OBJECTIVE: To investigate the role of α7-nicotinic acetylcholine receptor (α7-nAChR) in the regulation of neuronal activity and expression of synapse-related proteins in the thalamic reticular nucleus (TRN) of insomnia rats treated by acupuncture. METHODS: A total of 36 male Sprague-Dawley (SD) rats of clean grade were randomly divided into a control group, a model group, an acupuncture group, and an acupuncture+antagonist group, with 9 rats in each group. The model group, the acupuncture group, and the acupuncture+antagonist group were treated with intraperitoneal injection of p-chlorophenylalanine (PCPA) to establish insomnia model. After successful modeling, the acupuncture group and the acupuncture+antagonist group received acupuncture at bilateral Neiguan (PC6) and Zusanli (ST36) once daily for 5 consecutive days. Thirty min before each acupuncture session, the acupuncture+antagonist group was intraperitoneally injected with methyllycaconitine citrate (MLA), an α7-nAChR antagonist, at a dosage of 5 mg/kg while the acupuncture group received the same volume of 0.9% sodium chloride solution. The rats' daytime spontaneous activity was observed. Neuronal discharge in the TRN was detected using neuroelectrophysiological methods. Immunofluorescence staining was used to detect parvalbumin-positive (PV⁺) neurons and co-expression of PV⁺ and postsynaptic density protein-95 (PSD-95) in the TRN. RESULTS: Compared with the control group, the model group showed increased daytime spontaneous activity (P<0.01); decreased average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); decreased neuronal discharge frequency (P<0.01), prolonged inter-discharge intervals (P<0.01) in the TRN; reduced number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.01). Compared with the model group, the acupuncture group showed decreased daytime spontaneous activity (P<0.01); increased average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); increased neuronal discharge frequency (P<0.01), shortened inter-discharge intervals (P<0.01) in the TRN; increased number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.05). Compared with the acupuncture group, the acupuncture+antagonist group exhibited increased daytime spontaneous activity (P<0.01); reduced average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); decreased neuronal discharge frequency (P<0.05), prolonged inter-discharge intervals (P<0.05) in the TRN; reduced number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.01). CONCLUSION α7-nAChR are involved in mediating the regulatory effect of acupuncture on circadian rhythm disturbances in PCPA-induced insomnia rats. Blocking α7-nAChR attenuates the activating effect of acupuncture on TRN neurons, and reduces the expression of PSD-95 protein on GABAergic neurons.
Animals ; Male ; Acupuncture Therapy ; alpha7 Nicotinic Acetylcholine Receptor/genetics* ; Rats, Sprague-Dawley ; Rats ; Sleep Initiation and Maintenance Disorders/physiopathology* ; Neurons/metabolism* ; Humans ; Thalamic Nuclei/physiopathology* ; Acupuncture Points ; Disks Large Homolog 4 Protein

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 T2DM 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 HepG2 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 HepG2 cells were incubated with insulin (10(-6) mol/L) for 36 h. Meanwhile, the expression of alpha7 nicotinic acetylcholine receptor (α7nAChR) protein was reduced without the change of acetylcholinesterase (AChE) activity. The level of interleukin-6 (IL-6) in the culture supernatant, the ratio of phosphorylated I-kappa-B kinase-β (IKκβ) 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 α7nAChR protein and inhibited AChE activity. These changes were also accompanied with the decrease of the ratio of pIKKβ 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 HepG2 cells. The mechanism may be related to the cholinergic anti-inflammatory pathway and the inhibition of AChE activity.
Berberine ; pharmacology ; Glucose ; metabolism ; Hep G2 Cells ; Humans ; Hypoglycemic Agents ; pharmacology ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; Insulin ; metabolism ; Insulin Resistance ; Interleukin-6 ; metabolism ; Transcription Factor RelA ; metabolism ; alpha7 Nicotinic Acetylcholine Receptor ; genetics ; metabolism

OBJECTIVETo investigate effect of Chaiqin Chengqi Decoction (, CQCQD) on changes of neuronal acetylcholine receptor alpha 7 (nAChRα7) of peritoneal macrophages in acute pancreatitis (AP).

METHODSEighteen 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.

RESULTSThe 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 (1228.3±419.2 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).

CONCLUSIONCQCQD is protective against L-arginine-induced AP through mechanisms involving nAChRα7 of peritoneal macrophages.

Acetylcholine ; pharmacology ; Acute Disease ; Animals ; Blotting, Western ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Interleukin-10 ; blood ; Interleukin-6 ; blood ; Macrophages, Peritoneal ; drug effects ; metabolism ; pathology ; Mice ; Neurons ; drug effects ; metabolism ; Pancreas ; drug effects ; pathology ; Pancreatitis ; blood ; drug therapy ; pathology ; RNA, Messenger ; genetics ; metabolism ; alpha7 Nicotinic Acetylcholine Receptor ; genetics ; metabolism

OBJECTIVETo investigate the influence of inhibited α7 neuronal nicotinic acetylcholine receptor (nAChR) by small interference RNA (siRNA) in SH-SY5Y cells and to explore the connection of these changes with the β-amyloid precursor protein (APP) metabolism and the pathogenesis of Alzheimer's disease (AD).

METHODSThe siRNA of α7 nAChR was transfected into SH-SY5Y cells, and the expression of α7 nAChR and two subtypes of β-secretases (BACE1 and BACE2) at mRNA and protein levels was studied by real-time PCR and Western blot, respectively. The variation of Aβ(1-42) content was detected by ELISA.

RESULTSAs compared with controls, the expression of α7 nAChR at mRNA and protein levels in the SH-SY5Y cells transfected with the α7 nAChR siRNA were decreased by 84% and 79% (P < 0.01), respectively. The expressions of BACE1 mRNA and protein levels was increased by 527% and 71% (P < 0.01), respectively, while the expression of BACE2 decreased by 58% and 75% (P < 0.01), respectively. The Aβ(1-42) content increased by 208% (P < 0.01).

CONCLUSIONSAn inhibited α7 nAChR mRNA induced by siRNA may markedly stimulate the production of Aβ through the mechanism of increased expression of BACE1 and inhibited expression of BACE2, which may be related to the pathogenesis of AD.

Amyloid Precursor Protein Secretases ; genetics ; metabolism ; Amyloid beta-Peptides ; metabolism ; Aspartic Acid Endopeptidases ; genetics ; metabolism ; Cell Line, Tumor ; Humans ; Neuroblastoma ; metabolism ; pathology ; Peptide Fragments ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, Nicotinic ; genetics ; metabolism ; Transfection ; alpha7 Nicotinic Acetylcholine Receptor

OBJECTIVETo investigate the influence of APP(SWE) on the expression of neuronal acetylcholine receptors (nAChRs) and its relationship with Alzheimer's disease (AD).

METHODSAPP(SWE), carried the Swedish family AD double mutants, were transfected into SH-SY5Y cells and primary cultured neurons from rat brains to build a cellular model of AD. The mRNA levels of APP and nAChRs, and the protein levels of total APP, αAPPs and nAChRs in the cultured cells were measured using real-time PCR and Western blot, respectively. The numbers of α3 nAChR were determined by receptor-[³H]epibatidine binding assay.

RESULTSIncreased expressions of Swedish 670/671 APP at mRNA and protein levels, and down-regulation of αAPPs were observed in both of the cultured neuronal cells transfected with APP(SWE). A significant increase of α7 nAChR expression at protein and mRNA levels was detected in the APP(SWE) transfected SH-SY5Y cells. On the other hand, after transfection with APP(SWE), the expressions of α3 nAChR at protein and mRNA levels in SH-SY5Y cells, and α4 nAChR at mRNA level in primary cultured neurons were inhibited. In addition, the numbers of receptor binding sites were deceased in SH-SY5Y cells overexpressing with APP(SWE).

CONCLUSIONOverexpression of APP(SWE) can decrease αAPPs and modify nAChRs by increasing expression of α7 nAChR and decreasing α3 and α4 nAChRs, which might play an important role in the pathogenesis of AD.

Alzheimer Disease ; genetics ; Amyloid Precursor Protein Secretases ; secretion ; Amyloid beta-Protein Precursor ; genetics ; metabolism ; physiology ; Animals ; Brain Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Cerebral Cortex ; cytology ; metabolism ; Down-Regulation ; Humans ; Neuroblastoma ; metabolism ; pathology ; Neurons ; cytology ; metabolism ; Plasmids ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Nicotinic ; genetics ; metabolism ; Transfection ; alpha7 Nicotinic Acetylcholine Receptor

OBJECTIVETo investigate the association between neuronal nicotinic acetylcholine receptor alpha 7 subunit (CHRNA7) gene and schizophrenia.

METHODSThe three polymorphisms rs2337980, rs1909884, rs883473 in CHRNA7 gene were detected based on PCR and polyacrylamide gel microarray in 129 schizophrenic trios. The results of genotyping were analyzed by haplotype relative risk analysis based on haplotype(HHRR), transmission disequilibrium test(TDT) and hyplotype analysis.

RESULTS(1)The HHRR analysis suggested that there was significant differences in rs2337980 allele frequencies between schizophrenia group and dummy control group(P= 0.017); (2)In TDT test, there may be transmission disequilibrium between rs2337980 and schizophrenia, the heterozygous parents excessively transferred the C allele to patients (P= 0.021); (3)The haplotype between rs2337980 and rs1909884 as well as the hyplotype among rs2337980, rs1909884 and rs883473 may have significant association with schizophrenia (global P= 0.034; global P= 0.027), the T-C and T-C-T hyplotype may have transmission disequilibrium with schizophrenia.

CONCLUSIONThere may be association between CHRNA7 gene polymorphisms and schizophrenia, the variant allele T in rs2337980 may have a protective effect to schizophrenia.

Adolescent ; Adult ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Haplotypes ; Humans ; Linkage Disequilibrium ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Receptors, Nicotinic ; genetics ; Schizophrenia ; genetics ; Young Adult ; alpha7 Nicotinic Acetylcholine Receptor