The present study employed UPLC-MS/MS to analyze and identify compounds in the raw powder of Tongluo Shenggu capsules. An HPLC method for the determination of vitexin content was established. The analysis of this drug was performed on a 30 ℃ thermostatic Acquity UPLC® BEH C18 (2.1 mm×100 mm,1.7 μm) column, with the mobile phase comprising 0.2% formic acid-methanol flowing at 0.3 mL /min in a gradient elution manner. Mass spectrometry was detected by ESI sources in both positive and negative ion modes for qualitative identification of chemical constituents. 12 flavonoid and 3 stilbenes compounds in the raw powder of Tongluo Shenggu capsules were successfully identified. Additionally, an HPLC method for the determination of vitexin content was established using a XBridge C18 column (4.6 mm × 250 mm, 5 µm) with a mobile phase of 0.05% glacial acetic acid in methanol for gradient elution, at a column temperature of 30 °C, a flow rate of 1.0 mL/min, and an injection volume of 20 μL. The method demonstrated good linearity in the concentration range of 10 µg/mL to 40 µg/mL (R=1.000) with an average recovery rate of 96.7%. The establishment of these methods provides a scientific basis for the quality control and development of the raw powder of Tongluo Shenggu capsules.

Angle class Ⅱ malocclusion is often characterized by mandibular retraction and lip incompetence,which affects the patient's lateral appearance and may even lead to upper airway stenosis.It can be classified into dental and skeletal types.For skeletal class Ⅱ malocclusion patients with mandibular retraction during the peak growth period,mandibular anterior guidance with a functional orthodontic appliance is generally considered as the optimal clinical treatment approach.At present,there remains a paucity of clinical reports on the clinical application of bracket-free clear aligners in mandibular anterior guidance,both domestically and internationally.This article presented a case of an adolescent patient with skeletal class Ⅱ malocclusion accompanied with deep overbite treated with bracket-free clear aligner for mandibular anterior guidance in combination with intermaxillary class Ⅱ traction.During the treatment,vertical correction involved anterior intrusion of the anterior teeth to improve the deep overbite,while horizontal correction included maxillary and mandibular expansion to coordinate the width of the dental arches,and asymmetric anterior guidance was used to correct the midline deviation.After 35 months of treatment,the patient's convex facial profile and mandibular retrusion were significantly improved.The subspinale-nasion-supramentale angle(ANB)was decreased from 6.8° to 3.9°,the overbite and overjet were normalized,and the bilateral canine and molar reached a neutral relationship.The mentolabial sulcus depth(Si-LiPg′)and the soft tissue thickness of pogonion to pogonion(Pm-Pm′)were decreased,resulting in a shallower mentolabial sulcus and a more harmonized lateral facial soft tissue profile.The mandibular incisor to mandibular plane angle(IMPA)was decreased from 116.6° to 110.7°,indicating retraction of the lower incisors during mandibular anterior guidance.In conclusion,the orthodontic strategy of mandibular advancement with clear aligners in skeletal class Ⅱ malocclusion patients can avoid excessive overcompensation of the upper and lower anterior teeth and shorten the orthodontic treatment cycle.

Objective:To explore the clinical and pathological features and survival outcomes of patients with early-onset intrahepatic cholangiocarcinoma (EOICC).Methods:This is a multicenter, retrospective cohort study. Data of 1 160 intrahepatic cholangiocarcinoma patients undergoing radical resection in 14 tertiary Grade A hospitals in China from January 2010 to November 2021 were retrospectively collected. The cohort included 632 males and 528 females, aged( M (IQR)) 61 (14) years (range: 22 to 93 years). ICC aged ≤50 years at the time of diagnosis was defined as EOICC and >50 years as late-onset intrahepatic cholangiocarcinoma (LOICC). Of these, there were 247 cases in the EOICC group and 913 cases in the LOICC. The clinical and pathological characteristics of both groups were analyzed and compared using the independent sample t-test, Mann-Whitney U test or Kaplan-Meier method. Univariate and multivariate Cox regression models for patient outcomes were constructed and forest graphed. Results:Compared with the patients in the LOICC group, patients in the EOICC group had lower carcinoembryonic antigen levels (2.5(4.0) μg/L vs. 3.1(5.2)μg/L, U=124 899, P=0.009) and CA19-9 level (63.4(524.7)U/ml vs. 77.9(611.3)U/ml, U=120 320, P=0.013), higher levels of ALT (29(35)U/L vs. 24(26)U/L, U=101 214, P=0.013), a lower score of the Eastern US Cooperative Oncology Group (0 score patients: 54.7% vs. 44.1%, χ2=12.472, P=0.014), higher TNM stage ( χ2=11.807, P=0.038), and proportion of lymph node dissection (62.3% vs. 54.1%, χ2=5.355, P=0.021). Patients in the two groups in sex, first diagnosis symptoms, intrahepatic bile duct stone history, nail protein, albumin, total bilirubin, transaminase, liver function Child-Pugh grade, T stage, stage, N stage, preoperative laparoscopic exploration proportion, tumor diameter, vascular invasion proportion, differentiation, margin, intraoperative bleeding, postoperative complications, postoperative hospital days were no statistical significance (all P>0.05). Patients in the EOICC group had better outcomes than the LOICC group (median survival time: 29.7 months vs. 25.0 months, 3-year overall survival: 45.1% vs. 37.8%, P=0.027). Conclusion:EOICC patients are better than LOICC patients in carcinoembryonic antigen, CA19-9, ALT, physical strength status and TNM stage, and the long-term prognosis is also better than LOICC patients.

Objective:To explore the clinical and pathological features and survival outcomes of patients with early-onset intrahepatic cholangiocarcinoma (EOICC).Methods:This is a multicenter, retrospective cohort study. Data of 1 160 intrahepatic cholangiocarcinoma patients undergoing radical resection in 14 tertiary Grade A hospitals in China from January 2010 to November 2021 were retrospectively collected. The cohort included 632 males and 528 females, aged( M (IQR)) 61 (14) years (range: 22 to 93 years). ICC aged ≤50 years at the time of diagnosis was defined as EOICC and >50 years as late-onset intrahepatic cholangiocarcinoma (LOICC). Of these, there were 247 cases in the EOICC group and 913 cases in the LOICC. The clinical and pathological characteristics of both groups were analyzed and compared using the independent sample t-test, Mann-Whitney U test or Kaplan-Meier method. Univariate and multivariate Cox regression models for patient outcomes were constructed and forest graphed. Results:Compared with the patients in the LOICC group, patients in the EOICC group had lower carcinoembryonic antigen levels (2.5(4.0) μg/L vs. 3.1(5.2)μg/L, U=124 899, P=0.009) and CA19-9 level (63.4(524.7)U/ml vs. 77.9(611.3)U/ml, U=120 320, P=0.013), higher levels of ALT (29(35)U/L vs. 24(26)U/L, U=101 214, P=0.013), a lower score of the Eastern US Cooperative Oncology Group (0 score patients: 54.7% vs. 44.1%, χ2=12.472, P=0.014), higher TNM stage ( χ2=11.807, P=0.038), and proportion of lymph node dissection (62.3% vs. 54.1%, χ2=5.355, P=0.021). Patients in the two groups in sex, first diagnosis symptoms, intrahepatic bile duct stone history, nail protein, albumin, total bilirubin, transaminase, liver function Child-Pugh grade, T stage, stage, N stage, preoperative laparoscopic exploration proportion, tumor diameter, vascular invasion proportion, differentiation, margin, intraoperative bleeding, postoperative complications, postoperative hospital days were no statistical significance (all P>0.05). Patients in the EOICC group had better outcomes than the LOICC group (median survival time: 29.7 months vs. 25.0 months, 3-year overall survival: 45.1% vs. 37.8%, P=0.027). Conclusion:EOICC patients are better than LOICC patients in carcinoembryonic antigen, CA19-9, ALT, physical strength status and TNM stage, and the long-term prognosis is also better than LOICC patients.

AIM:The aim of this study was to investigate the effects and mechanism of high fat on autolyso-somes in hepatoma cells before and after serum deprivation.METHODS:HepG2 cells were intervened with 1 mmol/L so-dium oleate to create a cell high-fat model.The gene expression of transcription factor EB(TFEB)in HepG2 cells was knocked down using TFEB small interfering RNA(TFEB-siRNA)transfection reagent.AMP-activated protein kinase(AMPK)inhibitor compound C(CC)was used to inhibit AMPK phosphorylation expression in HepG2 cells.The expres-sion of nuclear and cytoplasmic TFEB,lysosome-associated membrane protein 1(LAMP1),microtubule-associated pro-tein light chain 3(LC3),autophagy adaptor protein(p62),AMPK,and p-AMPK proteins in each group was analyzed through Western blot experiments.Lipid metabolism and liver function damage in each group were analyzed using total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),and aspartate aminotransferase(AST)assay kits.The accumulation of lipid droplets in each group of cells was analyzed through oil red O staining.RESULTS:(1)Sodium oleate intervention resulted in a concentration-dependent decrease in the protein expression levels of LAMP1,LC3-Ⅱ/LC3-Ⅰ,and nuclear TFEB,while increasing the protein expression level of p62(P＜0.01).(2)Compared to the NC group,the sodium oleate group showed decreased expression of LAMP1,LC3-Ⅱ/LC3-Ⅰ,nuclear TFEB,and AMPK phosphorylation levels,with an increase in p62 expression.Compared to the sodium oleate group,the sodium oleate+serum deprivation combined intervention group showed increased nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,AMPK phosphorylation levels,and decreased p62 expression levels(P＜0.05).(3)The levels of TC,TG,LDL-C,ALT,and AST were increased in the sodium oleate group compared to the NC group.Serum deprivation reduced the number of lipid droplets induced by sodium oleate in HepG2 cells and decreased the levels of TC,TG,LDL-C,ALT,and AST compared to the sodium oleate group(P＜0.05).(4)Knockdown of TFEB did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,TC,TG,LDL-C,ALT and AST compared to the so-dium oleate group.(5)Inhibition of AMPK phosphorylation did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,and AMPK phosphorylation in the sodium oleate+serum deprivation group com-pared to the sodium oleate group.CONCLUSION:Serum deprivation improves sodium oleate-induced lipid metabolism damage in HepG2 cells through the autophagolysosome pathway mediated by AMPK-TFEB.

AIM:The aim of this study was to investigate the effects and mechanism of high fat on autolyso-somes in hepatoma cells before and after serum deprivation.METHODS:HepG2 cells were intervened with 1 mmol/L so-dium oleate to create a cell high-fat model.The gene expression of transcription factor EB(TFEB)in HepG2 cells was knocked down using TFEB small interfering RNA(TFEB-siRNA)transfection reagent.AMP-activated protein kinase(AMPK)inhibitor compound C(CC)was used to inhibit AMPK phosphorylation expression in HepG2 cells.The expres-sion of nuclear and cytoplasmic TFEB,lysosome-associated membrane protein 1(LAMP1),microtubule-associated pro-tein light chain 3(LC3),autophagy adaptor protein(p62),AMPK,and p-AMPK proteins in each group was analyzed through Western blot experiments.Lipid metabolism and liver function damage in each group were analyzed using total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),and aspartate aminotransferase(AST)assay kits.The accumulation of lipid droplets in each group of cells was analyzed through oil red O staining.RESULTS:(1)Sodium oleate intervention resulted in a concentration-dependent decrease in the protein expression levels of LAMP1,LC3-Ⅱ/LC3-Ⅰ,and nuclear TFEB,while increasing the protein expression level of p62(P＜0.01).(2)Compared to the NC group,the sodium oleate group showed decreased expression of LAMP1,LC3-Ⅱ/LC3-Ⅰ,nuclear TFEB,and AMPK phosphorylation levels,with an increase in p62 expression.Compared to the sodium oleate group,the sodium oleate+serum deprivation combined intervention group showed increased nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,AMPK phosphorylation levels,and decreased p62 expression levels(P＜0.05).(3)The levels of TC,TG,LDL-C,ALT,and AST were increased in the sodium oleate group compared to the NC group.Serum deprivation reduced the number of lipid droplets induced by sodium oleate in HepG2 cells and decreased the levels of TC,TG,LDL-C,ALT,and AST compared to the sodium oleate group(P＜0.05).(4)Knockdown of TFEB did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,TC,TG,LDL-C,ALT and AST compared to the so-dium oleate group.(5)Inhibition of AMPK phosphorylation did not result in significant changes in the levels of nuclear TFEB,LAMP1,LC3-Ⅱ/LC3-Ⅰ,p62,and AMPK phosphorylation in the sodium oleate+serum deprivation group com-pared to the sodium oleate group.CONCLUSION:Serum deprivation improves sodium oleate-induced lipid metabolism damage in HepG2 cells through the autophagolysosome pathway mediated by AMPK-TFEB.

Methods: In this study, 267 matched pairs of AIS and controls were recruited. The participants underwent EMG measurements at their first presentation and did not receive any treatment for 6 months at which point they underwent EMG and radiographs. Early curve progression was defined as >5° in Cobb angle at 6 months. The root mean square of the EMG (rms-EMG) signal was recorded with the participants in sitting and back extension. The rms-EMG ratio at the upper end vertebrae, apical vertebrae (AV), and lower end vertebrae (LEV) of the major curve was calculated. Results: The rms-EMG ratio in the scoliosis cohort was high compared with that in the controls (sitting: 1.2±0.3 vs. 1.0±0.1, p<0.01; back extension: 1.1±0.2 vs. 1.0±0.1, p<0.01). An AV rms-EMG ratio in back extension, with a cutoff threshold of ≥1.5 in the major thoracic curve and ≥1.3 in the major lumbar curve, was a risk factor for early curve progression after 6 months without treatment (odds ratio, 4.1; 95% confidence interval, 2.8–5.9; p<0.01). Increases in side deviation (SD) (distance between the AV and the central sacral line) were related to a higher rms-EMG ratio in LEV of the major thoracic curve (baseline: rs=0.2, p=0.03; 6 months: rs=0.3, p<0.01). Conclusions An EMG discrepancy was detected in the scoliosis cohort, which was related to increases in SD in the major thoracic curve. The AV rms-EMG ratio in back extension was correlated with curve progression after 6 months of no treatment.

To develop a medication reminder based on Android platform, which can be used to set up personalized reminders quickly and record the patient' s medication time-stamp and related information. In addition, the effect of using the reminder on medication compliance was studied. 14 subjects participated in the evaluation of medica-tion compliance. The differences of dosage and medication rate between the group using and without using reminder were investigated. The dosage and medication rate were got by the pill counting. The Wilcoxon matched -pairs signed rank test showed that the dosage and the medication rate of the group using reminder were higher than that without using reminder significantly(Z= -2. 93, P =0. 003 ). The results demonstrated that the use of the re-minder can improve the medication dose and rate significantly,indicate that the reminder might be a new technical support for clinical research on medication compliance, and also might reduce the workload for medical staff and patients.

Extracellular matrix (ECM) keeps cell's shape, protects and nourishes cells; it plays a great role in cell proliferation and differentiation. Therefore, ECM is very important in cell and tissue engineering. In this study, after primary mouse osteoblasts and fibroblasts maintained at confluence in vitro were removed, their ECM coated on cell culture plate was prepared, and bone morphogenetic proteins 2 (BMP-2) was detected in the osteoblasts ECM. MC3T3-E1 preosteoblasts cells were seeded on cell culture plates covered with fibroblasts ECM and osteoblasts ECM respectively. The proliferative activity of the cells cultured on fibroblasts ECM was higher than that on osteoblasts ECM and the control group. The alkaline phosphatase activity, relative protein levels of BMP-2 and osteopontin, secreted calcium of the cells cultured on osteoblasts ECM were all the highest. The results indicate that the two different ECMs produced in vitro had different bioactivities, the fibroblasts ECM coated on cell culture plates could accelerate MC3T3-E1 cells proliferation, and the osteoblasts ECM could promote cells osteogenic differentiation.
3T3 Cells ; Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Bone Morphogenetic Protein 2 ; metabolism ; Calcium ; metabolism ; Cell Differentiation ; physiology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Matrix ; metabolism ; physiology ; Mice ; Osteoblasts ; cytology ; Osteopontin ; metabolism

BACKGROUND: Seed cell exerting its function is required to depend on the extracellular matrix in tissue engineering, so that biocompatible material is important to be selected. OBJECTIVE: To prepare a novel composite scaffolds of chitosan/collagen/nano-hydroxyapatite (HA-CS/Col/nHAP) and to optimize the technology of tissue engineered-stents according to the circumstances of cell adhesion.METHODS: Chitosan was modified by hyaluronate acid. The structure was observed by differential scanning calorimetry and the Fourier transformed infrared spectroscopy. Three composites of HA-CS/Col/nHAP according to different ratio of chitosan and collagen solution (1: 2; 1: 1 and 2: 1) were prepared. The composite scaffolds were co-cultured with osteoblast MC3T3-E1, and the proliferation and cell growth curve were measured by CCK-8 method. RESULTS AND CONCLUSION: Hyaluronic acid and chitosan were crosslinked with amide linkage. Pore size was on the range from 50 μm to 250 μm. Porosity was increased with increased collagen level and elastic modulus, but density was reduced. Increased collagen content was beneficial for cell adhesion and proliferation on stent in the primary phase of cell co-culture. However, from day 10, no significant difference was determined among three samples. At the beginning of cell culture, cells adhered to the airspace insides the composite scaffolds. In the following days, cells grew in a colony manner, and cell-cell junction could be easily observed. These indicate that HA-CS/Col /nHAP composite scaffolds can improve the adhesion and proliferation of osteoblast. The ratio of chitosan to collagen volume at 1: 1 was optimal.