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

BACKGROUNDThe antagonistic actions of anticholinesterase drugs on non-depolarizing muscle relaxants are theoretically related to the activity of acetylcholinesterase (AChE) in the neuromuscular junction (NMJ). However, till date the changes of AChE activity in the NMJ during sepsis have not been directly investigated. We aimed to investigate the effects of sepsis on the antagonistic actions of neostigmine on rocuronium (Roc) and the underlying changes of AChE activity in the NMJ in a rat model of cecal ligation and puncture (CLP).

METHODSA total of 28 male adult Sprague-Dawley rats were randomized to undergo a sham surgery (the sham group, n = 12) or CLP (the septic group, n = 16). After 24 h, the time-response curves of the antagonistic actions of 0.1 or 0.5 μmol/L of neostigmine on Roc (10 μmol/L)-depressed diaphragm twitch tension were measured. Meanwhile, the activity of AChE in the NMJ was detected using a modified Karnovsky and Roots method. The mRNA levels of the primary transcript and the type T transcript of AChE (AChET) in the diaphragm were determined by real-time reverse transcription-polymerase chain reaction.

RESULTSFour of 16 rats in the septic group died within 24 h. The time-response curves of both two concentrations of neostigmine in the septic group showed significant upward shifts from those in the sham group (P < 0.001 for 0.1 μmol/L; P = 0.009 for 0.5 μmol/L). Meanwhile, the average optical density of AChE in the NMJ in the septic group was significantly lower than that in the sham group (0.517 ± 0.045 vs. 1.047 ± 0.087, P < 0.001). The AChE and AChETmRNA expression levels in the septic group were significantly lower than those in the sham group (P = 0.002 for AChE; P = 0.001 for AChET).

CONCLUSIONSSepsis strengthened the antagonistic actions of neostigmine on Roc-depressed twitch tension of the diaphragm by inhibiting the activity of AChE in the NMJ. The reduced content of AChE might be one of the possible causes of the decreased AChE activity in the NMJ.

Acetylcholinesterase ; metabolism ; Androstanols ; pharmacology ; Animals ; Cecum ; injuries ; Cholinesterase Inhibitors ; pharmacology ; Diaphragm ; drug effects ; metabolism ; Disease Models, Animal ; Ligation ; Male ; Neostigmine ; pharmacology ; Neuromuscular Junction ; enzymology ; Neuromuscular Nondepolarizing Agents ; pharmacology ; Punctures ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sepsis ; physiopathology

BACKGROUNDMuscles present different responses to muscle relaxants, a mechanism of importance in surgeries requiring facial nerve evoked electromyography under general anaesthesia. The non-depolarizing muscle relaxants have multiple reaction formats in the neuromuscular junction, in which pre-synaptic quantal release of acetylcholine was one of the important mechanisms. This study was to compare the pre-synaptic quantal release of acetylcholine from the neuromuscular junctions innervated by normal/damaged facial nerves and somatic nerve under the effect of rocuronium in rats in vitro.

METHODSAcute right-sided facial nerve injury was induced by nerve crush axotomies. Both sided facial nerve connected orbicularis oris strips and tibial nerve connected gastrocnemius strips were isolated to measure endplate potentials (EPP) and miniature endplate potentials (MEPP) using an intracellular microelectrode gauge under different rocuronium concentrations. Then, the pre-synaptic quantal releases of acetylcholine were calculated by the ratios of the EPPs and the MEPPs, and compared among the damaged or normal facial nerve innervated orbicularis oris and tibial nerve innervated gastrocnemius.

RESULTSThe EPP/MEPP ratios of the three neuromuscular junctions decreased in a dose dependent manner with the increase of the rocuronium concentration. With the concentrations of rocuronium being 5 µg/ml, 7.5 µg/ml and 10 µg/ml, the decrease of the EPP/MEPP ratio in the damaged facial nerve group was greater than that in the normal facial nerve group. The decrease in the somatic nerve group was the biggest, with significant differences.

CONCLUSIONSRocuronium presented different levels of inhibition on the pre-synaptic quantal release of acetylcholine in the three groups of neuromuscular junctions. The levels of the inhibition showed the following sequence: somatic nerve > damaged facial nerve > normal facial nerve. The difference may be one of the reasons causing the different sensitivities to rocuronium among the muscles innervated by the normal/injured facial nerves and the somatic nerve. The results may provide some information for the proper usage of muscle relaxants in surgeries requiring electromyographic monitoring for the pre-surgically impaired facial nerves.

Acetylcholine ; metabolism ; Androstanols ; pharmacology ; Animals ; Facial Nerve ; drug effects ; metabolism ; Male ; Neuromuscular Junction ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo determine the effects of atracurium pretreatment with magnesium on speed of onset, duration, and recovery of neuromuscular block.

METHODSThirty patients who were undergoing elective gynecologic laparoscopic examination and treatments under general anesthesia were randomized into magnesium group (n = 15) and control group (n = 15). Before induction of general anesthesia, patients in magnesium group intravenously received MgSO4 30 mg/kg in saline within 5 minutes, and patients in control group received the same volume of saline without MgSO4. In both groups, the train-of-four (TOF) responses to stimuli of the ulnar nerve were measured at intervals of 12 seconds. Anesthesia was induced with Fentanyl and Propofol through target controlled infusion (TCI), and tracheal intubation was performed with 0.5 mg/kg atracurium after stabilization of the electromyography recording. The onset time of muscle relaxation, clinical duration of action, recovery index, and recovery time were recorded. To determine serum magnesium and calcium levels, blood samples were collected before MgSO4/saline infusion and at the end of operation. Haemodynamic changes and other responses during induction were also recorded.

RESULTSThe onset time from the end of injection to maximum neuromuscular blockade was significantly shorter in magnesium group than in control group (P < 0.01). Duration of relaxant action, recovery index, and recovery time in magnesium group were significantly prolonged than in control group (P < 0.01). Serum magnesium level significantly decreased after management (P < 0.01), and there was also a decrease trend in magnesium group. No change of serum calcium levels in both groups was observed. No adverse event was reported.

CONCLUSIONPrior administration of magnesium sulphate can increase the onset speed of atracurium and prolong the duration of atracurium-induced neuromuscular blockade.

Adolescent ; Adult ; Anesthesia Recovery Period ; Anesthesia, General ; Atracurium ; pharmacology ; Drug Synergism ; Female ; Humans ; Magnesium Sulfate ; pharmacology ; Middle Aged ; Neuromuscular Blockade ; Neuromuscular Junction ; drug effects ; Neuromuscular Nondepolarizing Agents ; pharmacology ; Time Factors ; Young Adult

Nitric oxide (NO) is a non-adrenergic, non-cholinergic neurotransmitter found in the enteric nervous system that plays a role in a variety of enteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to be dependent on the manner by which inflammation is caused. However, this observed alteration has not been reported with acetic acid-induced colitis. Therefore, the purpose of the current study was to investigate changes in nitrergic neuromuscular transmission in experimental colitis in a rat model. Distal colitis was induced by intracolonic administration of 4% acetic acid in the rat. Animals were sacrificed at 4 h and 48 h postacetic acid treatment. Myeloperoxidase activity was significantly increased in the acetic acid-treated groups. However, the response to 60 mM KCl was not significantly different in the three groups studied. The amplitude of phasic contractions was increased by Nomega-nitro-L-arginine methyl ester (L-NAME) in the normal control group, but not in the acetic acid-treated groups. Spontaneous contractions disappeared during electrical field stimulation (EFS) in normal group. However, for the colitis groups, these contractions initially disappeared, and then reappeared during EFS. Moreover, the observed disappearance was diminished by L-NAME; this suggests that these responses were NO-mediated. In addition, the number of NADPH-diaphorase positive nerve cell bodies, in the myenteric plexus, was not altered in the distal colon; whereas the area of NADPH-diaphorase positive fibers, in the circular muscle layer, was decreased in the acetic acidtreated groups. These results suggest that NO-mediated inhibitory neural input, to the circular muscle, was decreased in the acetic acid-treated groups.
Acetic Acid/toxicity ; Animals ; Colitis/chemically induced/*pathology/*physiopathology ; Colon/drug effects/enzymology/*innervation/pathology ; Indicators and Reagents/toxicity ; Male ; Muscle Contraction/drug effects ; Muscle, Smooth/drug effects/metabolism ; Myenteric Plexus/pathology ; NADPH Dehydrogenase/metabolism ; NG-Nitroarginine Methyl Ester/pharmacology ; Neuromuscular Junction/drug effects/*metabolism ; Nitrergic Neurons/drug effects/*metabolism ; Nitric Oxide/*metabolism ; Peroxidase/metabolism ; Potassium Chloride/pharmacology ; Rats ; Rats, Sprague-Dawley

In this study, we tested the hypothesis that volatile anesthetic enhancement of muscle relaxation is the result of combined drug effects on the nicotinic acetylcholine receptors. The poly A m RNA from muscle by isolation were microinjected into Xenopus oocytes for receptor expression. Concentration-effect curves for the inhibition of Ach-induced currents were established for vecuronium, rocuranium, and isoflurane. Subsequently, inhibitory effects of NDMRs were studied in the presence of the isoflurane at a concentration equivalent to half the concentration producing a 50% inhibition alone. All tested drugs produced rapid and readily reversible concentration-dependent inhibition. The 50% inhibitory concentration values were 889 micromol/L (95% CI: 711-1214 micromol). 33.4 micromol (95% CI: 27.1-41.7 nmol) and 9.2 nmol (95% CI: 7.9-12.3 nmol) for isoflurane. rocuranium and vecuronium, respectively. Coapplication of isoflurane significantly enhanced the inhibitory effects of rocuranium and vecuronium, and it was especially so at low concentration of NMDRs. Isoflurane increases the potency of NDMRs, possibly by enhancing antagonist affinity at the receptor site.
Androstanols/*pharmacology ; Anesthetics, Inhalation/pharmacology ; Drug Synergism ; Isoflurane/*pharmacology ; Neuromuscular Blocking Agents/*pharmacology ; Neuromuscular Junction/drug effects ; Neuromuscular Nondepolarizing Agents/*pharmacology ; Oocytes ; Receptors, Nicotinic/*drug effects ; Vecuronium Bromide/pharmacology ; Xenopus laevis

In this study, we tested the hypothesis that volatile anesthetic enhancement of muscle relaxation is the result of combined drug effects on the nicotinic acetylcholine receptors. The poly A m RNA from muscle by isolation were microinjected into Xenopus oocytes for receptor expression. Concentration-effect curves for the inhibition of Ach-induced currents were established for vecuronium, rocuranium, and isoflurane. Subsequently, inhibitory effects of NDMRs were studied in the presence of the isoflurane at a concentration equivalent to half the concentration producing a 50% inhibition alone. All tested drugs produced rapid and readily reversible concentration-dependent inhibition. The 50% inhibitory concentration values were 889 micromol/L (95% CI: 711-1214 micromol). 33.4 micromol (95% CI: 27.1-41.7 nmol) and 9.2 nmol (95% CI: 7.9-12.3 nmol) for isoflurane. rocuranium and vecuronium, respectively. Coapplication of isoflurane significantly enhanced the inhibitory effects of rocuranium and vecuronium, and it was especially so at low concentration of NMDRs. Isoflurane increases the potency of NDMRs, possibly by enhancing antagonist affinity at the receptor site.
Androstanols ; pharmacology ; Anesthetics, Inhalation ; pharmacology ; Animals ; Drug Synergism ; Female ; Isoflurane ; pharmacology ; Neuromuscular Blocking Agents ; pharmacology ; Neuromuscular Junction ; drug effects ; Neuromuscular Nondepolarizing Agents ; pharmacology ; Oocytes ; Receptors, Nicotinic ; drug effects ; Vecuronium Bromide ; pharmacology ; Xenopus laevis

OBJECTIVETo study the neuromuscular function and its relation with the occurrence of myasthenia in rats poisoned by dimethoate (D), phoxim (P), methomyl (M), M + D and M + P respectively.

METHODSThe stimulation single fiber electromyography(SSFEMG) at different stimulus frequencies(5, 10 and 20 Hz) was used. The whole blood cholinesterase (ChE) activity was measured 1 h before and after poisoning.

RESULTS(1) Myasthenia occurred in 5 out of 9.5 out of 10.5 out of 5, and 8 rats poisoned by D, P, M + D, and M + P, respectively. (2) The average mean consecutive differences(MCD) at 5, 10, and 20 Hz in myasthenic rats were significantly higher than those of poisoned rats without myasthenia and the control ones. (3) SSFEMG changes at 5, 10 and 20 Hz were significantly consistent with the clinical manifestation of myasthenia, especially at 10 Hz and 20 Hz. (4) ChE activity was significantly lower in rats with P or D poisoning while ChE inhibition was of no difference in rats with M, M + D, and M + P poisoning. In the D poisoning and P poisoning groups, there was no significant difference in ChE inhibition between the rats with and without myasthenia.

CONCLUSIONMuscle weakness was associated with neuromuscular transmission dysfunction, but not well correlated with ChE inhibition. The SSFEMG with stimulus frequency at 10 Hz or 20 Hz could be used to detect the neuromuscular dysfunction during myasthenia induced by organophosphate insecticides and their mixtures poisoning.

Animals ; Cholinesterase Inhibitors ; poisoning ; Dimethoate ; poisoning ; Electromyography ; Insecticides ; poisoning ; Methomyl ; poisoning ; Muscle Weakness ; chemically induced ; Myasthenia Gravis ; chemically induced ; physiopathology ; Neuromuscular Junction ; drug effects ; physiology ; Organothiophosphorus Compounds ; poisoning ; Rats ; Synaptic Transmission ; drug effects

OBJECTIVETo study the effects of improving the neuromuscular transmission (NMT), "non-AChE-reactivating effects", by oximes in treating acute isocarbophos poisoning.

METHODSThe effect of pralidoxime chloride(PAM-Cl) on the neuromuscular transmission(NMT) in rats exposed to isocarbophos was studied by using the stimulation single fiber electromyography (SSFEMG) to determine the single fiber action potential.

RESULTSAfter the rats exposed to isocarbophos were treated by PAM-Cl, the mean consecutive difference(MCD) value [(25.99 +/- 5.84) microsecond] of single fiber action potential was significantly lower than that before PAM-Cl treatment [(33.21 +/- 4.09) microsecond, (P < 0.01)], but no AChE reactivation in blood and gastrocnemius was observed.

CONCLUSIONPAM-Cl has "non-AChE-reactivating effects", it could markedly improve isocarbophos-induced NMT block of gastrocnemius.

Acute Disease ; Animals ; Antidotes ; pharmacology ; Cholinesterase Inhibitors ; poisoning ; Insecticides ; poisoning ; Malathion ; poisoning ; Neuromuscular Junction ; drug effects ; physiology ; Pralidoxime Compounds ; pharmacology ; Rats ; Synaptic Transmission ; drug effects

HWTX-I is a peptide neurotoxin purified from the crude venom of the Chinese bird Spider Selenocosmia Huwena, which has analyesic activity. rHWTX-I expressed by P. pastoris and secreted to culture supernatant was first precipitated by (NH4)2SO4, then it was isolated and desalted by ultrofiltration following by ion exchange chromatography of CM column, after reverse phase HPLC of C18 column and vacuum drying, the pure HWTX-I protein was obtained which was proved to be recombinant HWTX-I by Tricine SDS-PAGE, MALDI-TOF mass spectrometry, amino acid composition analysis, the N-terminal amino acid sequence and its biological activity. The final yield of the purified HWTX-I was about 80 mg/L accounting for 23.6% of its total secretory proteins.
Amino Acid Sequence ; Animals ; Chromatography, High Pressure Liquid ; methods ; Chromatography, Ion Exchange ; methods ; Culture Media ; Gene Expression ; Hydrogen-Ion Concentration ; Male ; Mice ; Molecular Sequence Data ; Neuromuscular Junction ; drug effects ; Neurotoxins ; genetics ; isolation & purification ; pharmacology ; Pichia ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; pharmacology ; Reptilian Proteins ; Seminiferous Tubules ; drug effects ; Sequence Analysis, Protein ; Spider Venoms ; genetics ; isolation & purification ; pharmacology ; Spiders ; Synaptic Transmission ; drug effects ; Time Factors