Objective: To explore the mechanism of intestinal tissue damage induced by macrophages activated by WNT2B high-expressed fibroblasts. Methods: This study involved biological information analysis, pathological tissue research and cell experimental research. The biological information of the colon tissue from the children with inflammatory bowel disease in previous study was analyzed again with single-cell sequencing. The pathological tissues were collected by colonoscopy from 10 children with Crohn's disease treated in the Department of Gastroenterology of Guangzhou Women and Children's Medical Center from July 2022 to September 2022. According to the findings of colonoscopy, tissues with obvious inflammation or ulceration were classified as the inflammatory group, while tissues with slight inflammation and no ulceration were classified as the non-inflammatory group. HE staining was performed to observe the pathological changes of the colon tissues. Macrophage infiltration and CXCL12 expression were detected by immunofluorescence. In terms of cell experiments, fibroblasts transfected with WNT2B plasmid or empty plasmid were co-cultured with salinomycin treated or non-treated macrophages, respectively; the expression of proteins through Wnt classical pathway were detected by western blotting. Macrophages treated with SKL2001 were used as the experimental group, and those with phosphate buffer as the control group. The expression and secretion of CXCL12 in macrophages were detected by quantitative Real-time PCR and enzyme-linked immunosorbent assay (ELISA). T-test or rank sum test were used for the comparison between groups. Results: Single-cell sequencing analysis suggested that macrophages were the main cells in inflammatory bowel disease colon tissue, and there was interaction between WNT2B high-expressed fibroblasts and macrophages. HE staining of the 10 patients ((9.3±3.8) years old, 7 males and 3 females) showed that the pathological score of colon tissue in the inflammatory group was higher than that in the non-inflammatory group (4 (3, 4) vs. 2 (1, 2) points, Z=3.05, P=0.002). Tissue immunofluorescence indicated that the number of infiltrating macrophages in the inflammatory group was significantly higher than that in the non-inflammatory group under high power field of view (72.8±10.4 vs.8.4±3.5, t=25.10, P<0.001), as well as the number of cells expressing CXCL12 (14.0±3.5 vs. 4.7±1.9, t=14.68, P<0.001). In cell experiments, western blotting suggested an elevated level of glycogen synthase kinase-3β phosphorylation in macrophages co-cultured with fibroblast transfected with WNT2B plasmid, and salinmycin could reverse this change. Real-time PCR suggested that the transcription level of CXCL12 in the experimental group was higher than that in the control group (6.42±0.04 vs. 1.00±0.03, t=183.00, P<0.001), as well as the expression and secretion of CXCL12 by ELISA ((465±34) vs. (77±9) ng/L, t=13.21, P=0.006). Conclusion: WNT2B high-expressed fibroblasts can secrete WNT2B protein and activate the Wnt classical signaling pathway thus enhancing the expression and secretion of CXCL12 in macrophages, inducing the development of intestinal inflammation of Crohn's disease.
Child ; Male ; Humans ; Female ; Child, Preschool ; Adolescent ; Crohn Disease ; Inflammatory Bowel Diseases ; Colon ; Inflammation ; Colonoscopy ; Glycoproteins ; Wnt Proteins

OBJECTIVE: This study was aimed at investigating the carrier rate of, and molecular variation in, α- and β-globin gene mutations in Hunan Province. METHODS: We recruited 25,946 individuals attending premarital screening from 42 districts and counties in all 14 cities of Hunan Province. Hematological screening was performed, and molecular parameters were assessed. RESULTS: The overall carrier rate of thalassemia was 7.1%, including 4.83% for α-thalassemia, 2.15% for β-thalassemia, and 0.12% for both α- and β-thalassemia. The highest carrier rate of thalassemia was in Yongzhou (14.57%). The most abundant genotype of α-thalassemia and β-thalassemia was -α ^3.7/αα (50.23%) and β ^IVS-II-654/β ^N (28.23%), respectively. Four α-globin mutations [CD108 (ACC>AAC), CAP +29 (G>C), Hb Agrinio and Hb Cervantes] and six β-globin mutations [CAP +8 (C>T), IVS-II-848 (C>T), -56 (G>C), beta nt-77 (G>C), codon 20/21 (-TGGA) and Hb Knossos] had not previously been identified in China. Furthermore, this study provides the first report of the carrier rates of abnormal hemoglobin variants and α-globin triplication in Hunan Province, which were 0.49% and 1.99%, respectively. CONCLUSION Our study demonstrates the high complexity and diversity of thalassemia gene mutations in the Hunan population. The results should facilitate genetic counselling and the prevention of severe thalassemia in this region.
Humans ; beta-Thalassemia/genetics* ; alpha-Thalassemia/genetics* ; Hemoglobinopathies/genetics* ; China/epidemiology* ; High-Throughput Nucleotide Sequencing

OBJECTIVE: To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD). METHODS: Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins. RESULTS: In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa. CONCLUSION Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.
Humans ; Mice ; Animals ; Caco-2 Cells ; beta Catenin/metabolism* ; Culture Media, Conditioned/pharmacology* ; Tight Junctions/metabolism* ; Intestinal Mucosa ; Inflammatory Bowel Diseases ; Tight Junction Proteins/metabolism* ; Inflammation/metabolism* ; Fibroblasts/metabolism* ; Mice, Inbred C57BL ; Glycoproteins/metabolism* ; Wnt Proteins/pharmacology* ; Frizzled Receptors/metabolism*

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

Objective: To explore the clinical value of von Willebrand Factor (vWF) and VITRO score (vWF:Ag/platelet count) in assessing disease progression in patients with HBV infection. Methods: Randomly collect relevant clinical data of 308 patients with HBV infection (including 154 cases of chronic hepatitis B, 66 cases of hepatitis B cirrhosis in compensatory period, 88 cases of hepatitis B cirrhosis in decompensated period) from December 1, 2018 to January 5, 2021 in the Second Affiliated Hospital of Chongqing Medical University. The vWF values are measured by a uniform optical method, and all data are included using a uniform standard. Analyze the difference and significance of plasma vWF level and VITRO score in chronic hepatitis B, hepatitis B cirrhosis in the compensatory phase and decompensated phase. Results: The plasma vWF level and VITRO score of the chronic hepatitis B group were (139.47±76.44) and (0.86±0.8), respectively, and the hepatitis B cirrhosis compensated group was (164.95±67.12 and 1.44±1.14), respectively. Hepatitis cirrhosis decompensated group were (317.48±103.32 and 6.81±4.98), respectively; plasma vWF level and VITRO score increased with the progression of HBV infection, and the difference was statistically significant (F=133.669,P=0.000F=137.598,P=0.000).The plasma vWF level and VITRO score in patients with hepatitis B cirrhosis were (185.65±85.07 and 2.3±2.37) in the Child-Pugh A group, (304.74±105.81 and 6.37±5.19) in the B grade group, and (369.48±73.238.28±5.38) in the C grade group; plasma vWF level and VITRO score in patients with hepatitis B cirrhosis increased with the increase of Child-Pugh grade, and the difference was statistically significant (F=60.236, P=0.000F=32.854, P=0.000). The area under the curve (AUC) of plasma vWF level and VITRO score for diagnosing the decompensated stage of hepatitis B cirrhosis were 0.897 [95% confidence interval (CI): 0.855-0.940, P＜0.01], 0.949 [95% CI: 0.916-0.982, P＜0.01). When the vWF level and VITRO score were taken as cut-off values of 238.5% and 1.65, respectively, the sensitivity of diagnosing the decompensated stage of hepatitis B cirrhosis was 79.5% and 94.3%, the specificity was 92.3% and 87.7%, and the positive predictive value was 80.5% and 94.3%, the negative predictive value was 91.9% and 97.5%, and the diagnostic accuracy was 88.6% and 89.3%. Among the patients with decompensated hepatitis B cirrhosis, the level of vWF in the group with gastrointestinal bleeding (367.24±68.29)% was significantly higher than that in the group without gastrointestinal bleeding (286.15±109.69)%, and the difference was statistically significant (P＜0.001) The VITRO score of the group with gastrointestinal bleeding (9.12±5.4) was significantly higher than that of the group without gastrointestinal bleeding (5.36±4.13), and the difference was statistically significant (P＜0.01). The vWF level in the spontaneous peritonitis group was (341.73±87.92)% higher than that in the non-spontaneous peritonitis group (296.32±111.74)%, and the difference was statistically significant (P＜0.05). There was no statistical difference in VITRO score between the two groups. significance. Conclusion: Plasma vWF level and VITRO score can evaluate the progression of liver disease and the degree of decompensation of liver cirrhosis in patients with HBV infection, and have a predictive effect on various complications after decompensation of liver cirrhosis, and have certain guiding significance for early intervention measures.
Disease Progression ; Gastrointestinal Hemorrhage/etiology* ; Hepatitis B/complications* ; Hepatitis B virus ; Hepatitis B, Chronic/diagnosis* ; Humans ; Liver Cirrhosis/virology* ; Peritonitis/complications* ; von Willebrand Factor/analysis*

The present situation of the clinical application of dog-days moxibustion (moxibustion applied in the three periods of the hot season) is summarized so as to provide the guide for the theoretic study and clinical application of dog-days moxibustion. The intervention time of dog-days moxibustion is on the 1st day of each of the three periods of the hot season. Simultaneously, the geographic factors are considered. The disorders of lung system are mostly dominant among the indications of dog-days moxibustion, complicated with spleen and stomach disorders as well as cold and deficiency syndromes/patterns. The acupoints are mainly selected from the front- points on the chest and the back- points on the back, in combination with the differentiation of diseases, symptoms/patterns and the disorder stages. The duration of treatment is ranged from 1 to 3 years. The clinical therapeutic effect is improved constantly along with the increase of treatment periods by years.
Acupuncture Points ; Acupuncture Therapy ; Humans ; Lung Diseases ; Moxibustion ; Seasons ; Splenic Diseases ; Stomach Diseases

Objective: The moisture absorption of Rhei Radix et Rhizoma (RRR) with different initial moisture content was studied under the conditions of relative humidity of 20%-85% and temperature of 5, 15, 25 and 35 ℃, respectively, so as to provide reference for the control of safe storage moisture and reasonable storage of RRR. Methods: RRR was stored at temperature of 5, 15, 25 and 35 ℃ and humidity of 45%, 60% and 75%, respectively. The samples were taken at different time points to determine its safe water activity with powder color and mildew as indicators. The isotherm adsorption data of RRR at 5, 15, 25 and 35 ℃ and water activity of 0.2-0.8 were obtained by static weighing method and six isotherm adsorption models, GAB, Oswin, Smith, Halsey, Henderso and Peleg, were used for fitting and evaluation. Results: The absolute safe water activity and relative safe water activity of RRR were 0.5 and 0.6, respectively. The adsorption isotherms of RRR at 5, 15, 25 and 35 ℃ were "S" type, which belonged to type II isotherm. Oswin model was the best fitting model and the model expression was: Meq=A[Aw/(1-Aw)B. According to the model, the absolute safe water content of RRR with different initial moisture content at 5 ℃ was 9.00%, 9.59%, 8.00%, 6.71% and relative safe water content was 10.17%, 10.89%, 9.20% and 8.07%, respectively; The absolute safe water content of RRR with different initial moisture content at 15 ℃ was 8.24%, 8.83%, 7.24%, 5.86% and relative safe water content was 9.57%, 10.17%, 8.59% and 7.20%, respectively; The absolute safe water content of that at 25 ℃ was 7.17%, 7.75%, 5.73%, 4.70% and the relative safe water content was 8.72%, 9.26%, 7.26% and 6.25%, respectively; The absolute safe water content of that at 35 ℃ was 8.00%, 8.45%, 6.53%, 5.21% and the relative safe water content was 9.74%, 9.85%, 8.40%, 7.27%, respectively. Conclusion: Oswin model can be used to predict the equilibrium moisture content of RRR in storage, which can provide reference for the control of safe moisture and scientific maintenance of RRR.

Objective: To study the correlation between the content changes of main medicinal ingredients and the color values of Rhei Radix et Rhizoma during storage based on the principle of chromaticity analysis,and to provide reference for studying on the mechanism of discoloration and improving the quality evaluation of Rhei Radix et Rhizoma. Method: Simulated accelerated test was adopted in this study, where Rhei Radix et Rhizoma was stored under high temperature(40±5)℃,high humidity RH(92.5±5)%and strong light(4 000±500)Lx conditions to accelerate its discoloration. For the samples taken at different time points,the color value was determined by spectrophotometer and the total contents of anthraquinone and free anthraquinones,sennoside A,B,catechin and gallic acid were determined by high performance liquid chromatography (HPLC). The correlation between the effective components and the color value of rhubarb was analyzed by SPSS software. Result: During the storage process,it was observed by the eye that the color of Rhei Radix et Rhizoma was significantly darker and darker in the simulated acceleration test. According to the analysis of the chromaticity value results,the changes of chromaticity values L^*and E^*ab of Rhei Radix et Rhizoma were significantly negatively correlated with free strontium content(P<0.05),and significantly positively correlated with catechin content(P<0.05),but there was no correlation with total anthraquinones and sennoside A. The chromaticity value a^* was significantly negatively correlated with gallic acid(P<0.05) and significantly positively correlated with sennoside B(P<0.05). Conclusion: There is a certain correlation between the change of color value and the content of six medicinal ingredients during Rhei Radix et Rhizoma storage.

The purpose of the present study was to investigate the effects and underlying mechanism of gonadotropin-releasing hormone agonist (GnRHa) controlled ovarian hyperstimulation (COH) on embryo implantation in mice. Forty female Kunming mice aged 9 weeks were randomly divided into two groups (control and COH groups). The COH group received intraperitoneal (i.p.) injections of aminocyclin acetate (GnRHa), human menopausal gonadotropin (HMG) and human chorionic gonadotropin (hCG), while the control group was given equal amount of physiological saline by i.p. injection. One male mouse and two female mice were put into the same cage at 16:00 on the hCG injection day, and on the fourth day of pregnancy, 10 mice from each group were killed. The levels of serum estradiol (E) and progesterone (P) were measured by radioimmunoassay; HE staining was used to observe the morphology of ovarian and endometrial tissues. The protein expression levels of endometrial leukemia inhibitory factor (LIF), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), heparin-binding epidermal growth factor-like growth factor (HB-EGF) and glycodelin A were detected by Western blot and immunohistochemistry. Ten mice from each group were sacrificed on the eighth day of pregnancy, and the status of the uterus and the average number of blastocysts were observed. The results showed that, compared with control group, the serum E level in COH group was significantly decreased (P < 0.05), while the P level was increased significantly (P < 0.05); the ovarian follicles at different developmental stages were rare, corpus lutea (CL) were visible and multiple, the endometrium was thinned, and the number of endometrial glands was reduced (P < 0.05); the contents of LIF, p-STAT3, HB-EGF and glycodelin A in the endometrium were decreased significantly (P < 0.05) on the fourth day of pregnancy; mouse blastocysts developed slowly and were decreased in number on the eighth day of pregnancy (P < 0.05). The above results suggest that GnRHa COH can affect embryo implantation in mice. The mechanism may be related to the imbalance of gonadal hormone, the changes in the structure of the endometrium and the expressions of LIF, p-STAT3, HB-EGF and glycodelin A in the implantation stage, which may lead to the decrease of endometrial receptivity and the abnormal dialogue between the embryo and the uterus.

OBJECTIVE To provide the basis for the medicinal quality standard of Chinese medicine processing accessories of rice vinegar through analysis with fingerprints of different kinds of origin and brand of rice vinegar.METHODS 33 batches of vinegar were collected.The mobile phase,column,elution,the mobile phase pH were investigated and the analysis method of HPLC fingerprint was optimazated,Among them,20 batch of rice vinegar fingerprint were chosen to perform the similari-ty,cluster analysis and principal component analysis.RESULTS The concentration of rice vinegar was diluted at 1:3 when it was injected.Chromatographic column was Thermo Hypersil GOLD(5 μm,250 mm×4.6 mm);the mobile phase was com-posed of methanol and 0.07％phosphoric acid aqueous solution and a gradient elution program at flow rate of 1 mL/min was applied.The column temperature maintained at 30 ℃.The injection volume was 10μL.Then,the separation of characteristic peaks was optimum.Rice vinegar was divided into black vinegar and white vinegar.Furthermore,there was a big difference between black vinegar and white vinegar.The white vinegar almost showed only one peak of acetic acid,so,it's impossible to distinguish between brewing white vinegar and formulated white vinegar by HPLC.Similarity evaluation showed that 20 batch of white vinegar's fingerprints had palpable difference.Besides,clustering analysis and principal component analysis splited them into several categories.Different types of Hengshun vinegar indicated high similarity.Hence it could be seen that the quality of rice vinegar from the same brand was uniform and stable because of similar brewing processes and similar materials.CONCLUSION There are varieties of rice vinegar on the market with extensive source and uneven quality.20 batches of black rice vinegar were chosen to compare the differences by HPLC fingerprints analysis,which provides reference for the medicinal quality control standard of traditional Chinese medicine processing accessories of rice vinegar.