Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway

Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae （Gegen）, has been reported to inhibit neuronal apoptosis and play an anti-inflammatory role in focal cerebral ischemia model rats. Recent findings regarding stroke pathophysiology have recognized that anti-inflammation is an important target for the treatment of ischemic stroke. The cholinergic anti-inflammatory pathway is a highly robust neural-immune mechanism for inflammation control. This study was to investigate whether activating the cholinergic anti-inflammatory pathway can be involved in the mechanism of inhibiting the inflammatory response during puerarin-induced cerebral ischemia/reperfusion in rats. Results showed that puerarin pretreatment （intravenous injection） re- duced the ischemic infarct volume, improved neurological deficit after cerebral ischemia/reperfusion and decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-a in brain tissue. Pretreatment with puerarin （intravenous injection） attenuated the inflammatory response in rats, which was accompanied by janus-activated kinase 2 （JAK2） and signal transducers and activators of transcription 3 （STAT3） activation and nuclear factor kappa B （NF-KB） inhibition. These observa- tions were inhibited by the alpha7 nicotinic acetylcholine receptor （a7nAchR） antagonist a-bungarotoxin （a-BGT）. In addition, puerarin pretreatment increased the expression of a7nAchR mRNA in ischemic cerebral tissue. These data demonstrate that puerarin pretreatment strongly protects the brain against cerebral ischemia/reperfusion injury and inhibits the inflammatory re- sponse. Our results also indicated that the anti-inflammatory effect of puerarin may partly be medi- ated through the activation of the cholinergic anti-inflammatory pathway.

Berberine Relieves Insulin Resistance via the Cholinergic Anti-inflammatory Pathway in HepG2 Cells

Berberine（BBR） is an isoquinoline alkaloid extracted from Rhizoma coptidis and has been used for treating type 2 diabetes mellitus（T2DM） in China. The development of T2 DM is often associated with insulin resistance and impaired glucose uptake in peripheral tissues. In this study, we examined whether BBR attenuated glucose uptake dysfunction through the cholinergic anti-inflammatory pathway in Hep G2 cells. Cellular glucose uptake, quantified by the 2-[N-（7-Nitrobenz-2-oxa-1,3-diazol-4-yl）-amino]-2-deoxy-D-glucose（2-NBDG）, was inhibited by 21% after Hep G2 cells were incubated with insulin（10-6 mol/L） for 36 h. Meanwhile, the expression of alpha7 nicotinic acetylcholine receptor（α7n ACh R） protein was reduced without the change of acetylcholinesterase（ACh E） activity. The level of interleukin-6（IL-6） in the culture supernatant, the ratio of phosphorylated I-kappa-B kinase-β（IKKβ） Ser181/IKKβ and the expression of nuclear factor-kappa B（NF-κB） p65 protein were also increased. However, the treatment with BBR enhanced the glucose uptake, increased the expression of α7n ACh R protein and inhibited ACh E activity. These changes were also accompanied with the decrease of the ratio of p IKKβ Ser181/IKKβ, NF-κB p65 expression and IL-6 level. Taken together, these results suggest that BBR could enhance glucose uptake, and relieve insulin resistance and inflammation in Hep G2 cells. The mechanism may be related to the cholinergic anti-inflammatory pathway and the inhibition of ACh E activity.

Electroacupuncture targeting the immune system to alleviate sepsis

Sepsis is a life-threatening inflammatory syndrome with high morbidity and mortality rates.However,options for sepsis are still limited to general treatment in intensive care units(ICUs),and effective therapies that improve sepsis survival are required.Immune disturbances play a vital role in the pathology of sepsis and are associated with protracted inflammation,susceptibility to infections,and death.Therefore,many investigators have focused on the potential benefits of immunomodulation therapy for sepsis.Electroacupuncture(EA)has been practiced in clinics for many years and has shown advantages in treating infectious diseases.Over the last few decades,our understanding of the efficacy and mechanisms of EA in sepsis has undergone considerable developments.We searched the literature regarding“CNKI,Wan Fang Data,VIP Database,PubMed,and Ingenta Connect”from 2010 to 2023,using the keywords“sepsis”“septic”and“electroacupuncture”and 336 sources were searched.Finally,we included 82 studies that targeted the immune system to determine EA’s anti-inflammatory and immunomodulatory effects on sepsis.In this review,we found that EA has clinical benefits in relieving septic inflammation,improving immune function,and attenuating related multi-organ injury through several mechanisms,such as activation of the cholinergic anti-inflammatory pathway(CAP),vagaladrenal axis,inhibition of the nuclear factor Kappa-B(NF-κB)signaling pathway,signal transducers and activators of transcription(STAT)signaling pathway,and improvement of immune cell function.Therefore,EA may be a promising complementary therapy for sepsis treatment.We also expect these data will contribute to further studies on EA in sepsis.

Electro-acupuncture regulates the cholinergic anti-inflammatory pathway in a rat model of chronic obstructive pulmonary disease

Objective： Acupuncture has a definite therapeutic effect on chronic obstructive pulmonary disease （COPD）, and the cholinergic anti-inflammatory pathway （CAP） has been shown to be involved in regula- tion of inflammation. In this study, we investigated whether electro-acupuncture （EA） affects the CAP in COPD,Methods： Sprague-Dawley rats were induced into COPD through exposure to cigarette smoke combined with lipopolysaccharide. EA treatment was applied at Zusanli （ST36） and Feishu （BL13） points for 30 min/d for 7 d. Seventy-two rats were randomly divided into six study groups, including normal, normal ＋ EA, normal ＋ α-bungarotoxin （α-BGT） （the antagonist of the nicotinic acetylcholine receptor α7 subunit （α7nAChR）） ＋ EA, COPD, COPD ＋ EA, and COPD ＋ α-BGT ＋ EA. Lung function, pathology and vagus nerve discharge were tested. The levels of acetylcholine （ACh）, acetylcholinesterase （ACHE）, interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-ct） in bronchoalveolar lavage fluid （BALF） and lung tissue were measured by enzyme-linked immunosorbent assay. The mRNA and protein expression and immunoreac- tivity of α7nAChR and its postreceptor inflammation signal pathway, including janus kinase 2 （JAK2）, sig- nal transducers and activators of transcription 3 （STAT3）, nuclear factor-KB （NF-KB）, were observed by quantitative reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry. Results： Compared with normal rats, there were a significant decline in lung function and discharge of the vagus nerve （P 〈 0.01）, a marked sign of lung inflammation and an increase of ACh, ACHE, IL-6 and TNF-α level in BALF or lung tissue （P 〈 0.05, P 〈 0.01 ） and higher expression of 0t7nAChR, JAK2, STAT3 and NF-αB （P 〈 0.05, P 〈 0.01） in the COPD rats. In rats receiving EA, the lung function and vagal discharge were enhanced （P 〈 0.01 ）, lung inflammation was improved and the levels of ACh, ACHE, IL-6 and TNF-α were decreased （P 〈 0.01）. Further, the expression of α7nAChR, JAK2, STAT3 and NF-κB was downregulated （P 〈 0.05, P 〈 0.01 ）. However, the above effects of EA were blocked in rats injected with α-BCT （P 〈 0.01 ）. Conclusion： EA treatment can reduce the lung inflammatory response and improve lung function in COPD, which may be related to its involvement in the regulation of CAP.

Dexmedetomidine attenuates inflammation and pancreatic injury in a rat model of experimental severe acute pancreatitis via cholinergic anti-inflammatory pathway

Background:Excessive inflammatory responses play a critical role in the development of severe acute pancreatitis(SAP),and controlling such inflammation is vital for managing this often fatal disease.Dexmedetomidine has been reported to possess protective properties in inflammatory diseases.Therefore,this study aimed to investigate whether dexmedetomidine pre-treatment exerts an anti-inflammatory effect in rats with SAP induced by sodium taurocholate,and if so,to determine the potential mechanism.Methods:SAP was induced with sodium taurocholate.Rats received an intraperitoneal injection of dexmedetomidine 30 min before sodium taurocholate administration.α-bungarotoxin,a selective alpha-7 nicotinic acetylcholine receptor(α7nAchR)antagonist,was injected intra-peritoneally 30 min before dexmedetomidine administration.The role of the vagus nerve was evaluated by performing unilateral cervical vagotomy before the administration of dexmedetomidine.Efferent discharge of the vagal nerve was recorded by the BL-420F Data Acquisition&Analysis System.Six hours after onset,serum pro-inflammatory cytokine(tumor necrosis factorα[TNF-α]and interleukin 6[IL-6])levels and amylase levels were determined using an enzyme-linked immunosorbent assay and an automated biochemical analyzer,respectively.Histopathological changes in the pancreas were observed after hematoxylin and eosin staining and scored according to Schmidt criteria.Results:Pre-treatment with dexmedetomidine significantly decreased serum levels of TNF-α,IL-6,and amylase,strongly alleviating pathological pancreatic injury in the rat model of SAP(TNF-α:174.2±30.2 vs.256.1±42.4 pg/ml;IL-6:293.3±46.8 vs.421.7±48.3 pg/ml;amylase:2102.3±165.3 vs.3186.4±245.2 U/L).However,the anti-inflammatory and pancreatic protective effects were abolished after vagotomy or pre-administration ofα-bungarotoxin.Dexmedetomidine also significantly increased the discharge frequency and amplitude of the cervical vagus nerve in the SAP rat model(discharge frequency:456.8±50.3 vs.332.4±25.1 Hz;discharge amplitude:33.4±5.3 vs.20.5±2.9μV).Conclusions:Dexmedetomidine administration attenuated the systemic inflammatory response and local pancreatic injury caused by SAP in rats through the cholinergic anti-inflammatory pathway involving vagus-andα7nAChR-dependent mechanisms.

Septic encephalopathy: When cytokines interact with acetylcholine in the brain

Sepsis-associated encephalopathy(SAE) is a brain dysfunction that occurs secondary to infection in the bo characterized by alteration of consciousness, ranging from delirium to coma, seizure or focal neurological signs. S involves a number of mechanisms, including neuroinflammation, in which the interaction between cytokines a acetylcholine results in neuronal loss and alterations in cholinergic signaling. Moreover, the interaction also occurs the periphery, accelerating a type of immunosuppressive state. Although its diagnosis is not specific in biochemis and imaging tests, it could potentiate severe outcomes, including increased mortality, cognitive decline, progress immunosuppression, cholinergic anti-inflammatory deficiency, and even metabolic and hydroelectrolyte imbalan Therefore, the bilateral communication between SAE and the multiple peripheral organs and especially the immu system should be emphasized in sepsis management.

Alpha-7 nicotinic acetylcholine receptor agonist treatment in a rat model of Huntington's disease and involvement of heme oxygenase-1

Neuroinflammation is a common element involved in the pathophysiology of neurodegenerative diseases.We recently reported that repeated alpha-7 nicotinic acetylcholine receptor（α7 n ACh R） activations by a potent agonist such as PHA 543613 in quinolinic acid-injured rats exhibited protective effects on neurons.To further investigate the underlying mechanism,we established rat models of early-stage Huntington＇s disease by injection of quinolinic acid into the right striatum and then intraperitoneally injected 12 mg/kg PHA 543613 or sterile water,twice a day during 4 days.Western blot assay results showed that the expression of heme oxygenase-1（HO-1）,the key component of the cholinergic anti-inflammatory pathway,in the right striatum of rat models of Huntington＇s disease subjected to intraperitoneal injection of PHA 543613 for 4 days was significantly increased compared to the control rats receiving intraperitoneal injection of sterile water,and that the increase in HO-1 expression was independent of change in α7 n ACh R expression.These findings suggest that HO-1 expression is unrelated to α7 n ACh R density and the increase in HO-1 expression likely contributes to α7 n ACh R activation-related neuroprotective effect in early-stage Huntington＇s disease.

Therapeutic potential of α7 nicotinic receptor agonists to regulate neuroinflammation in neurodegenerative diseases

Neurodegenerative diseases, such as Alzheimer＇s, Parkinson＇s and Huntington＇s diseases, are all character- ized by a component of innate immunity called neuroinflammation. Neuronal loss and neuroinflammation are two phenomena closely linked. Hence, the neuroinflammation is a relevant target for the management of the neurodegenerative diseases given that, to date, there is no treatment to stop neuronal loss. Several studies have investigated the potential effects of activators of alpha 7 nicotinic acetylcholine receptors in animal models of neurodegenerative diseases. These receptors are widely distributed in the central nervous system. After activation, they seem to mediate the cholinergic anti-inflammatory pathway in the brain. This anti-inflammatory pathway, first described in periphery, regulates activation of microglial cells considered as the resident macrophage population of the central nervous system. In this article, we shortly review the agonists of the alpha 7 nicotinic acetylcholine receptors that have been evaluated in vivo and we focused on the selective positive allosteric modulators of these receptors. These compounds represent a key element to enhance receptor activity only in the presence of the endogenous agonist.

TaVNS reduces inflammatory responses in a L-NAME-induced rat model of pre-eclampsia

Pre-eclampsia is characterized by an excessive maternal inflammatory response.The cholinergic antiinflammatory pathway(CAP)has been shown as the efferent arm of a vagal reflex with the potential to limit inflammatory responses.Therefore,in this study,the CAP regulation through the nervous vagal stimulation(VNS)reduced the severity of NG-nitro-L-arginine methyl ester(L-NAME)-induced pre-eclampsia was determined in a rat model.Rats were given 125 mg/kg/day of L-NAME via subcutaneous injection on gestational day(GD)10–16.In addition,the rats were treated by active or sham electrical stimulation once a day during GD 13–19.Systolic blood pressure(SBP),urinary albumin,and pregnancy outcomes were documented for each rat.The average fetal weights and crown-rump length(CRL)as well as the placental weights of rats in both control and experimental groups were recorded onthe 13th day,16th day and 20th day of gestation.Subsequently,placentas were collected from the rats on GD20 to measure the level of cytokines.In addition,qRT-PCR and Western blot analysis were used to detect the mRNA and protein expression ofα7 nicotinic acetylcholine receptor(α7nAChR)and nuclear factor-κB(NF-κB),respectively.Immunohistochemistry assays were also carried out to determine the location and level ofα7nAChR and NF-κB in placentas.CAP regulation through the transcutaneous auricular nerve stimulation alleviated the clinical symptoms in the rats after L-NAME induction,including hypertension,proteinuria,fetal growth retardation and fetal death.In addition,TaVNS also increasedα7nAChR expression,reduced NF-κB p65 expression,and reversed LNAME-induced proinflammatory cytokines in the placenta tissues,including tumor necrosis factor-alpha(TNF-α),high mobility group box 1(HMGB-1)and interleukin-6(IL-6).The findings of this study showed that TaVNS might be used as a promising tool to attenuate pre-eclampsia-like symptoms.In addition,the protective effect of TaVNS was associated with the improvement ofα7nAChR expression and the inhibition of inflammatory reactions at the maternal-fetal interface through activating cholinergic anti-inflammation pathway.

Medicinal nicotine in COVID-19 acute respiratory distress syndrome,the new corticosteroid

The cholinergic anti-inflammatory pathway(CAP)refers to the anti-inflammatory effects mediated by the parasympathetic nervous system.Existence of this pathway was first demonstrated when acetylcholinesterase inhibitors showed benefits in animal models of sepsis.CAP functions via the vagus nerve.The systemic antiinflammatory effects of CAP converges on theα7 nicotinic acetylcholine receptor on splenic macrophages,leading to suppression of pro-inflammatory cytokines and simultaneous stimulation of anti-inflammatory cytokines,including interleukin 10.CAP offers a novel mechanism to mitigate inflammation.Electrical vagal nerve stimulation has shown benefits in patients suffering from rheumatoid arthritis.Direct agonists like nicotine and GTS-1 have also demonstrated antiinflammatory properties in models of sepsis and acute respiratory distress syndrome,as have acetylcholinesterase inhibitors like Galantamine and Physostigmine.Experience with coronavirus disease 2019(COVID-19)induced acute respiratory distress syndrome indicates that immunomodulators have a protective role in patient outcomes.Dexamethasone is the only medication currently in use that has shown to improve clinical outcomes.This is likely due to the suppression of what is referred to as a cytokine storm,which is implicated in the lethality of viral pneumonia.Nicotine transdermal patch activates CAP and harvests its anti-inflammatory potential by means of an easily administered depot delivery mechanism.It could prove to be a promising,safe and inexpensive additional tool in the currently limited armamentarium at our disposal for management of COVID-19 induced acute hypoxic respiratory failure.

Effect of Shensong Yangxin on the Progression of Paroxysmal Atrial Fibrillation is Correlated with Regulation of Autonomic Nerve Activity

Background： Shensong Yangxin （SSYX）, a traditional Chinese herbal medicine, has long been used clinically to treat arrhythmias in China. However, the mechanism of SSYX on atrial fibrillation （AF） is unknown. In this study, we tested the hypothesis that the effect of SSYX on the progression of paroxysmal AF is correlated with the regulation of autonomic nerve activity. Methods： Eighteen mongrel dogs were randomly divided into control group （n = 6）, pacing group （n = 6）, and pacing ＋ SSYX group （n = 6）. The control group was implanted with pacemakers without pacing; the pacing group was implanted with pacemakers with long-term intermittent atrial pacing; the pacing ＋ SSYX group underwent long-term intermittent atrial pacing and SSYX oral administration. Results： Compared to the pacing group, the parameters of heart rate variability were lower after 8 weeks in the pacing ＋ SSYX group （low-frequency [LF] component： 20.85± 3.14 vs. 15.3±1.89 ms2, P =0.004; LF component/high-frequency component： 1.34 ± 0.33 vs. 0.77± 0.15, P 〈 0.001 ）. The atrial effective refractory period （AERP） was shorter and the dispersion of the AERP was higher after 8 weeks in the pacing group, while the changes were suppressed by SSYX intake. The dogs in the pacing group had more episodes and longer durations of AF than that in the pacing ＋ SSYX group. SSYX markedly inhibited the increase in sympathetic nerves and upregulation of tumor necrosis factor-alpha and interleukin-6 expression in the pacing ＋ SSYX group. Furthermore, SSYX suppressed the decrease of acetylcholine and α7 nicotinic acetylcholine receptor protein induced by long-term intermittent atrial pacing. Conclusions： SSYX substantially prevents atrial electrical remodeling and the progression of AF. These effects of SSYX may have association with regulating the imbalance of autonomic nerve activity and the cholinergic anti-inflammatory pathway.

Changes of Neuronal Acetylcholine Receptor Alpha 7 of Peritoneal Macrophage in Experimental Acute Pancreatitis Treated by Chaiqin Chengqi Decoction(柴芩承气汤)

Objective：To investigate effect of Chaiqin Chengqi Decoction（柴芩承气汤,CQCQD） on changes of neuronal acetylcholine receptor alpha 7（nAChRα7） of peritoneal macrophages in acute pancreatitis（AP）.Methods：Eighteen Kunming mice were equally randomized into the control group,AP group and CQCQD treatment group.AP was induced by two intraperitoneal injections of 4 g/kg L-arginine at 1 h apart,while control mice received saline injections.At 72 h after the first injection of L-arginine,mice in the treatment group were intragastrically administered 0.1 mL/10 g CQCQD every 2 h for 3 times,whilst mice in the other two groups received the same amount of saline feeding.Mice were sacrificed by cervical dislocation 2 h after the last feeding of either CQCQD or saline.Peritoneal macrophages were collected for determination of nAChRα7 mRNA and protein expression.Serum was collected for detection of interleukin-6（IL-6）,IL-10 and acetylcholine（ACh）levels,and pancreas was for histopathology analysis.Results：The CQCQD treatment significantly ameliorated the severity of AP as evidenced by reducing the pancreatic histopathology score（4.5 ± 0.5 vs.6.2 ± 1.7,P〈0.05）and the serum IL-6 levels（1228.31419.2 pg/mL vs.1589.6 ±337.3 pg/mL,P〈0.05）.The mRNA and protein expression of nAChRα7 of the peritoneal macrophages in the AP group were similar to the control group（P〉0.05）,but were significantly up-regulated after the CQCQD treatment（P〈0.05）.The serum ACh levels in the AP group were significantly lower than those in the control group（3.1 ± 0.6 μg/mL vs 4.8 ± 0.7 μg/mL P〈0.05）,but were significantly increased after the CQCQD treatment（5.6±1.5 μg/mL vs 3.1 ±0.6 μg/mL,P〈0.05）.Conclusion：CQCQD is protective against L-arginine-induced AP through mechanisms involving nAChR α 7 of peritoneal macrophages.

Interacting with α7 nAChR is a new mechanism for AChE to enhance the inflammatory response in macrophages

Acetylcholine(ACh)regulates inflammation viaα7 nicotinic acetylcholine receptor(α7 nAChR).Acetylcholinesterase(AChE),an enzyme hydrolyzing ACh,is expressed in immune cells suggesting non-classical function in inflammatory responses.Here,the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages.In lipopolysaccharide-induced inflammatory processes,AChE was upregulated by the binding of NF-κB onto the ACHE promotor.Conversely,the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration,which was in contrast to that of applied AChE inhibitors.AChEmt,a DNA construct without enzymatic activity,was adopted to identify the protein role of AChE in immune system.Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration.The co-localization ofα7 nAChR and AChE was found in macrophases,suggesting the potential interaction ofα7 nAChR and AChE.Besides,immunoprecipitation showed a close association ofα7 nAChR and AChE protein in cell membrane.Hence,the novel function of AChE in macrophage by interacting withα7 nAChR was determined.Together with hydrolysis of ACh,AChE plays a direct role in the regulation of inflammatory response.As such,AChE could serve as a novel target to treat age-related diseases by antiinflammatory responses.

Therapeutic Effects of Electroacupuncture at ST36 Acupoint on Sodium-Taurocholate-Induced Severe Acute Pancreatitis

Objective： To explore the protective effects and mechanisms of electroacupuncture （EA） at Zusanli （ST36） acupoint in rats with severe acute pancreatitis （SAP）. Methods： Sixty-six male Sprague- Dawley rats were randomly assigned to three groups of 22 each： a SAP model group （SAP group）, a sham- operated group （sham group） and a EA at ST36 acupoint group （EA group）. A rat model of SAP was induced by pancreatic duct injection with 3.5% sodium taurocholate. EA was performed at ST36 acupoint for 30 min after induction of SAP and 30 min before sacrificed. The rats were killed at 3 h （n=7）, 6 h （n=7） and 12 h （n=8） after operation, and blood samples were taken for the measurement of tumor necrosis factor-alpha （TNF-α）, interleukin-6 （IL-6） and acetylcholine （Ach） by enzyme-linked immunosorbnent assay. The pathological changes of pancreatic tissue, volume of ascites and pancreatic weight/body weight ratio were measured. Results The serum concentrations of TNF-α and IL-6 in the EA group were significantly lower than in the SAP group at 3, 6 and 12 h after the operation （P〈0.05）. Serum Ach in the EA group was significantly higher than in the SAP group at various time points after operation （P〈0.05）. The other parameters were clearly improved after treatment with EA. Conclusion： EA at ST36 acupoint might have a therapeutic effect in rats with SAP through activating the cholinergic anti-inflammatory pathway.