This study investigated the mechanism by which Jianpi Bushen Yiqi Decoction promotes acetylcholine receptor(AChR) clustering in myasthenia gravis through the Agrin/low-density lipoprotein receptor-related protein 4(LRP4)/muscle-specific receptor tyrosine kinases(MuSK) signaling pathway. A total of 114 female C57BL/6J mice were divided into the normal group, modeling group, and solvent control group. The normal group and the solvent control group were immunized with phosphate-buffered saline(PBS), while the modeling group was established as an experimental autoimmune myasthenia gravis(EAMG) model using the murine-derived AChR-α subunit R97-116 peptide fragment. After successful modeling, the mice were randomly assigned to the model group, the low-, medium-, and high-dose Jianpi Bushen Yiqi Decoction groups, and the prednisone group. After four weeks of continuous treatment, muscle strength was assessed using Lennon scores and grip strength tests. Immunofluorescence staining was conducted on differentiated C2C12 myotubes incubated with a drug-containing serum to observe the number of AChR clusters. The integrity of AChR on myofilaments in mouse gastrocnemius muscles was further assessed by immunofluorescence staining. Hematoxylin-Eosin(HE)staining was applied to examine pathological changes in the gastrocnemius muscles of EAMG mice treated with Jianpi Bushen Yiqi Decoction. Western blot was utilized to detect the expression of key proteins in the Agrin/LRP4/MuSK signaling pathway in both C2C12 myotubes and mouse gastrocnemius muscles. The results demonstrated that compared to the model group, the prednisone group exhibited a significant decrease in the body weights of mice, whereas no significant differences in the body weights of mice were observed among the low-, medium-, and high-dose Jianpi Bushen Yiqi Decoction groups. All treatment groups showed significantly improved grip strength and Lennon scores. Additionally, the formula promoted AChR clustering on myotubes and enhanced AChR integrity in gastrocnemius myofilaments and reduced inflammatory infiltration between muscle tissue and fibrous hyperplasia. Furthermore, Jianpi Bushen Yiqi Decoction upregulated the protein expression of AChRα1, Agrin, and p-MuSK in C2C12 myotubes and increased the protein expression of AChRα1, Agrin, MuSK, p-MuSK, LRP4, and docking protein 7(Dok-7)in gastrocnemius tissue. In conclusion, Jianpi Bushen Yiqi Decoction may promote AChR clustering by targeting key proteins in the Agrin/LRP4/MuSK signaling pathway, thereby improving neuromuscular junction function and enhancing muscle strength.
Animals ; Agrin/genetics* ; Mice ; Drugs, Chinese Herbal/administration & dosage* ; Signal Transduction/drug effects* ; Receptors, Cholinergic/genetics* ; Female ; Mice, Inbred C57BL ; Receptor Protein-Tyrosine Kinases/genetics* ; Neuromuscular Junction/metabolism* ; Myasthenia Gravis, Autoimmune, Experimental/physiopathology* ; Humans ; LDL-Receptor Related Proteins

OBJECTIVE: To explore the genetic basis for a female patient featuring unstable head upright and hypotonia of limbs. METHODS: The child was examined clinically. Peripheral blood samples of the child, her parents and siblings were collected. Genomic DNA was extracted and subjected to next generation sequencing (NGS). Suspected variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: DNA sequencing found that the patient has carried a de novo heterozygous c.354C>A (p.N118K) variant of the CHRND gene, which was not found in her parents and sibling. Bioinformatics analysis predicted that the variant was likely to be pathogenic. Literature review suggested that the phenotype of the patient was very similar to previously reported ones. CONCLUSION The child was diagnosed with slow-channel congenital myasthenic syndrome (SCCMS) type 3A caused by heterozygous variant of the CHRND gene. NGS has provided a powerful tool for the diagnosis of such disorders.
Child ; Female ; Genetic Testing ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Humans ; Mutation ; Myasthenic Syndromes, Congenital ; genetics ; pathology ; Receptors, Cholinergic ; genetics

Epilepsy is a chronic neurological disorder, which is not only related to the imbalance between excitatory glutamic neurons and inhibitory GABAergic neurons, but also related to abnormal central cholinergic regulation. This article summarizes the scientific background and experimental data about cholinergic dysfunction in epilepsy from both cellular and network levels, further discusses the exact role of cholinergic system in epilepsy. In the cellular level, several types of epilepsy are believed to be associated with aberrant metabotropic muscarinic receptors in several different brain areas, while the mutations of ionotropic nicotinic receptors have been reported to result in a specific type of epilepsy-autosomal dominant nocturnal frontal lobe epilepsy. In the network level, cholinergic projection neurons as well as their interaction with other neurons may regulate the development of epilepsy, especially the cholinergic circuit from basal forebrain to hippocampus, while cholinergic local interneurons have not been reported to be associated with epilepsy. With the development of optogenetics and other techniques, dissect and regulate cholinergic related epilepsy circuit has become a hotspot of epilepsy research.
Acetylcholine ; physiology ; Basal Forebrain ; pathology ; Brain Chemistry ; genetics ; physiology ; Cholinergic Neurons ; chemistry ; classification ; pathology ; physiology ; Epilepsy ; genetics ; pathology ; physiopathology ; Epilepsy, Frontal Lobe ; genetics ; GABAergic Neurons ; physiology ; Hippocampus ; pathology ; Humans ; Mutation ; genetics ; physiology ; Neurons ; Non-Neuronal Cholinergic System ; genetics ; physiology ; Receptors, Muscarinic ; genetics ; physiology ; Receptors, Nicotinic ; genetics ; physiology ; Synaptic Transmission ; genetics ; physiology

OBJECTIVE: To determine the role of miR-155 in the pathogenesis of generalized myasthenia gravis (GMG) and the effect of dexamethasone (DXM) on miR-155. METHODS: The expression of miR-155 in B cells from the GMG patients and healthy controls was analyzed by qPCR. The B cells were cultured with DXM and PBS. The B cell proliferation was examined by MTT; CD80 and CD86 frequencies were detected by flow cytometry; and anti-AChRIgG and isotypes anti-AChR-IgG1, 2, 3 in the supernatant were detected by ELISA. RESULTS: qPCR revealed that the expression of miR-155 in the B cells was much higher than that in the controls, and the miR155 expression decreased after DXM treatment. flow cytometry showed that there was no significant difference in the proliferation and the expressions of CD80 and CD86 in the B cells between the DXM group and the PBS group. The concentration of anti-AChR-IgG1 was obviously lower in the DXM group than in the PBS group, but the concentration of anti-AChRIgG, anti-AChR-IgG2, and anti-AchR-IgG3 was similar. CONCLUSION high expression of miR-155 may be associated with myasthenia gravis progression. DXM may disturb the antibody class switch of B cells by suppressing the expression of miR-155 and improve the symptom of MG patients.
Adult ; B-Lymphocytes ; cytology ; immunology ; metabolism ; B7-1 Antigen ; metabolism ; Cell Proliferation ; Cells, Cultured ; Dexamethasone ; therapeutic use ; Female ; Humans ; Immunoglobulin G ; immunology ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Myasthenia Gravis ; drug therapy ; genetics ; immunology ; Receptors, Cholinergic ; immunology ; Tetraspanin 28 ; metabolism ; Young Adult

OBJECTIVE: To explore the role of IL-21 in the pathogenesis of myasthenia gravis (MG) and its influence on the the class switch of anti-AChR antibodies. METHODS: Blood was taken from 26 patients and 18 healthy controls, and the expression of IL-21R mRNA in peripheral blood mononuclear cells (PBMCs) was detected by RT-PCR. The expression of IL-21R on B lymphocytes was measured by flow cytometry, while the concentrations of serum IL-21 and the levels of anti-AChR-IgG and its isotype IgG(1), IgG(2), and IgG(3) were tested by ELISA. RESULTS: The serum concentration of IL-21 in the MG group was higher than that in the control group (31.686±8.499 pg/mL, 15.147±6.366 pg/mL) and the difference was significant (P<0.01). IL-21R mRNA expressed on PBMCs in the MG group was higher than that in the control group (0.139±0.052, 0.101±0.022), and the difference was significant (P<0.05). There was no difference between ocular MG and generalized MG subgroup (P>0.05). Compared with the control group, the expression of IL-21R on B lymphocytes also increased in the MG group (P<0.05). In the anti-AChR-Ab positive MG group, the serum concentration of IL-21 showed positive correlation with anti-AChR-IgG(P<0.05),but no correlation with its isotype IgG(1), IgG(2), and IgG(3), respectively(P>0.05). Expression of IL-21R mRNA in the PBMCs showed no correlation with the level of serum anti-AChR-IgG and its isotype IgG(1), IgG(2), and IgG(3), respectively(P>0.05); however the expression of IL-21R in B lymphocytes showed positive correlation with anti-AChR-IgG and it's isotype IgG(1) and IgG(3) (P<0.05,P<0.01,P<0.05), but no correlation with IgG(2) (P>0.05). CONCLUSION IL-21 might induce the class switch of anti-AChR antibodies to IgG(1) and IgG(3) isotype through IL-21R on B lymphocytes which promotes the pathogenesis of the MG.
Adolescent ; Adult ; Autoantibodies ; classification ; immunology ; Female ; Humans ; Immunoglobulin Class Switching ; immunology ; Immunoglobulin G ; classification ; Interleukins ; blood ; genetics ; Male ; Middle Aged ; Myasthenia Gravis ; blood ; immunology ; RNA, Messenger ; blood ; genetics ; Receptors, Cholinergic ; immunology ; Receptors, Interleukin-21 ; blood ; genetics ; Young Adult

OBJECTIVETo detect over-expression of AChR-gamma mRNA in rhabdomyosarcoma tissues by duplex RT-PCR and discuss its potential in diagnosis of rhabdomyosarcoma.

METHODSDuplex RT-PCR was applied to the simultaneous detection of AChR-alpha and gamma subunit messenger RNA in 17 cases of rhabdomyosarcoma (9 ERMS, 6 ARMS, 2 PRMS). 20 cases of non-rhabdomyosarcomous small round cell tumors (6 poorly differentiated synovial sarcomas, 6 ES/PNET, 6 lymphomas, 2 neuroblastomas) and three normal muscle samples were also detected for AChR-alpha and gamma mRNA by the same method.

RESULTSAChR-alpha and AChR-gamma mRNA were expressed in all the cases of rhabdomyosarcoma. The rate of quantity in both transcripts was AChR-gamma/AChR-alpha >or= 1, but the rate for three normal muscle samples was < 1. Cases of non-rhabdomyosarcomous small round cell tumors were all negative for AChR-gamma.

CONCLUSIONAChR-gamma mRNA expression detected by molecular genetic methods is useful in diagnosis and differential diagnosis of rhabdomyosarcoma.

Diagnosis, Differential ; Humans ; Protein Subunits ; RNA, Messenger ; analysis ; Receptors, Cholinergic ; genetics ; Receptors, Nicotinic ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Rhabdomyosarcoma ; diagnosis