Single-Cell Transcriptional Profiling Reveals Low-Level Tragus Stimulation Improves Sepsis-Induced Myocardial Dysfunction by Promoting M2 Macrophage Polarization.

Background: Sepsis can lead to multiple organ damage, of which the heart is one of the most vulnerable organs. Vagal nerve stimulation can reduce myocardial injury in sepsis and improve survival rates. However, the potential impact of low-level tragus stimulation and disparate cell populations on sepsis-induced myocardial dysfunction remains undetermined. Methods: A cardiac single-cell transcriptomic approach was used for characterizing cardiac cell populations that form the heart. Single-cell mRNA sequencing data were used for selecting all cardiac macrophages from CD45+ cells. Then, echocardiography, western blot, flow cytometry, immunofluorescence, and immunohistochemistry were performed to verify the single-cell mRNA sequencing results. Results: Using single-cell mRNA sequencing data, we uncovered the multiple cell populations contributing to myocardial injury in sepsis under low-level tragus stimulation, thereby illustrating a comprehensive map of the cardiac cellular landscape. Pseudotiming analysis in single-cell sequencing showed that low-level vagal nerve stimulation played an anti-inflammatory role by promoting cardiac monocytes into M2 macrophages, which significantly increased α7nAChR expression in heart tissues. Echocardiography assessment indicated that low-level vagal nerve stimulation could also improve cardiac functions in mice with sepsis-induced myocardial dysfunction. In addition, the heart tissues of mice from the sepsis group with low-level tragus stimulation had significantly lower interleukin-1ß expression levels than those from the sepsis group. Flow cytometry analysis showed that different acetylcholine concentrations promoted cardiac monocytes into M2 macrophages in in vitro experiments. Conclusion: Low-level tragus stimulation could improve sepsis-induced myocardial dysfunction by promoting cardiac monocytes to M2 macrophages.

The nonopioid cholinergic agonist GTS-21 mitigates morphine-induced aggravation of burn injury pain together with inhibition of spinal microglia activation in young rats.

BACKGROUND: Repetitive opioid use does not always alleviate basal pain, procedural pain, or both after burn injury. Mitigation of burn injury-site pain can be achieved by GTS-21 stimulation of α7-acetylcholine nicotinic receptors (α7AChRs) and reduced microglia activation in rat. We tested the hypothesis that morphine exaggerates burn injury-site pain and GTS-21 alleviates both morphine-induced aggravated burn injury pain and microglia activation. METHODS: Young rats with dorsal paw burn injury or sham-burn received intraperitoneal saline, morphine, GTS-21, or a combination twice daily for 14 days. Ipsilateral plantar pain thresholds were tested every other day before morning drugs from days 0-20. Spinal microglia activation, evidenced as pain-transducer (tumour necrosis factor-α [TNF-α], interleukin [IL]-6, IL-1ß, nuclear factor kappa B [NF-κB], Toll-like receptor 4 [TLR4]) expression, was examined using immunohistochemistry and immunoblot. In cultured microglia, morphine-induced cytokine expression was measured (quantitative polymerase chain reaction/enzyme-linked immunosorbent assay [qPCR/ELISA]). RESULTS: Morphine aggravated allodynia at day 5 in sham-burn (P=0.039, n=8-11) but significantly aggravated burn injury site allodynia by day 3 (P=0.010, n=8-11). Microgliosis paralleled nociceptive behaviour changes where burn injury with morphine had highest microgliosis compared with burn injury, morphine alone, or controls (number of cells per field [SD]: 33.8 [2.4], 18.0 [4.1], 8.2 [1.9], and 4.8 [2.0], respectively; P<0.001, n=4-5]. GTS-21 reversed the morphine-induced pain component in sham-burn and burn injury rats together with reduced microgliosis and spinal pain-transducer expression (TNF-α, IL-6, IL-1ß, NF-κB, and TLR4). Morphine-exposed microglial cells showed increased cytokine expression, which was mitigated by GTS-21. CONCLUSIONS: Morphine or burn injury alone increases pain together with microgliosis and pain-transducer expression. Morphine administration augments burn injury-site nociception sooner and aggravated spinal microgliosis and inflammatory pain-transducer expression. GTS-21 has the potential to treat morphine-induced pain in burn injury.

Alpha7 nicotinic acetylcholine receptor expression in Sorafenib-resistant Hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC), the most prevalent kind of liver cancer, remains one of the world's main causes of death. The alpha7 nicotinic acetylcholine receptor (α7nAchR) has been recognized to be overexpressed in malignancies and chemoresistance. Since little is known about the role of α7nAchR expression in drug-resistant cells, this study was designed to investigate the effect of α7nAchR suppression in combination with Sorafenib (SOR) on SOR-resistant HCC cells. First, SOR-resistant HCC cells were generated. To suppress the expression of α7nAchR, cells were treated with SOR following siRNA transfection. qRT-PCR was used to examine the expression of α7nAchR and apoptotic genes by evaluating the IC50 of SOR and the combination of α7nAchR siRNA and SOR on the survival of resistant cells. Moreover, apoptosis, autophagy, and cell cycle analysis for resistant HCC cells were performed using flow cytometry. Cell migration and colony formation assays were also used for further confirmation. Our results suggest that inhibiting α7nAchR can lead resistant HCC cells to become sensitive. Furthermore, when siRNA and SOR were treated together, HCC-resistant cells showed a considerable reduction in α7nAchR mRNA gene expression. In addition, when α7nAchR was downregulated in combination with SOR, migration and colony formation were inhibited. Apoptosis was triggered by modulating the expression of apoptotic target genes, and cell cycle arrest was observed in the G2-M and subG1 phases. Overexpression of α7nAchR in SOR-resistant HCC cells suggests that it might be a therapeutic target for HCC cell resistance therapy.

Transcutaneous Vagus Nerve Stimulation Ameliorates the Phenotype of Heart Failure With Preserved Ejection Fraction Through Its Anti-Inflammatory Effects.

BACKGROUND: A systemic proinflammatory state plays a central role in the development of heart failure with preserved ejection fraction (HFpEF). Low-level transcutaneous vagus nerve stimulation (LLTS) suppresses inflammation in animals and humans, mediated by an α7nAchR (alpha7 nicotinic acetylcholine receptor)-dependent pathway. We examined the effects of LLTS on cardiac function, inflammation, and fibrosis in the presence of α7nAchR pharmacological blockade in a rat model of HFpEF. METHODS: Dahl salt-sensitive rats at 7 weeks of age were treated with high-salt diet for 6 weeks to induce HFpEF, followed by 4 weeks of (1) LLTS, (2) LLTS with the α7nAchR blocker methyllycaconitine, (3) sham, and (4) olmesartan. Blood pressure, cardiac function by echocardiography, heart rate variability, and serum cytokines were measured at 13 and 17 weeks of age. Cardiac fibrosis, inflammatory cell infiltration, and gene expression were determined at 17 weeks. RESULTS: LLTS attenuated the increase in blood pressure; improved cardiac function; decreased inflammatory cytokines, macrophage infiltration, and fibrosis; and improved survival compared with other groups. Methyllycaconitine attenuated these effects, whereas olmesartan did not improve cardiac function or fibrosis despite maintaining similar blood pressure as LLTS. Heart rate variability was similarly improved in the LLTS and LLTS plus methyllycaconitine groups but remained low in the other groups. LLTS reversed the dysregulated inflammatory signaling pathways in HFpEF hearts. CONCLUSIONS: Neuromodulation with LLTS improved cardiac function in a rat model of HFpEF through its anti-inflammatory and antifibrotic effects. These results provide the basis for further clinical trials in humans.

Survey of immunopharmacological effects of botulinum toxin in cell signaling of bronchial smooth muscle cells in allergic asthma.

BACKGROUND: Asthma is a lung disease that has influenced more than 350 million people worldwide. Airway smooth muscle (ASM) spasm leads to airway hyperresponsiveness (AHR) and bronchial obstruction, which are clinical manifestations of an asthma attack. Botulinum toxin (BTX) is a bacteria toxin that acts as muscle relaxant and may have therapeutic effects on AHR and asthma. OBJECTIVE: In this study, the effect of BTX on AHR and related gene expressions was evaluated. MATERIAL AND METHODS: An asthma mice model was developed which was treated with BTX in two ways: intranasally (IN) and via nebulization (N) (0.01, 0.1, and 1 U/mL and 10 U/mL, respectively) on days 25, 27 and 29. AHR was evaluated on days 24, 26, 28, and 30, and gene expressions were evaluated for TrkA, TrkB, M1-M5, α7nAChR, TNF-α, and extracellular signal-regulated kinase 2 (ERK2) proteins. For histopathology of the lungs, perivascular and peribronchial inflammation, production of mucus, and goblet cell hyperplasia were studied. RESULTS: On day 24, treatment with BTX (for all doses) had no significant effect on AHR, but on days 26 and 28, AHR was decreased and this continued up to day 30 for all treated groups. Treatment with BTX had no significant effect on the gene expressions of TrkA, TrkB, M1-M5, α7nAChR, TNF-α, and ERK2 proteins, perivascular inflammation, peribronchial inflammation, hyperplasia of the goblet cell and production of mucus. Besides, mice administered with 10 mg/mL BTX perished. The BTX therapy controlled asthma attacks by decreasing AHR and relaxation of ASMs. CONCLUSION: However, BTX had no significant effect on airway inflammation and production of mucus. While using BTX, it is necessary to prescribe safe doses in order to prevent adverse reactions.

Acetylcholine receptor agonists provide cardioprotection in doxorubicin-induced cardiotoxicity via modulating muscarinic M2 and α7 nicotinic receptor expression.

The balance between cardiac sympathetic and parasympathetic activities has been intricately linked to mitochondrial function, cellular oxidative status, and immunomodulation in healthy and diseased myocardium. Cardiac autonomic neuropathy, along with the associated mitochondrial and cellular dysfunction, is an important pathophysiological feature of doxorubicin-induced cardiotoxicity (DIC). We tested the hypothesis that autonomic modulation by activation of acetylcholine receptors (AChR) effectively attenuates DIC. Rats were divided into control (0.9% sodium chloride solution) and doxorubicin groups (DOX, 3 mg/kg/d, 6 doses). Rats in the DOX group were equally subdivided into 4 interventional groups and treated for 30 days: vehicle, α7 nicotinic receptor agonist (PNU: PNU-282987, 3 mg/kg/d), muscarinic receptor agonist (BET: bethanechol, 12 mg/kg/d), and combined α7nAChR and mAChR agonists group (COM). Cardiac biochemical and functional analyses were done. The results show that AChR agonists protected the heart against DIC via improving mitochondrial and cardiac function, which was accompanied by reducing mitochondrial oxidative damage, apoptosis, and inflammation. Strikingly, PNU and BET exerted cardioprotection through different molecular pathways. PNU-mediated α7nAChR activation promoted mitochondrial fusion via upregulation of Mfn1-2 and attenuated DOX-induced autophagy. Contrarily, activation of mAChR by BET attenuated mitochondrial fission and mitophagy. The in vitro experiments confirmed the cytoprotective effects of AChR activation in DOX-treated H9c2 cells without compromising the anticancer effect of DOX in cancer cells. In conclusion, α7nAChR and mAChR agonists exerted cardioprotection against DIC via rebalancing autonomic function, improving mitochondrial function, reducing oxidative stress, and decreased cardiomyocyte apoptosis and inflammation, leading to improved cardiac function.

α7-nAChR Activation Has an Opposite Effect on Healing of Covered and Uncovered Wounds.

The α7 nicotinic acetylcholine receptor (α7-nAChR) is associated with inflammation, re-epithelialization, and angiogenesis in wound healing process. A recent study demonstrated that PNU-282987, a selective agonist of α7-nAChR, accelerates the repair of diabetic excisional wounds. Whether α7-nAChR activation promotes non-diabetic wounds healing is unknown. The aim of this study was to evaluate the effects of α7-nAChR activation on non-diabetic wound healing. The effects were evaluated in two wound models. In the first model, the wound was covered with a semi-permeable transparent dressing. In the second model, the wound was left uncovered. In both models, the mice were randomly assigned to two treatment groups: saline or PNU282987 (25 mice in each group). In covered wounds, we found that α7-nAChR activation inhibited re-epithelialization, angiogenesis, and epithelial cells proliferation, promoted neo-epithelial detachment, and suppressed neutrophil infiltration and the expression of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). However, in uncovered wounds, we observed that α7-nAChR activation promoted re-epithelialization and angiogenesis, inhibited neutrophil infiltration and the expression of high mobility group box (HMGB)-1, epidermal growth factor (EGF), and VEGF. In conclusion, this data demonstrated that α7-nAChR activation inhibited wound healing in covered wounds but played an opposite role in uncovered wounds. The opposite effect might be primarily due to inhibition of inflammation.

α7 Nicotinic Acetylcholine Receptor Stimulation Attenuates Neuroinflammation through JAK2-STAT3 Activation in Murine Models of Intracerebral Hemorrhage.

Accounting for high mortality and morbidity rates, intracerebral hemorrhage (ICH) remains one of the most detrimental stroke subtypes lacking a specific therapy. Neuroinflammation contributes to ICH-induced brain injury and is associated with unfavorable outcomes. This study aimed to evaluate whether α7 nicotinic acetylcholine receptor (α7nAChR) stimulation ameliorates neuroinflammation after ICH. Male CD-1 mice and Sprague-Dawley were subjected to intracerebral injection of autologous blood or bacterial collagenase. ICH animals received either α7nAChR agonist PHA-543613 alone or combined with α7nAChR antagonist methyllycaconitine (MLA) or Janus kinase 2 (JAK2) antagonist AG490. Neurobehavioral deficits were evaluated at 24 hours, 72 hours, and 10 weeks after ICH induction. Perihematomal expressions of JAK2, signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor-α (TNF-α), and myeloperoxidase (MPO) were quantified via Western blot. Histologic volumetric analysis of brain tissues was conducted after 10 weeks following ICH induction. PHA-543613 improved short-term neurobehavioral (sensorimotor) deficits and increased activated perihematomal JAK2 and STAT3 expressions while decreasing TNF-α and MPO expressions after ICH. MLA reversed these treatment effects. PHA-543613 also improved long-term neurobehavioral (sensorimotor, learning, and memory) deficits and ameliorated brain atrophy after ICH. These treatment effects were reduced by AG490. α7nAChR stimulation reduced neuroinflammation via activation of the JAK2-STAT3 pathway, thereby ameliorating the short- and long-term sequelae after ICH.