Solid-phase extraction with the functionalization of calcium-sensing receptors onto magnetic microspheres as an affinity probe can capture ligands selectively from herbal extract.

Magnetic solid phase extraction with the functionalization of protein onto micro- or nano-particles as a probe is favorable for the discovery of new drugs from complicated natural products. Herein, we aimed to develop a rapid method by immobilizing halogenated alkane dehalogenase (Halo)-tagged calcium-sensing receptor (CaSR) directly out of crude cell lysates onto the surface of magnetic microspheres (MM) with no need to purify protein. Thereby we achieved CaSR-functionalized MM for revealing adsorption characteristics of agonist neomycin and screening ligands from herbal medicine Radix Astragali (RA). About 43.87 mg CaSR could be immobilized per 1 g MM within 30 min, and the acquired CaSR-functionalized MM showed good stability and activity for 4 weeks. The maximum adsorption capacity of neomycin on CaSR-functionalized MM was determined as 4.70 × 10-4 ~ 3.96 × 10-4 mol/g within 277 ~ 310 K, and its adsorption isotherm characteristics described best by the Temkin model were further validated using isothermal titration calorimetry. It was inferred that CaSR's affinity for neomycin was driven by electrostatic forces in a spontaneous process when the system reached an equilibrium state. Moreover, the ligands from the RA extract were screened, three of which were assigned as astragaloside IV, ononin, and calycosin based on HPLC-MS. Our findings demonstrated that the functionalization of a receptor onto magnetic materials designed as an affinity probe has the capability to recognize its agonist and capture the ligands selectively from complex matrices like herbs.

A rapid approach to capture the potential bioactive compounds from Rhizoma Drynariae, utilizing disease-associated mutation in calcium sensing receptor to alter the binding affinity for agonists.

Rhizoma Drynariae (RD) was used clinically to treat osteoporosis in China due to stimulating bone formation and inhibiting bone resorption, however, the bioactive constituents with the dual effect on bone are still unknown exactly. Disease-causing mutations in calcium sensing receptor (CaSR) can alter parathyroid hormone secretion and affect Ca2+ release from bone and Ca2+ reabsorption from kidney, which gives an indication that CaSR is a potential target for developing therapeutics to manage osteoporosis. Herein, a chromatographic approach was established, by immobilizing the mutant CaSR onto the surface of silica gels as stationary phase in a one-step procedure and then adding the different amino acids into mobile phase as competitors, for exploring the binding features of the known agonists and further screening ligands from RD. The mutant CaSR-coated column was prepared rapidly without the complicated purification and separation of the receptor, which had the large capacity of 13.1 mg CaSR /g silica gels and kept a good stability and specificity for at least 35 days. The CaSR mutation can weaken the binding affinities for three agonists, and the largest decreases occurred on the mutational site Thr151Met for neomycin, on the two sites of Asn118Lys and Glu191Lys for gentamicin-C, and on the site Phe612Ser for kanamycin, which gained new insights into their structure-function relationship. The potential bioactive compounds from RD were screened using the mutant CaSR-coated column and were recognized as coumaric acid 4-O-ß-D-glucopyranoside, caffeic acid, and naringin using UPLC-MS. Among them, naringin targeting CaSR gives a possible explanation that RD could manage osteoporosis. These results indicated that, such a rapid and simple method, utilizing disease-associated mutation in CaSR to alter the binding affinity for agonists, can be applied in capturing the potential bioactive compounds efficiently from complex matrices like herb medicines.

Extracellular domain of human calcium sensing receptor immobilized to silica beads as biomaterial: A rapid chromatographic method for recognizing ligands from complex matrix 'Shuangdan'.

Human calcium-sensing receptor (CaSR), a member of the G-protein-coupled receptor superfamily (GPCR), has been a therapeutic target for developing new drugs against calciotropic disorders and non-calciotropic diseases. The highly efficient methodologies for pursuing novel ligands/drugs remained a challenge due to the redundant purification processes of membrane protein in some widely-used methods including NMR, X-ray crystallography, Fluorescence Titration Spectroscopy, and Circular Dichroism. Herein, extracellular domain (ECD) of CaSR as its functional fragment was used to develop a rapid chromatographic method, which involved the synthesis of stationary phase material based on the site-specific covalent reaction of Halogenated alkane dehalogenase (Halo)-tagged ECD of CaSR in cell lysate with 6-chlorocaproic acid modified silica beads, the use of the immobilized CaSR column for revealing the interaction of three known agonists with CaSR and further screening ligands from complex matrix like Chinese herb medicine 'Shuangdan'. The immobilized CaSR column was prepared rapidly without the protein purification and retained a good stability and specificity for at least 35 days. It was revealed that one type of binding sites occurred on CaSR with the binding affinity of neomycin > gentamicin-C / kanamycin, presumably which related to the number of structural amino groups attached. This method allowed for recognizing specifically novel ligands from 'Shuangdan', demonstrating one type of binding sites on CaSR with the binding affinity of gallic acid > caffeic acid > paeonol. These results indicated that, the immobilization of a representative extracellular domain of CaSR to silica beads as biomaterial is feasible to develop a new rapid method, which can be successfully applied in screening novel ligands efficiently from complex matrices.

Insights into the development of pentylenetetrazole-induced epileptic seizures from dynamic metabolomic changes.

Epilepsy is often considered to be a progressive neurological disease, and the nature of this progression remains unclear. Understanding the overall and common metabolic changes of epileptic seizures can provide novel clues for their control and prevention. Herein, a chronic kindling animal model was established to obtain generalized tonic-clonic seizures via the repeated injections of pentylenetetrazole (PTZ) at subconvulsive dose. Dynamic metabolomic changes in plasma and urine from PTZ-kindled rats at the different kindling phases were explored using NMR-based metabolomics, in combination with behavioral assessment, brain neurotransmitter measurement, electroencephalography and histopathology. The increased levels of glucose, lactate, glutamate, creatine and creatinine, together with the decreased levels of pyruvate, citrate and succinate, ketone bodies, asparagine, alanine, leucine, valine and isoleucine in plasma and/or urine were involved in the development and progression of seizures. These altered metabolites reflected the pathophysiological processes including the compromised energy metabolism, the disturbed amino acid metabolism, the peripheral inflammation and changes in gut microbiota functions. NMR-based metabolomics could provide brain disease information by the dynamic plasma and urinary metabolic changes during chronic epileptic seizures, yielding classification of seizure stages and profound insights into controlling epilepsy via targeting deficient energy metabolism.

Revealing the Antiepileptic Effect of α-Asaronol on Pentylenetetrazole-Induced Seizure Rats Using NMR-Based Metabolomics.

α-Asaronol from Acorus tatarinowii (known as "Shichangpu" in Traditional Chinese medicine) has been proved to possess more efficient antiepileptic activity and lower toxicity than α-asarone (namely "Xixinnaojiaonang" as an antiepileptic drug in China) in our previous study. However, the molecular mechanism of α-asaronol against epilepsy needs to be known if to become a novel antiepileptic medicine. Nuclear magnetic resonance (NMR)-based metabolomics was applied to investigate the metabolic patterns of plasma and the brain tissue extract from pentylenetetrazole (PTZ)-induced seizure rats when treated with α-asaronol or α-asarone. The results showed that α-asaronol can regulate the metabolomic level of epileptic rats to normal to some extent, and four metabolic pathways were associated with the antiepileptic effect of α-asaronol, including alanine, aspartate, and glutamate metabolism; synthesis and degradation of ketone bodies; glutamine and glutamate metabolism; and glycine, serine, and threonine metabolism. It was concluded that α-asaronol plays a vital role in enhancing energy metabolism, regulating the balance of excitatory and inhibitory neurotransmitters, and inhibiting cell membrane damage to prevent the occurrence of epilepsy. These findings are of great significance in developing α-asaronol into a promising antiepileptic drug derived from Traditional Chinese medicine.

Immobilized beta2-adrenergic receptor: A powerful chromatographic platform for drug discovery and evaluation of drug-like property for natural products.

Drug discovery based on natural products like medicinal herbs remains challenging due to the technique limitations for rapidly screening and validating leads. To address the challenges, we employ the immobilized ß2- adrenergic recepotor (ß2-AR), an identified target of asthma, as the stationary phase in chromatographic column to screen compounds extracted from Stemonae Radix, Playtycodonis Radix, and Glycyrrhizae Radix et Rhizoma. To analyze binding properties of the extracted compounds to the immobilized receptors, we measured their retention behavior in the receptor chromatography and compared with six clinical asthma drugs. We identified tuberostemonine, platycodin D, and glycyrrhizic acid as the potential leads against asthma by our ß2-AR chromatography coupled with mass spectrum (MS). The association constants of the three compounds to ß2-AR were 2.85 × 10-5, 2.55 × 10-4, and 4.07 × 10-6 M with the dissociation rate constants of 6.91 ± 0.35, 11.88 ± 0.60, and 9.49 ± 0.64 min-1, respectively. Tuberostemonine, a pentacyclic Stemona alkaloids, presented the most optimum values of binding efficiency index (BEI) and surface efficiency index (SEI) as close to the diagonal of SEI-BEI optimization plane when it is compared with platycodin D, glycyrrhizic and the six clinical drugs. Our results suggest that tuberostemonine is a promising natural product to be developed for treating asthma because it exhibits better drug-like binding properties to ß2-AR than the clinical drugs. As such, we demonstrate a chromatographic strategy to identify bioactive natural products based on the ß2-AR immobilization, which can be widely adopted to screen natural products from mixture of herbal extracts.

[Community of Benthic Diatoms and Their Relationship with Aquatic Environmental Factors in the Tangwang River, China].

To reasonably evaluate the eco-environmental health of the Tangwang River, which is a tributary of the Songhua River in China, community structures of periphyton and cleanliness of the benthic diatom at 24 sampling sites were investigated using McNaughton's dominance index, clustering, and ecotype analysis, while the relationship between the environmental factors and the diatom communities were studied by principal component analysis, Spearman correlation test, and redundancy analysis, in August 2018 (flood season). A total of 99 species or variants of benthic diatoms have been identified, indicating that there were abundant diatoms in the Tangwang River. Achnanthidium minutissimum and other diatoms that can be used as clean water indicators were dominant species in the Tangwang River, which indicates that the eco-environmental quality of the Tangwang River was relatively healthy in the flood season. Of these, the dominant degree of A. minutissimum was 0.32, making it the absolute dominant species in Tangwang River. Sampling sites can be divided into three groups based on clustering analysis. The dominant species of group 1 and group 2 were mainly clean species, indicating that the two groups were in a relatively healthy state. Nitzschia palea, Ulnaria ulna, and other diatoms that can be used as eutrophication indicators were the dominant species of group 3, indicating that group 3 was less healthy than the other two groups. From groups 1 and 2, the results from ecotype analysis showed a decrease in the proportion of polyoxybiontic diatoms and an increase in the proportion of α-mesosaphrobe diatoms, polysaprobe diatoms, oligo-mesotrophic diatoms, mesotrophic diatoms, meso-eutrophic diatoms, and eutrophic diatoms. Compared to the other two groups, the results from ecotype analysis showed a significant increase in the proportion of α-mesosaphrobe diatoms, polysaprobe diatoms, eutrophic diatoms and hypereutrophic diatoms in group 3. The predominant aquatic influencing factors of diatom community structures for the Tangwang River were permanganate index, total nitrogen (TN), and ammonia nitrogen (NH4+-N), of which permanganate index was the main factor for group 2, while TN and NH4+-N were the main factors for group 3. As a result, the eco-environmental quality of the Tangwang River was good, and the benthic diatom was found to be an effective indicator of the nutritional conditions and saprophytic status.

The Impacts of Land Use Patterns on Water Quality in a Trans-Boundary River Basin in Northeast China Based on Eco-Functional Regionalization.

The relationships between land use patterns and water quality in trans-boundary watersheds remain elusive due to the heterogeneous natural environment. We assess the impact of land use patterns on water quality at different eco-functional regions in the Songhua River basin during two hydrological seasons in 2016. The partial least square regression indicated that agricultural activities associated with most water quality pollutants in the region with a relative higher runoff depth and lower altitude. Intensive grazing had negative impacts on water quality in plain areas with low runoff depth. Forest was related negatively with degraded water quality in mountainous high flow region. Patch density and edge density had major impacts on water quality contaminants especially in mountainous high flow region; Contagion was related with non-point source pollutants in mountainous normal flow region; landscape shape index was an effective indicator for anions in some eco-regions in high flow season; Shannon's diversity index contributed to degraded water quality in each eco-region, indicating the variation of landscape heterogeneity influenced water quality regardless of natural environment. The results provide a regional based approach of identifying the impact of land use patterns on water quality in order to improve water pollution control and land use management.

Assessment of surface water quality using a growing hierarchical self-organizing map: a case study of the Songhua River Basin, northeastern China, from 2011 to 2015.

The analysis of a large number of multidimensional surface water monitoring data for extracting potential information plays an important role in water quality management. In this study, growing hierarchical self-organizing map (GHSOM) was applied to a water quality assessment of the Songhua River Basin in China using 22 water quality parameters monitored monthly from 13 monitoring sites from 2011 to 2015 (14,782 observations). The spatial and temporal features and correlation between the water quality parameters were explored, and the major contaminants were identified. The results showed that the downstream of the Second Songhua River had the worst water quality of the Songhua River Basin. The upstream and midstream of Nenjiang River and the Second Songhua River had the best. The major contaminants of the Songhua River were chemical oxygen demand (COD), ammonia nitrogen (NH3-N), total phosphorus (TP), and fecal coliform (FC). In the Songhua River, the water pollution at downstream has been gradually eased in years. However, FC and biochemical oxygen demand (BOD5) showed growth over time. The component planes showed that three sets of parameters had positive correlations with each other. GHSOM was found to have advantages over self-organizing maps and hierarchical clustering analysis as follows: (1) automatically generating the necessary neurons, (2) intuitively exhibiting the hierarchical inheritance relationship between the original data, and (3) depicting the boundaries of the classification much more clearly. Therefore, the application of GHSOM in water quality assessments, especially with large amounts of monitoring data, enables the extraction of more information and provides strong support for water quality management.

Effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes.

The effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes and the possible mechanisms were investigated in this study. 3T3-L1 preadipocytes were cultured in vitro and treated with different concentrations of ghrelin. Proliferation of 3T3-L1 preadipocytes was evaluated by MTT method and mRNA levels of c-myc and thymidine kinase were detected by RT-PCR. Morphological changes of 3T3-L1 preadipocytes were observed and cell differentiation was measured by oil red O staining. The mRNA levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and CAAT/enhancer binding protein (C/EBPalpha) in the cells at different differentiation stages were detected by RT-PCR. The results showed that ghrelin at concentrations of 10(-7) to 10(-15) mol/L could significantly promote preadipocyte proliferation (P<0.05), with the most pronounced effect observed at 10(-11) mol/L (P<0.01). Treatment of 3T3-L1 preadipocytes with ghrelin significantly increased the mRNA levels of c-myc and thymidine kinase (P<0.01). Morphological findings demonstrated that the great amount of lipid droplets appeared in the 3T3-L1 preadipocytes treated with ghrelin. Ghrelin could morphologically induce the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Ghrelin significantly increased the mRNA levels of PPARgamma and C/EBPalpha during the differentiation, when compared with control group (P<0.05). The mRNA levels of PPARgamma and C/EBPalpha were obviously up-regulated with the differentiation of preadipocytes after the treatment of ghrelin. There were significant difference in the mRNA levels of PPARgamma and C/EBPalpha on day 2 and day 8 of the differentiation of 3T3-L1 preadipocytes (P<0.01). In conclusion, ghrelin could promote the proliferation and differentiation of 3T3-L1 preadipocytes by increasing the mRNA levels of PPARgamma and C/EBPalpha and therefore enhance the sensitivity of adipocytes against insulin.

Evaluation of atrial septal defect using real-time three-dimensional echocardiography: comparison with surgical findings.

The present study evaluated the application of three dimensional echocardigraphy (3DE) in the diagnosis of atrial septal defect (ASD) and the measurement of its size by 3DE and compared the size with surgical findings. Two-dimensional and real-time three dimensional echocardiography (RT3DE) was performed in 26 patients with atrial septal defect, and the echocardiographic data were compared with the surgical findings. Significant correlation was found between defect diameter by RT3DE and that measured during surgery (r=0.77, P<0.001). The defect area changed significantly during cardiac cycle. Percentage change in defect size during cardiac cycle ranged from 6%-70%. Our study showed that the size and morphology of atrial septal defect obtained with RT3DE correlate well with surgical findings. Therefore, RT3DE is a feasible and accurate non-invasive imaging tool for assessment of atrial septal size and dynamic changes.

[Effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes and its possible mechanisms].

OBJECTIVE: To investigate the effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocyte, and study the possible mechanisms. METHODS: 3T3-L1 preadipocytes were cultured in vitro. The proliferation potentials of 3T3-L1 preadipocytes that were treated with different concentrations of ghrelin were evaluated by MTT methods. The levels of c-myc and thymidine kinase mRNA were detected using RT-PCR. 3T3-L1 preadipocytes were differentiated into the matured adipocytes with insulin (INS) or ghrelin. The morphological changes of 3T3-L1 adipocytes were observed and the differentiation rate was assayed by oil-red O staining. Total RNA was extracted from adipocytes at various times, and the levels of peroxisome proliferation activated receptor gamma (PPARgamma) and CAAT/enhancer binding protein(C/EBPalpha) mRNA expressions were detected using RT-PCR. RESULTS: Ghrelin at concentrations of 10(-7) to 10(-15) mol/L significantly stimulated preadipocyte proliferation (p<0.05). The levels of c-myc and thymidine kinase mRNA significantly increased in 3T3-L1 preadipocytes with 10(-9) mol/L and 10(-11) mol/L ghrelin treatment (p<0.01). The 3T3-L1 preadipocytes treated with 10(-11) mol/L ghrelin had lots of lipid droplets in the cytoplasma, but the differentiation rate was lower than those treated with INS. Ghrelin of 10(-11) mol/L significantly increased the mRNA expression of PPARgamma and C/EBPalpha in the course of 3T3-L1 preadipocyte differentiation, compared with the normal control group (p<0.05). The PPARgamma and C/EBPalpha mRNA expression increased with the prolonged differentiation of preadipocytes induced by ghrelin or INS. There were significant differences in the levels of PPARgamma and C/EBPalpha mRNA expression between the 2nd and 8th days of differentiation(p<0.01). CONCLUSIONS: Ghrelin promotes the proliferation and differentiation of 3T3-L1 preadipocytes. The proliferation of 3T3-L1 preadipocytes induced by ghrelin may be associated with increased c-myc levels. Ghrelin may promote differentiation of 3T3-L1 preadipocytes by increasing mRNA expression of PPARgamma and C/EBPalpha, thus enhances the sensitivity of adipocytes to INS.

Effects of Ghrelin on the Proliferation and Differentiation of 3T3-L1 Preadipocytes / 华中科技大学学报（医学）（英德文版）

The effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes and the possible mechanisms were investigated in this study.3T3-L1 preadipocytes were cultured in vitro and treated with different concentrations of ghrelin.Proliferation of 3T3-L1 preadipocytes was evaluated by MTT method and mRNA levels of c-myc and thymidine kinase were detected by RT-PCR.Morphological changes of 3T3-L1 preadipocytes were observed and cell differentiation was measured by oil red O staining.The mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ) and CAAT/enhancer binding protein (C/EBPα) in the cells at different differentiation stages were detected by RT-PCR.The results showed that ghrelin at concentrations of 10-7 to 10-15 mol/L could significantly promote preadipocyte proliferation (P<0.05),with the most pronounced effect observed at 1011mol/L (P<0.01).Treatment of 3T3-L1 preadipocytes with ghrelin significantly in-creased the mRNA levels of c-myc and thymidine kinase (P<0.01).Morphological findings demonstrated that the great amount of lipid droplets appeared in the 3T3-L1 preadipocytes treated with ghrelin.Ghrelin could morphologically induce the differentiation of 3T3-L1 preadipocytes into mature adipocytes.Ghrelin significantly increased the mRNA levels of PPART and C/EBPα during the differentiation,when compared with control group (P<0.05).The mRNA levels of PPARγ and C/EBPα were obviously up-regulated with the differentiation of preadipocytes after the treatment of ghrelin.There were significant difference in the mRNA levels of PPARγ and C/EBPα on day 2 and day 8 of the differentiation of 3T3-L1 preadipocytes (P<0.01).In conclusion,ghrelin could promote the proliferation and differentiation of 3T3-L1 preadipocytes by increasing the mRNA levels of PPARγ and C/EBPα and therefore enhance the sensitivity of adipocytes against insulin.

Evaluation of Atrial Septal Defect Using Real-time Three-dimensional Echocardiography:Comparison with Surgical Findings / 华中科技大学学报（医学）（英德文版）

The present study evaluated the application of three dimensional echocardigraphy (3DE) in the diagnosis of atrial septal defect (ASD) and the measurement of its size by 3DE and compared the size with surgical findings.Two-dimensional and real-time three dimensional echocardiography (RT3DE) was performed in 26 patients with atrial septal defect,and the echocardiographic data were compared with the surgical findings.Significant correlation was found between defect diameter by RT3DE and that measured during surgery (r=0.77,P<0.001).The defect area changed significantly during cardiac cycle.Percentage change in defect size during cardiac cycle ranged from 6%-70%.Our study showed that the size and morphology of atrial septal defect obtained with RT3DE correlate well with surgical findings.Therefore,RT3DE is a feasible and accurate non-invasive imaging tool for assessment of atrial septal size and dynamic changes.

[Expression of fibrillin-1 in congenital bicuspid aortic valves].

OBJECTIVE: To detect the expression of fibrillin-1 in congenital bicuspid aortic valves, and to investigate the molecular mechanism of congenital bicuspid aortic valves. METHODS: Specimens of aortic valve were obtained from 12 pediatric patients with congenital bicuspid aortic valve, 11 boys and 1 girl, aged 16.7 (10 - 18), including 5 cases of aortic stenosis (AS), 8 of aortic insufficiency (AI), and 1 of AS and AI, undergoing valve replacement, 8 children who died accidentally without cardiovascular system and collagen system diseases, 6 boys and 2 girls, aged 9.1 (1 - 17), collected in autopsy [normal (tricuspid) aortic valve controls], and 18 pediatric patients of rheumatic valvular heart disease with diseased tricuspid aortic valves who underwent aortic valve replacement, 13 boys and 5 girls, aged 16.5 (12 - 18) (rheumatic valvular heart disease controls). HE staining and light microscopy were conducted. Immunohistochemistry was used to detect the expression of fibrillin-1 in the aortic valves. RESULTS: Microscopy showed that the tissue structure of the congenital bicuspid aortic valves was unclear with hyperplasia of fibrous tissue. The grey degree value of fibrillin-1 of the congenital bicuspid aortic valve group was 170 +/- 10, significantly lower than those of normal aortic valve group and diseased tricuspid aortic valve group (126 +/- 8 and 73 +/- 16 respectively, both P < 0.05). There were not significant difference in the grey degree value of fibrillin-1 among the patients of congenital bicuspid aortic valves with AS, AI, and AS + AI (167 +/- 6, 171 +/- 8, and 168 +/- 6 respectively). CONCLUSION: The expression of fibrillin-1 is significantly reduced in congenital bicuspid aortic valves which may contribute to the morphological changes of the aortic valve leaflets and their resultant functional failure in congenital bicuspid aortic valves.

A clinicopathological study on aortic valves in children.

In order to investigate the clinicopathological characteristics of aortic valve disease in children, all the native surgically excised aortic valves obtained between January 2003 and December 2005 were studied macroscopically and microscopically. The patients' medical records were reviewed and the clinical information was extracted. According to preoperative echocardiography, intraoperative assessment, and postoperative pathology, combined with clinical symptoms and signs, aortic valve diseases were divided into three categories: aortic stenosis (AS), aortic insufficiency (AI), and aortic stenosis with insufficiency (AS-AI). The etiology was determined according to the macroscopic, microscopic and clinical findings. The results showed that among 70 aortic valves, patient age ranged from 6 to 18 years, with a mean of 15.4 years, and there were 56 boys and 14 girls (male: female=4:1). Forty-four children only had pure aortic valve disease, and the other 26 children had aortic valve disease associated with other heart valve diseases. There were 5 cases of AS (7.14%), 60 cases of AI (85.71%) and 5 cases of AS-AI (7.14%). The causes were congenital aortic valve malformation (32 cases, 45.71%), rheumatic disease (28 cases, 40%), infective endocarditis (7 cases, 10%), Marfan syndrome (2 cases, 2.86%), and undetermined (1 case, 1.43%). It was concluded that the common causes of aortic valve disease in order of frequency in children were congenital aortic valve malformation, rheumatic disease, infective endocarditis, and Marfan syndrome. AI was more common in children with aortic valve disease. Compared with adult patients, congenital bicuspid aortic valve in children was often AI. Histologically, the leaflets of congenital bicuspid aortic valve were mainly myxomatous, fibrosis and calcification less seen. AI was frequently found in rheumatic disease, mostly associated with other heart valve diseases. Macroscopic and microscopic examinations together with clinical information, echocardiographic findings and operative details were important in evaluating the etiology of aortic valve disease.

Histopathological study of congenital aortic valve malformations in 32 children.

The histopathological characteristics of congenital aortic valve malformations in children were investigated. All the native surgically excised aortic valves from 32 pediatric patients suffering from symptomatic aortic valve dysfunction due to congenital aortic valve malformations between January 2003 and December 2005 were studied macroscopically and microscopically. The patients' medical records were reviewed and the clinical information was extracted. The diagnosis was made by the clinical presentation, preoperative echocardiography, intraoperative examination, and postoperative histopathological study, excluding rheumatic or degenerative aortic valve diseases, infective endocarditis and primary connective tissue disorders, e.g. Marfan syndrome. Among 32 children with congenital aortic valve malformations, the age was ranged from six to 18 years, with a mean of 14.9 years, and there were 27 boys and five girls (male: female = 5.4:1). There were five cases of aortic stenosis (AS, 15.62%), 25 cases of aortic insufficiency (AI, 78.13%) and two cases of AS-AI (6.25%), without other valve diseases. Twenty cases still had other congenital heart diseases: ventricular septal defect (19 cases), patent ductus arteriosus (two cases), double-chambered right ventricle (one case), aneurysm of the right anterior aortic sinus of valsalva (three cases). Histopathological examination indicated that the cusps became thickening with unequal size, irregular shape (coiling and prolapse edge), enhanced hardness, and partly calcification. Microscopic investigation revealed the unsharp structure of valve tissue, fibrosis, myxomatous, reduced collagen fiber, rupture of elastic fibers, different degrees of infiltration of inflammatory cells, secondary calcareous and lipid deposit, and secondary fibrosis. Congenital aortic valve malformations in children involve males more than females, mostly associated with other congenital heart diseases. Aortic insufficiency is more common in children with congenital aortic valve malformations. Histopathologically, the leaflets of aortic valve are mainly myxomatous, thickening with unequal size, irregular shape (coiling and prolapse edge), reduced collagen fiber, rupture of elastic fibers, without small vessel proliferation and inflammatory cell infiltration, fibrosis and calcification rarely seen.