7R in the dorsal horn of the lumbar spinal cord during MA. Mice injected intrathecally with a selective antagonist of P2X7R and P2X7R knockout (KO) mice both presented attenuated progression of MA. Double-immunofluorescent labeling demonstrated that P2X7R-positive cells were mostly co-expressed with Iba1 (a microglia marker). Our results suggest that P2X7R plays an important role in the development of MA and could be used as a cellular target for treating PDN.

Involvement of Interleukin-10 in Analgesia of Electroacupuncture on Incision Pain.

OBJECTIVE: Postincision pain often occurs after surgery and is an emergency to be treated in clinic. Electroacupuncture (EA) is a Chinese traditional treatment widely used to cure acute or chronic pain, but its mechanism is not clear. Interleukin-10 (IL-10) is a powerful anti-inflammatory cytokine that shows neuroprotective effects in inflammation and injury in the CNS. The present study attempts to reveal that IL-10 is crucial for EA analgesia on postincision pain. METHODS: A model of incision pain was established in C57BL/6J mice. The pain threshold was detected by behavioral test, and the expression of IL-10 and its receptor was detected by an immunohistochemical method. C-fiber-evoked field potentials were recorded by in vivo analysis. RESULTS: The mechanical allodynia induced by paw incision was significantly inhibited by pretreatment of EA in mice. Intrathecal injection of IL-10 neutralizing antibody (2 µg/10 µL) but not intraplantar injection (10 µg/10 µL) reversed the analgesia of EA. The upregulations of IL-10 mRNA and protein were induced by EA at 6 h and 1 d after incision, respectively. Spinal long-term potentiation (LTP), a substrate for central sensitization, was also suppressed by EA with IL-10. IL-10 recombinant protein (1 µg/10 µL, i.t.) mimicked the analgesia of EA on mechanical allodynia and inhibition on the spinal LTP. Posttreatment of EA after incision also transitorily relieved the mechanical allodynia, which can be blocked by spinal IL-10 antibody. IL-10 and its receptor, IL-10RA, are predominantly expressed in the superficial spinal astrocytes. CONCLUSIONS: These results suggested that pretreatment of EA effectively prevented postincision pain and IL-10 in spinal astrocytes was critical for the analgesia of EA and central sensitization.

[Roles of CX3CR1 in mediation of post-incision induced mechanical pain hypersensitivity: Effects of acupuncture-combined anesthesia].

Post-incision pain often occurs after surgery and is emergent to be treated in clinic. It hinders the rehabilitation of patients and easily leads to various types of postoperative complications. Acupuncture-combined anesthesia (ACA) is the combination of traditional acupuncture and modern anesthesia, which means acupuncture is applied at acupoints with general anesthesia. It was testified that ACA strengthened the analgesic effect and reduced the occurrence of postoperative pain, but its mechanism was not clear. Numerous reports have shown that chemokine receptor CX3CR1 is involved in the development and progression of many pathological pains. The present study was aimed to reveal whether ACA played the analgesic roles in the post-incision pain by affecting CX3CR1. A model of toe incision pain was established in C57BL/6J mice. The pain threshold was detected by behavioral test, and the expression of CX3CR1 protein was detected by immunohistochemical method and Western blot. The results showed that the significant mechanical allodynia and thermal hyperalgesia were induced by paw incision in the mice. Mechanical allodynia was significantly suppressed by ACA, but thermal hyperalgesia was not changed. CX3CR1 was mainly expressed in microglia in the spinal cord dorsal horn, and its protein level was significantly increased at 3 d after incision compared with that of naïve C57BL/6J mice. ACA did not affect CX3CR1 protein expression at 3 d after incision in the toe incision model mice. Paw withdrawal threshold was significantly increased at 3 d after incision in CX3CR1 knockout (KO) mice compared with that in the C57BL/6J mice. But the analgesic effect of ACA was disappeared in CX3CR1 KO mice. Accordingly, it was also blocked when neutralizing antibody of CX3CR1 was intrathecally injected (i.t.) 1 h before ACA in the C57BL/6J mice. These results suggest that CX3CR1 in microglia is involved in post-incision pain and analgesia of ACA.

Spinal synaptic scaffolding protein Homer 1b/c regulates CREB phosphorylation and c-fos activation induced by inflammatory pain in rats.

Previous studies have shown that spinal Homer 1b/c plays an important role in the maintenance of chronic inflammatory pain induced by complete Freund's adjuvant (CFA). This study investigated the possible mechanism underlying Homer 1b/c mediating CFA-induced inflammatory pain. Chronic inflammation was induced by CFA injection into the left hind ankle of the rat. Homer 1b/c antisense or missense oligonucleotides were administered intrathecally (10µg/10µl) from 5 to 8 days following the onset of inflammation. Immunohistochemistry was conducted to detect the expression of phosphorylated cAMP response element binding protein (pCREB) and Fos protein in the spinal dorsal horn. Intrathecal administration of Homer 1b/c antisense oligonucleotides not only markedly reduced the expression of Homer 1b/c protein, but also attenuated CFA-induced inflammation, spinal CREB phosphorylation, and Fos expression. These results demonstrate for the first time that Homer 1b/c regulates CREB phosphorylation and c-fos activation in the spinal dorsal horn during the maintenance of chronic inflammatory pain, suggesting that Homer 1b/c may be involved in the development of CFA-induced inflammation.

Peripheral TGF-ß1 signaling is a critical event in bone cancer-induced hyperalgesia in rodents.

Pain is the most common symptom of bone cancer. TGF-ß, a major bone-derived growth factor, is largely released by osteoclast bone resorption during the progression of bone cancer and contributes to proliferation, angiogenesis, immunosuppression, invasion, and metastasis. Here, we further show that TGF-ß1 is critical for bone cancer-induced pain sensitization. We found that, after the progression of bone cancer, TGF-ß1 was highly expressed in tumor-bearing bone, and the expression of its receptors, TGFßRI and TGFßRII, was significantly increased in the DRG in a rat model of bone cancer pain that is based on intratibia inoculation of Walker 256 mammary gland carcinoma cells. The blockade of TGF-ß receptors by the TGFßRI antagonist SD-208 robustly suppressed bone cancer-induced thermal hyperalgesia on post-tumor day 14 (PTD 14). Peripheral injection of TGF-ß1 directly induced thermal hyperalgesia in intact rats and wide-type mice, but not in Trpv1(-/-) mice. Whole-cell patch-clamp recordings from DRG neurons showed that transient receptor potential vanilloid (TRPV1) sensitivity was significantly enhanced on PTD 14. Extracellular application of TGF-ß1 significantly potentiated TRPV1 currents and increased [Ca(2+)]i in DRG neurons. Pharmacological studies revealed that the TGF-ß1 sensitization of TRPV1 and the induction of thermal hyperalgesia required the TGF-ßR-mediated Smad-independent PKCε and TGF-ß activating kinase 1-p38 pathways. These findings suggest that TGF-ß1 signaling contributes to bone cancer pain via the upregulation and sensitization of TRPV1 in primary sensory neurons and that therapeutic targeting of TGF-ß1 may ameliorate the bone cancer pain in advanced cancer.

Estrogen in the anterior cingulate cortex contributes to pain-related aversion.

The rostral anterior cingulate cortex (rACC) is a key structure of pain affect. Whether and how estrogen in the rACC regulates pain-related negative emotion remains unclear. Behaviorally, using formalin-induced conditioned place aversion (F-CPA) in rats, which is believed to reflect the pain-related negative emotion, we found that estrogen receptor (ER) inhibitor ICI 182, 780 (ICI, 7α,17ß-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol) or inhibitor of aromatase androstatrienedione into the rACC completely blocked F-CPA in either sex. An analogous effect was also observed in ovariectomy rats. Furthermore, exogenous estrogen in the absence of a formalin noxious stimulus was sufficient to elicit CPA (E-CPA) in both sexes by activating the membrane estrogen receptors (mERs) and N-methyl-D-aspartic acid (NMDA) receptors (NMDARs). Electrophysiologically, we demonstrated that estrogen acutely enhanced the glutamatergic excitatory postsynaptic currents (EPSCs) in rACC slices by increasing the ratio of NMDA-EPSCs to α-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid -EPSCs and presynaptic glutamate release. Interestingly, a brief exposure to estrogen elicited a persistent enhancement of NMDA-EPSCs, and this NMDA-long-term potentiation required the activation of the mERs, protein kinase A and NMDAR subunit NR2B. Finally, estrogen induced rapid dendritic spine formation in cultured rACC neurons. These results suggest that estrogen in the rACC, as a neuromodulator, drives affective pain via facilitating NMDA receptor-mediated synaptic transmission.

Estrogen facilitates spinal cord synaptic transmission via membrane-bound estrogen receptors: implications for pain hypersensitivity.

Recent evidence suggests that estrogen is synthesized in the spinal dorsal horn and plays a role in nociceptive processes. However, the cellular and molecular mechanisms underlying these effects remain unclear. Using electrophysiological, biochemical, and morphological techniques, we here demonstrate that 17ß-estradiol (E2), a major form of estrogen, can directly modulate spinal cord synaptic transmission by 1) enhancing NMDA receptor-mediated synaptic transmission in dorsal horn neurons, 2) increasing glutamate release from primary afferent terminals, 3) increasing dendritic spine density in cultured spinal cord dorsal horn neurons, and 4) potentiating spinal cord long term potentiation (LTP) evoked by high frequency stimulation (HFS) of Lissauer's tract. Notably, E2-BSA, a ligand that acts only on membrane estrogen receptors, can mimic E2-induced facilitation of HFS-LTP, suggesting a nongenomic action of this neurosteroid. Consistently, cell surface biotinylation demonstrated that three types of ERs (ERα, ERß, and GPER1) are localized on the plasma membrane of dorsal horn neurons. Furthermore, the ERα and ERß antagonist ICI 182,780 completely abrogates the E2-induced facilitation of LTP. ERß (but not ERα) activation can recapitulate E2-induced persistent increases in synaptic transmission (NMDA-dependent) and dendritic spine density, indicating a critical role of ERß in spinal synaptic plasticity. E2 also increases the phosphorylation of ERK, PKA, and NR2B, and spinal HFS-LTP is prevented by blockade of PKA, ERK, or NR2B activation. Finally, HFS increases E2 release in spinal cord slices, which can be prevented by aromatase inhibitor androstatrienedione, suggesting activity-dependent local synthesis and release of endogenous E2.

Involvement of estrogen in rapid pain modulation in the rat spinal cord.

The pivotal role of estrogens in the pain sensitivity has been investigated in many ways. Traditionally, it is ascribed to the slow genomic changes mediated by classical nuclear estrogen receptors (ER), ERα and ERß, depending on peripheral estrogens. Recently, it has become clear that estrogens can also signal through membrane ERs (mERs), such as G-protein-coupled ER1 (GPER1), mediating the non-genomic effects. However, the spinal specific role played by ERs and the underlying cellular mechanisms remain elusive. The present study investigated the rapid estrogenic regulation of nociception at the spinal level. Spinal administration of 17ß-estradiol (E2), the most potent natural estrogen, acutely produced a remarkable mechanical allodynia and thermal hyperalgesia without significant differences among male, female and ovariectomized (Ovx) rats. E2-induced the pro-nociceptive effects were partially abrogated by ICI 182,780 (ERs antagonist), and mimicked by E2-BSA (a mER agonist). Inhibition of local E2 synthesis by 1,4,6-Androstatrien-3,17-dione (ATD, a potent irreversible aromatase inhibitor), or blockade of ERs by ICI 182,780 produced an inhibitory effect on the late phase of formalin nociceptive responses. Notably, lumbar puncture injection of G15 (a selective GPER1 antagonist) resulted in similar but more efficient inhibition of formalin nociceptive responses as compared with ICI 182,780. At the cellular level, the amplitude and decay time of spontaneous inhibitory postsynaptic currents were attenuated by short E2 or E2-BSA treatment in spinal slices. These results indicate that estrogen acutely facilitates nociceptive transmission in the spinal cord via activation of membrane-bound estrogen receptors.

Activation of glycine site and GluN2B subunit of NMDA receptors is necessary for ERK/CREB signaling cascade in rostral anterior cingulate cortex in rats: implications for affective pain.

OBJECTIVE: The rostral anterior cingulate cortex (rACC) is implicated in processing the emotional component of pain. N-methyl-D-aspartate receptors (NMDARs) are highly expressed in the rACC and mediate pain-related affect by activating a signaling pathway that involves cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and/or extracellular regulated kinase (ERK)/cAMP-response element-binding protein (CREB). The present study investigated the contributions of the NMDAR glycine site and GluN2B subunit to the activation of ERK and CREB both in vitro and in vivo in rat rACC. METHODS: Immunohistochemistry and Western blot analysis were used to separately assess the expression of phospho-ERK (pERK) and phospho-CREB (pCREB) in vitro and in vivo. Double immunostaining was also used to determine the colocalization of pERK and pCREB. RESULTS: Both bath application of NMDA in brain slices in vitro and intraplantar injection of formalin into the rat hindpaw in vivo induced significant up-regulation of pERK and pCREB in the rACC, which was inhibited by the NMDAR antagonist DL-2-amino-5-phospho-novaleric acid. Selective blockade of the NMDAR GluN2B subunit and the glycine-binding site, or degradation of endogenous D-serine, a co-agonist for the glycine site, significantly decreased the up-regulation of pERK and pCREB expression in the rACC. Further, the activated ERK predominantly colocalized with CREB. CONCLUSION: Either the glycine site or the GluN2B subunit of NMDARs participates in the phosphorylation of ERK and CREB induced by bath application of NMDA in brain slices or hindpaw injection of 5% formalin in rats, and these might be fundamental molecular mechanisms underlying pain affect.

Targeting A-type K(+) channels in primary sensory neurons for bone cancer pain in a rat model.

Cancer pain is one of the most severe types of chronic pain, and the most common cancer pain is bone cancer pain. The treatment of bone cancer pain remains a clinical challenge. Here, we report firstly that A-type K(+) channels in dorsal root ganglion (DRG) are involved in the neuropathy of rat bone cancer pain and are a new target for diclofenac, a nonsteroidal anti-inflammatory drug that can be used for therapy for this distinct pain. There are dynamically functional changes of the A-type K(+) channels in DRG neurons during bone cancer pain. The A-type K(+) currents that mainly express in isolectin B4-positive small DRG neurons are increased on post-tumor day 14 (PTD 14), then faded but still remained at a higher level on PTD 21. Correspondingly, the expression levels of A-type K(+) channel Kv1.4, Kv3.4, and Kv4.3 showed time-dependent changes during bone cancer pain. Diclofenac enhances A-type K(+) currents in the DRG neurons and attenuates bone cancer pain in a dose-dependent manner. The analgesic effect of diclofenac can be reversed or prevented by A-type K(+) channel blocker 4-AP or pandinotoxin-Kα, also by siRNA targeted against rat Kv1.4 or Kv4.3. Repeated diclofenac administration decreased soft tissue swelling adjacent to the tumor and attenuated bone destruction. These results indicate that peripheral A-type K(+) channels were involved in the neuropathy of rat bone cancer pain. Targeting A-type K(+) channels in primary sensory neurons may provide a novel mechanism-based therapeutic strategy for bone cancer pain.

Involvement of lysophosphatidic acid in bone cancer pain by potentiation of TRPV1 via PKCε pathway in dorsal root ganglion neurons.

BACKGROUND: It has been demonstrated that lysophosphatidic acid (LPA) released from injury tissue and transient receptor potential vanilloid 1 (TRPV1) receptor are implicated in the induction of chronic pain. In the present study we examined whether an interaction between LPA receptor LPA(1) and TRPV1 in dorsal root ganglion (DRG) neurons contributes to the development of bone cancer pain. RESULTS: Bone cancer was established by injection of mammary gland carcinoma cells into the rat tibia. Following the development of bone cancer pain, the TRPV1 expression and capsaicin-evoked currents were up-regulated in rat DRG neurons at L(4-6) segments. Immunohistochemistry staining revealed a high co-localization of LPA(1) with TRPV1 in DRG neurons. In isolated DRG neurons, whole-cell patch recording showed that capsaicin-induced currents were potentiated by LPA in a dose-dependent manner. The potentiation was blocked by either LPA(1) antagonist, protein kinase C (PKC) inhibitor or PKCε inhibitor, but not by protein kinase A (PKA) inhibitor or Rho inhibitor. In the behavioral tests, both mechanical allodynia and thermal hyperalgesia in bone cancer rats were attenuated by LPA(1) antagonist. CONCLUSION: LPA potentiates TRPV1 current via a PKCε-dependent pathway in DRG neurons of rats with bone cancer, which may be a novel peripheral mechanism underlying the induction of bone cancer pain.

Prevention of paclitaxel-induced allodynia by minocycline: Effect on loss of peripheral nerve fibers and infiltration of macrophages in rats.

BACKGROUND: Although paclitaxel is a frontline antineoplastic agent for treatment of solid tumors, the paclitaxel-evoked pain syndrome is a serious problem for patients. There is currently no valid drug to prevent or treat the paclitaxel-induced allodynia, partly due to lack of understanding regarding the cellular mechanism. Studies have shown that minocycline, an inhibitor of microglia/macrophage, prevented neuropathic pain and promoted neuronal survival in animal models of neurodegenerative disease. Recently, Cata et al also reported that minocycline inhibited allodynia induced by low-dose paclitaxel (2 mg/kg) in rats, but the mechanism is still unclear. RESULTS: Here, we investigate by immunohistochemistry the change of intraepidermal nerve fiber (IENF) in the hind paw glabrous skin, expression of macrophage and activating transcription factor 3 (ATF3) in DRG at different time points after moderate-dose paclitaxel treatment (cumulative dose 24 mg/kg; 3 × 8 mg/kg) in rats. Moreover, we observe the effect of minocycline on the IENF, macrophages and ATF3. The results showed that moderate-dose paclitaxel induced a persisted, gradual mechanical allodynia, which was accompanied by the loss of IENF in the hind paw glabrous skin and up-regulation of macrophages and ATF3 in DRG in rats. The expressions of ATF3 mainly focus on the NF200-positive cells. More importantly, we observed that pretreatment of minocycline at dose of 30 mg/kg or 50 mg/kg, but not 5 mg/kg, prevented paclitaxel-evoked allodynia. The evidence from immunohistochemistry showed that 30 mg/kg minocycline rescued the degeneration of IENF, attenuated infiltration of macrophages and up-regulation of ATF3 induced by paclitaxel treatment in rats. CONCLUSIONS: Minocycline prevents paclitaxel-evoked allodynia, likely due to its inhibition on loss of IENF, infiltration of macrophages and up-regulation of ATF3 in rats. The finding might provide potential target for preventing paclitaxel-induced neuropathic pain.

The sensitization of peripheral C-fibers to lysophosphatidic acid in bone cancer pain.

AIMS: Lysophosphatidic acid (LPA) is released from injured tissue and cancer cells and is involved in the induction of neuropathic pain. The present study explores whether LPA plays a role in the development of osteocarcinoma-induced pain. MAIN METHODS: The bone cancer model was established using the Walker 256 mammary gland carcinoma cell line, and cancer-related behavioral and physiological changes were observed using von Frey, X-ray and immunohistochemical methods. The role of LPA in the bone cancer model and related mechanisms were examined by using in vitro single fiber recording and western blot. KEY FINDINGS: Rats exhibited severe hyperalgesia 2weeks after the cancer cell implantation. Several changes were observed at this time point including: ipsilateral dorsal root ganglion (DRG) neurons were labeled by injured neurons marker ATF3; LPA(1) receptor expression in DRG neurons was increased; sural C-fibers were more sensitive to LPA stimuli, and this response could be blocked by LPA receptor and substance P receptor antagonists. SIGNIFICANCE: These data indicate that LPA is involved in the induction of bone cancer pain through mechanisms of peripheral C-fibers sensitization. LPA and its downstream molecules possibly are promising therapeutic targets for treatment of cancer pain.

Involvement of microglial P2X7 receptors and downstream signaling pathways in long-term potentiation of spinal nociceptive responses.

Tetanic stimulation of the sciatic nerve (TSS) produces long-term potentiation (LTP) of C-fiber-evoked field potentials in the spinal cord. This potentiation is considered to be a substrate for long-lasting sensitization in the spinal pain pathway. Because microglia have previously been shown to regulate the induction of spinal LTP, we hypothesize that P2X7 receptors (P2X7R), which are predominantly expressed in microglia and participate in the communication between microglia and neurons, may play a role in this induction. This study investigated the potential roles of P2X7Rs in spinal LTP and persistent pain induced by TSS in rats. OxATP or BBG, a P2X7R antagonist, prevented the induction of spinal LTP both in vivo and in spinal cord slices in vitro and alleviated mechanical allodynia. Down-regulation of P2X7Rs with P2X7-siRNA blocked the induction of spinal LTP and inhibited mechanical allodynia. Double immunofluorescence showed colocalization of P2X7Rs with the microglial marker OX-42, but not with the astrocytic marker GFAP or the neuronal marker NeuN. Intrathecal injection of BBG suppressed the up-regulation of microglial P2X7Rs and increased expression of Fos in the spinal superficial dorsal horn. Further, pre-administration of BBG inhibited increased expression of the microglial marker Iba-1, phosphorylated p38 (p-p38), interleukin 1ß (IL-1ß) and GluR1 following TSS. Pre-administration of the IL-1 receptor antagonist (IL-1ra) blocked both the induction of spinal LTP and the up-regulation of GluR1. These results suggest that microglial P2X7Rs and its downstream signaling pathways play a pivotal role in the induction of spinal LTP and persistent pain induced by TSS.