Animals ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; In Vitro Techniques ; Mice ; Myocarditis ; drug therapy ; virology ; Phytotherapy ; methods ; Virus Diseases ; drug therapy

AIMTo study whether store-operated Ca2+ channel (SOC) is present in rat colonic smooth muscle cells.

METHODSIntracellular Ca2+ ([Ca2+]i) changes induced by thapsigargin- or caffeine-activated SOC channel were measured in enzymatically dissociated rat colonic smooth muscle cells with the fluorescent indicator Fura-2/AM.

RESULTSIn the absence of external Ca2+ , the sarco-endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (1 micromol/L) and ryanodine receptor (RyR) activator caffeine both transiently elevated [Ca2+]i from (68.32 +/- 3.43) nmol/L to (240.85 +/- 12.65 ) nmol/L, (481.25 +/- 34.77) nmol/L. A subsequent reintroduction of Ca2+ into the extracellular solution resulted in [Ca2+]i further elevated to (457.55 +/- 19.80) nmol/L, (1005.93 +/- 54.62) nmol/L; (643.88 +/- 34.65) nmol/L, (920.16 +/- 43.25) nmol/L, respectively. And the elevated response was blocked by lanthanum (1 mmol/L), but was insensitive to L-type voltage calcium channels blocker verapamil and membrane depolarization.

CONCLUSIONSOC activated by store depletion are present in rat colonic smooth muscle cells. And we further prove the existence of such Ca2+ channels in excitable cells.

Animals ; Caffeine ; pharmacology ; Calcium ; metabolism ; Calcium Channels ; physiology ; Colon ; cytology ; Fura-2 ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Rats ; Rats, Wistar ; Thapsigargin ; pharmacology

Contraction of smooth muscle cells is triggered by an increase in cytosolic Ca(2+) upon agonist stimulation. Ca(2+) influx across the plasma membrane constitutes a major component of the agonist-induced response in smooth muscle cells. Traditionally, voltage-operated Ca(2+) channel (VOCC) is considered as the channel mediating the Ca(2+) entry. However, this view has been challenged by recent discoveries, which demonstrated that other types of ion channels, such as store-operated and/or receptor-operated Ca(2+) channels (SOCC and/or ROCC), also participate in Ca(2+) response induced by agonists in smooth muscle cells. SOCC is defined as the channel activated in response to the depletion of the internal Ca(2+) stores, an event secondary to G protein coupled receptor or receptor tyrosine kinase stimulation. The Ca(2+) flow mediated by SOCC is termed as capacitative Ca(2+) entry (CCE). Previous study from other group has demonstrated that VOCC played a predominant role in ACh-induced contraction of distal colon smooth muscle in guinea pig. However, whether SOCC participates in the agonist-induced contractile response in this particular tissue is unknown. The present study was performed to investigate the role of CCE in ACh-induced mechanical activity of distal colon smooth muscle in rats. The contractile function of the smooth muscle was assessed by measuring isometric force of isolated rat distal colon rings. We showed that both high extracellular K(+) (40 mmol/L) and ACh (5 mumol/L) evoked striking contraction of the smooth muscle. The contractile responses were almost abolished by removal of extracellular Ca(2+) with ethylene glycol-bis(2-aminoethylether)-N,N,N',N' tetraacetic acid (EGTA), suggesting a critical contribution of extracellular source of Ca(2+) to the contraction. Verapamil (5 mumol/L), an L-type VOCC blocker, significantly attenuated, but didn't completely eliminate the high K(+)- and ACh-induced contraction (74% and 41% for high K(+) and ACh, respectively), indicating that additional channels might be involved in the contractile mechanism. Furthermore, ACh only induced transient contractions in the absence of extracellular Ca(2+). Readmission of Ca(2+) into the extracellular compartment resulted in a significant and sustained increase in the tension of the smooth muscle. This response was not affected by verapamil (5 mumol/L) and Cd(2+) (5 mumol/L), both of which efficiently block VOCC at the doses. However, La(3+), a known inhibitor of SOCC, significantly suppressed the Ca(2+) readdition-induced contraction in a dose-dependent manner. On the basis of these results, we conclude that contraction of smooth muscle in the distal colon is regulated by multiple Ca(2+) channels. In addition to VOCC-mediated Ca(2+) influx, SOCC-mediated CCE participates in agonist-induced contractile response of distal colon smooth muscle in rats.
Acetylcholine ; physiology ; Animals ; Calcium ; metabolism ; Calcium Channels ; physiology ; Colon ; physiology ; Female ; Male ; Muscle Contraction ; physiology ; Muscle, Smooth ; physiology ; Myocytes, Smooth Muscle ; physiology ; Rats ; Rats, Sprague-Dawley ; Verapamil ; pharmacology

The effect of bombesin (BOM) on non-cholinergic excitatory synaptic transmission of the guinea pig inferior mesenteric ganglion (IMG) was investigated by intracellular recording. Repetitive stimulation of the colon nerves (1 ms, 25 Hz, 4 s) elicited a burst of action potentials, which was followed by a long-lasting depolarization in 74.3% (52/70) of the IMG neurons. The depolarization was not blocked by nicotinic (d-tubocurarine, 100 micromol/L) and muscarinic (atropine, 1 micromol/L) antagonists, but was eliminated in a low Ca(2+)/high Mg(2+) Krebs solution, indicating that the depolarization was due to the release of non-cholinergic transmitters. Superfusing the ganglia with BOM (10 micromol/L, 1 min) induced a slow depolarization in 66.5% (109/164) neurons tested. The BOM response was not appreciably changed in low Ca(2+)/high Mg(2+) Krebs solution (n=6, P>0.05), suggesting that BOM depolarized the neurons by acting directly on the postsynaptic membrane rather than via a release of other endogenous depolarizing substances. In a total of 102 cells that exhibited late slow excitatory postsynaptic potential (ls-EPSP), superfusion of the ganglia with BOM produced a membrane depolarization in 82 neurons (80%), while the remaining 20 cells (20%) exhibited no response to BOM. In 18 neurons with ls-EPSP, 4 (22%) neurons were sensitive to both BOM and SP; 6 (33%) and 5 (28%) neurons were only sensitive to BOM and SP, respectively. The remaining 3 (17%) neurons were insensitive to both BOM and SP. Membrane resistance (Rm) had no apparent change in 47.3%, 59.5 % of the neurons tested during the ls-EPSP (n=55) and BOM depolarization (n=84), respectively, but had a marked decrease in 38.2%, 27.4%, and a marked increase in the remaining 14.5%, 13.1% of the neurons. However, when the Rm change accompanying ls-EPSP was compared with that accompanying BOM depolarization (n=20) in the same neuron, the changes in Rm were always parallel. Moreover, ls-EPSP (n=6) and BOM depolarization (n=8) were all augmented by conditioning hyperpolarization. The extrapolated values of the reversal potentials of ls-EPSP and BOM depolarization were 46.0+/-8.0 and 50.0+/-7.0 mV (n=8, P>0.05), respectively. In 14 BOM-sensitive neurons, a ls-EPSP was elicited by repetitive colon nerve stimulation. Superfusion of BOM (10 micromol/L) in these cells initially caused a large depolarization and then membrane potential gradually subsided to resting level in the continuous presence of BOM. Stimulation of the presynaptic nerves at this time failed to elicit a detecable ls-EPSP in 2 neurons and induced a much smaller one in 10 cells, while the ls-EPSP in the remaining 2 neurons was not appreciably affected. On the other hand, prolonged superfusion of BOM had no effect on the amplitude and duration of ls-EPSP in 6 BOM-insensititive neurons studied (P>0.05). The amplitude and duration of SP-induced depolarization were not altered by prolonged superfusion of BOM (n=4, P>0.05) Superfusion of tyr(4) D-phe(12) bombesin (1 micromol/L, 10 15 min), a BOM receptor antagonist, did not cause any noticeable changes in passive membrane properties nor block nicotinic f-EPSPs, but markedly suppressed (n=5) or completely abolished (n=11) BOM depolarization in all 16 neurons tested Similarly, tyr(4) D-phe(12) bombesin partially or completely antagonized the ls-EPSP in 9 out of a total of BOM sensitive neurons (n=11). The ls-EPSP elicited in the remaining two neurons was insignificantly affected by this drug. However, following 10 20 min of wash with Krebs solution the ls-EPSP was reversed. In contrast, superfusion of the ganglia with tyr(4) D-phe(12) bombesin did not change the amplitude and duration (P>0.05) of ls-EPSP in 10 BOM-insensitive cells. Similarly, the amplitude and duration of SP-induced depolarization were not appreciably affected by tyr(4) D-phe(12) bombesin (n=6, P>0.05). In conclusion, our results indicate that BOM may be another transmitter mediating the ls-EPSP in the guinea pig IMG and that there is no cross-desensitization of BOM receptors and SP receptors.
Action Potentials ; drug effects ; physiology ; Animals ; Bombesin ; pharmacology ; Electric Stimulation ; Excitatory Postsynaptic Potentials ; drug effects ; physiology ; Female ; Ganglia, Sympathetic ; drug effects ; physiology ; Guinea Pigs ; In Vitro Techniques ; Male ; Synaptic Transmission ; drug effects

OBJECTIVETo detect the presence of endothelial injury in patients with severe acute respiratory syndrome (SARS) via enhanced levels of tissue-type plasminogen activator (t-PA) and soluble thrombomodulin (sTM).

METHODSCase patients were from Xuanwu Hospital (Capital University of Medical Sciences, Beijing, China), and all of them met clinical criteria for SARS. Healthy controls were some of the hospital employees. Endothelial injury bio-markers tPA and sTM were detected by commercial ELISA-methods.

RESULTSClassic plasma markers of endothelial injury, tPA and sTM significantly elevated in SARS patients in comparison to controls [t-PA: 1.48 +/- 0.16 nmol/L versus 0.25 +/- 0.03 nmol/L (P<0.0001), and sTM: 0.26 +/- 0.06 nmol/L versus 0.14 +/- 0.02 nmol/L (P<0.05)]. The only patient who died had extremely high levels of these endothelial injury markers (t-PA: 2.77 nmol/L and sTM: 1.01 nmol/L). The likelihood ratio analysis indicated the excellent discriminating power for SARS at the optimal cut-point of 0.49 nmol/L for tPA and 0.20 nmol/L for sTM, respectively. Significant numerical correlations were found among these endothelial injury markers in SARS patients. The numerical coefficient of correlation Pearson r between t-PA and sTM was 0.5867 (P<0.05).

CONCLUSIONIncreased plasma concentrations of tPA and sTM in patients with SARS suggest the possibility of endothelial injury. SARS patients might need anticoagulant therapy or fibrinolytic therapy in order to reverse intraalveolar coagulation, microthrombi formation, alveolar and interstitial fibrin deposition. It may not only provide a useful treatment and prognostic index but also allow a further understanding of the pathological condition of the disease.

Adult ; Biomarkers ; blood ; Case-Control Studies ; China ; Female ; Humans ; Male ; Prognosis ; Severe Acute Respiratory Syndrome ; blood ; Thrombomodulin ; blood ; Tissue Plasminogen Activator ; blood

In this paper， through consulting relevant records in materia medica， medical and prescription books， and combining with modern literature， the name， origin， producing area， collection and processing of Gentianae Macrophyllae Radix in famous classical formulas from The Catalogue of Ancient Famous Classical Formulas （The First Batch） was systematically sorted out and textual research was carried out， in order to provide a basis for the development of the famous classical formulas containing Gentianae macrophyllae Radix. After textual research， it was found that Gentianae Macrophyllae Radix was the rectification of name in the past dynasties. In addition， there were other names such as Qinjiao， Qingua and Qinzhua. Gentiana macrophylla， G. straminea， G. dahurica and G. siphonantha were the main origin of this herb in ancient literature. Among them， G. macrophylla is the mainstream. In the Southern and Northern dynasties， G. straminea and G. macrophylla produced in northern Sichuan were recommended as the best. In the early Tang dynasty， G. macrophylla from the Liupan Mountain area at the border of Shanxi and Gansu provinces was the mainstream. During the Northern Song dynasty， G. siphonantha from Linxia and Qilian Mountain of Gansu province and G. macrophylla from eastern Shaanxi province were two new producing areas. In the Ming and Qing dynasties， the abundant base and production areas of Gentianae Macrophyllae Radix were gradually formed. In the past dynasties， harvesting was carried out in spring and autumn， and stored mainly by aeration drying or shade drying treatment. The processing methods are mainly the raw products after the net selection， cutting and drying， in addition to the frying， processing with wine and milk. G. macrophylla is recommended as the first choice for the herbal medicine involved in the famous classical formulas. Among them， wild products produced in Gansu and Shaanxi are the best， and raw products are recommended to be used. At the same time， it is suggested that G. siphonantha should be added to the subsequent edition of Chinese Pharmacopoeia as one of origins of Gentianae Macrophyllae Radix.

Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics* ; Child ; China/epidemiology* ; Cryptorchidism/genetics* ; Disorders of Sex Development/genetics* ; Female ; Genital Diseases, Male ; Genotype ; Humans ; Hypospadias/genetics* ; Male ; Membrane Proteins/genetics* ; Penis/abnormalities* ; Phenotype ; Retrospective Studies ; Steroid 21-Hydroxylase/genetics*

Objective: To analyze the clinical epidemiological characteristics including composition of pathogens , clinical characteristics, and disease prognosis acute bacterial meningitis (ABM) in Chinese children. Methods: A retrospective analysis was performed on the clinical and laboratory data of 1 610 children <15 years of age with ABM in 33 tertiary hospitals in China from January 2019 to December 2020. Patients were divided into different groups according to age,<28 days group, 28 days to <3 months group, 3 months to <1 year group, 1-<5 years of age group, 5-<15 years of age group; etiology confirmed group and clinically diagnosed group according to etiology diagnosis. Non-numeric variables were analyzed with the Chi-square test or Fisher's exact test, while non-normal distrituction numeric variables were compared with nonparametric test. Results: Among 1 610 children with ABM, 955 were male and 650 were female (5 cases were not provided with gender information), and the age of onset was 1.5 (0.5, 5.5) months. There were 588 cases age from <28 days, 462 cases age from 28 days to <3 months, 302 cases age from 3 months to <1 year of age group, 156 cases in the 1-<5 years of age and 101 cases in the 5-<15 years of age. The detection rates were 38.8% (95/245) and 31.5% (70/222) of Escherichia coli and 27.8% (68/245) and 35.1% (78/222) of Streptococcus agalactiae in infants younger than 28 days of age and 28 days to 3 months of age; the detection rates of Streptococcus pneumonia, Escherichia coli, and Streptococcus agalactiae were 34.3% (61/178), 14.0% (25/178) and 13.5% (24/178) in the 3 months of age to <1 year of age group; the dominant pathogens were Streptococcus pneumoniae and the detection rate were 67.9% (74/109) and 44.4% (16/36) in the 1-<5 years of age and 5-<15 years of age . There were 9.7% (19/195) strains of Escherichia coli producing ultra-broad-spectrum β-lactamases. The positive rates of cerebrospinal fluid (CSF) culture and blood culture were 32.2% (515/1 598) and 25.0% (400/1 598), while 38.2% (126/330)and 25.3% (21/83) in CSF metagenomics next generation sequencing and Streptococcus pneumoniae antigen detection. There were 4.3% (32/790) cases of which CSF white blood cell counts were normal in etiology confirmed group. Among 1 610 children with ABM, main intracranial imaging complications were subdural effusion and (or) empyema in 349 cases (21.7%), hydrocephalus in 233 cases (14.5%), brain abscess in 178 cases (11.1%), and other cerebrovascular diseases, including encephalomalacia, cerebral infarction, and encephalatrophy, in 174 cases (10.8%). Among the 166 cases (10.3%) with unfavorable outcome, 32 cases (2.0%) died among whom 24 cases died before 1 year of age, and 37 cases (2.3%) had recurrence among whom 25 cases had recurrence within 3 weeks. The incidences of subdural effusion and (or) empyema, brain abscess and ependymitis in the etiology confirmed group were significantly higher than those in the clinically diagnosed group (26.2% (207/790) vs. 17.3% (142/820), 13.0% (103/790) vs. 9.1% (75/820), 4.6% (36/790) vs. 2.7% (22/820), χ²=18.71, 6.20, 4.07, all P<0.05), but there was no significant difference in the unfavorable outcomes, mortility, and recurrence between these 2 groups (all P>0.05). Conclusions: The onset age of ABM in children is usually within 1 year of age, especially <3 months. The common pathogens in infants <3 months of age are Escherichia coli and Streptococcus agalactiae, and the dominant pathogen in infant ≥3 months is Streptococcus pneumoniae. Subdural effusion and (or) empyema and hydrocephalus are common complications. ABM should not be excluded even if CSF white blood cell counts is within normal range. Standardized bacteriological examination should be paid more attention to increase the pathogenic detection rate. Non-culture CSF detection methods may facilitate the pathogenic diagnosis.
Adolescent ; Brain Abscess ; Child ; Child, Preschool ; Escherichia coli ; Female ; Humans ; Hydrocephalus ; Infant ; Infant, Newborn ; Male ; Meningitis, Bacterial/epidemiology* ; Retrospective Studies ; Streptococcus agalactiae ; Streptococcus pneumoniae ; Subdural Effusion ; beta-Lactamases