BACKGROUND:Ursolic acid can promote the directed differentiation of bone marrow mesenchymal stem cells into osteoblasts.However,there are few reports on whether ursolic acid has osteogenic effect on human periodontal ligament stem cells. OBJECTIVE:To investigate the effect of ursolic acid on proliferation and osteogenic differentiation of human periodontal ligament stem cells. METHODS:The human periodontal ligament stem cells were isolated and cultured.Passage 3 cells were selected and treated with ordinary medium containing different concentrations(0,1,2,4,6,8 μmol/L)of ursolic acid.After intervention for 1,3,5,7 days,the cell proliferation was detected by CCK-8 assay and the appropriate intervention concentration was screened.Passage 3 human periodontal ligament stem cells were treated with osteogenic induction solution containing 0,1,2,4 μmol/L ursolic acid,respectively.After 7 days of intervention,the mRNA expressions of alkaline phosphatase,Runx2,and osteocalcin were detected by qRT-PCR.After 14 days of intervention,the formation of mineralized nodules was observed by alizarin red staining.Passage 3 human periodontal ligament stem cells were taken and the control group was added with osteogenic induction solution;the ursolic acid group and the antagonist group were added with osteogenic induction solution containing ursolic acid(2 μmol/L)and the bone morphogenetic protein signaling pathway antagonist Noggin,respectively.The ursolic acid+antagonist group was added with osteogenic induction solution containing ursolic acid(2 μmol/L)and Noggin,the inhibitor of bone morphogenetic protein signaling pathway,and cultured for 7 days.qRT-PCR and western blot assay were used to detect the mRNA and protein expressions of bone morphogenetic protein 2,Smad1,osteopontin,and Runx2. RESULTS AND CONCLUSION:(1)1,2,4 μmol/L ursolic acid could promote the proliferation of human periodontal ligament stem cells.6,8 μmol/L ursolic acid could inhibit the proliferation of human periodontal ligament stem cells,and 1,2,4 μmol/L ursolic acid was selected to intervene in subsequent experiments.(2)Compared with 0 μmol/L,1,2,4 μmol/L ursolic acid could promote the expression of alkaline phosphatase,Runx2,and osteocalcin mRNA and the formation of mineralized nodules(P<0.05),and the effect of 2 μmol/L ursolic acid was the most significant.(3)Compared with the control group,the mRNA and protein expressions of bone morphogenetic protein 2,Smad1,osteopontin,and Runx2 in the ursolic acid group were increased(P<0.05),while mRNA and protein expressions of the above indexes were decreased in the antagonist group(P<0.05).Compared with the ursolic acid group,mRNA and protein expressions of above indexes were decreased in ursolic acid+antagonist group(P<0.05).(4)The results indicate that ursolic acid promotes osteogenic differentiation of human periodontal ligament stem cells through bone morphogenetic protein signaling pathway.

Objective:To evaluate the role of O-sialoglycoprotein endopeptidase (OSGEP) in hepatic ischemia-reperfusion injury (HIRI) and the relationship with oxidative stress in mice.Methods:Experiment Ⅰ Twenty-four SPF healthy male C57BL/6 mice, 12 wild-type and 12 OSGEP knockdown, aged 6-8 weeks, weighing 18-22 g, were divided into 4 groups ( n=6 each) by the random number table method: wild-type shamoperation group (Sham group), wild-type HIRI group (HIRI group), OSGEP knockdown+ sham operation group (Sham+ KD group) and OSGEP knockdown+ HIRI group (HIRI+ KD group). Ischemia-reperfusion model was prepared by blocking the hepatic artery and portal vein for 60 min followed by reperfusion in anesthetized animals, the blood vessels were only exposed without occlusion in Sham group and Sham+ KD group, and the blood vessels were clamped for 60 min followed by reperfusion in HIRI group and HIRI+ KD group. The mice were sacrificed after 6-h reperfusion to extract liver tissue samples for microscopic examination of histopathological changes (with an optical microscope after HE staining) which were evaluated using Suzuki score and for determination of the serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), level of reactive oxygen species (ROS) (using the DCFH-DA fluorescent probe method), contents of malondialdehyde (MDA) and glutathione(GSH) in liver tissues (using a colorimetric method) and expression of OSGEP (using Western blot). Experiment Ⅱ The well-growing AML12 cells were divided into 4 groups ( n=30 each) using a random number table method: control group (C group), oxygen-glucose deprivation/restoration (OGD/R) group, OGD/R+ OSGEP knockdown group (OGD/R+ KD group), and OGD/R+ OSGEP knockdown negative control group (OGD/R+ NC group). Group C was cultured under normal conditions. Group OGD/R was subjected to O 2-glucose deprivation for 6 h followed by restoration of O 2-glucose supply for 24 h in OGD/R group. In OGD/R+ KD group, stable transfection of AML12 cells with OSGEP knockdown was performed prior to the experiment, and the other procedures were the same as those previously described. The cell survival rate was measured by the CCK-8 assay, the release of lactate dehydrogenase (LDH) was measured, the DCFH-DA method was used to detect the levels of ROS, and the contents of MDA and GSH were determined using a colorimetric method. Results:Experiment Ⅰ Compared with Sham group, the expression of OSGEP was significantly down-regulated, the serum concentrations of AST and ALT, Suzuki score, levels of ROS and content of MDA were increased, and the GSH content was decreased in HIRI group ( P<0.05), and no significant change was found in each parameter in Sham+ KD group ( P>0.05). Compared with HIRI group, the serum concentrations of AST and ALT, Suzuki score, levels of ROS and content of MDA were significantly increased, and the GSH content was decreased in HIRI+ KD group ( P<0.05). Experiment Ⅱ Compared with group C, the expression of OSGEP was significantly down-regulated, the cell survival rate and GSH content were decreased, and the release of LDH, levels of ROS and content of MDA were increased in group OGD/R ( P<0.05). Compared with OGD/R group, the cell survival rate and GSH content were significantly decreased, and the release of LDH, levels of ROS and content of MDA were increased in OGD/R+ KD group ( P<0.05), and no significant change was found in each parameter in OGD/R+ NC group ( P>0.05). Conclusions:OSGEP plays an endogenous protective role in HIRI by inhibiting oxidative stress in mice.

Objective To investigate the current status and awareness of pharmaceutical services in hospitals in Guizhou province and to provide a reference for exploring and carrying out pharmaceutical service fees.Methods The questionnaire was designed by the"wjx.cn"website.Three kinds of questionnaires were designed for pharmacists,doctors,nurses,and patients as the research objects,with corresponding differences in some questions,and promoted on WeChat,Dingxiangyuan,and other network platforms.Results A total of 655 questionnaires were collected,and 639 valid questionnaires were recovered,with an effective recovery rate of 97.56%.324 pharmacists(50.70%),82 doctors and nurses(12.83%),233 patients(36.46%)were surveyed.The average approval score of these three groups of respondents on pharmaceutical service fees was 4.67,4.23,and 4.22,respectively(full score:5).Conclusions Overall,pharmacists'professional services have received support from medical staff and patients.However,patients'pharmaceutical service projects currently focus on dispensing services.The recognition of pharmacists'work and the public's awareness of pharmaceutical services can be improved by enhancing the professional ability of pharmacists,strengthening publicity and guidance,and exploring"Internet+pharmaceutical services",etc.,to promote the sustainable development of pharmaceutical services.

Objective To study the expression of SWI/SNF-related,matrix-associated,actin-depend-ent regulator of chromatin,subfamily A,member 4(SMARCA4)/Brahma-related gene 1,V-raf murine sarco-ma viral oncogene homolog B(BRAF),P53,programmed cell death protein-1(PD-1),and programmed death-ligand 1(PD-L1),and changes in the expression of BRAF and neurotrophic tyrosine receptor kinase(NTRK)in the patients with colorectal cancer in Tibet,thereby providing a basis for targeted therapy and immunotherapy for this disease in Tibet.Methods A total of 64 patients with colorectal cancer resected in the Tibet Autonomous Region People's Hospital from January 2015 to July 2021 were enrolled in this study.The expression of SMARCA4,BRAF,P53,PD-1,and PD-L1 was detected by immunohistochemical staining.The gene fusion involving NTRK1,NTRK2,and NTRK3 was detected by fluorescence in situ hybridization,and the BRAF V600E gene mutation by polymerase chain reaction.Results The 64 patients with colorectal cancer were at a male-to-female ratio of 1.21∶1,with the mean age of(56.59±13.27)years.The tumors were located in the co-lon in 46(71.88%)patients and in the rectum in 18(28.12%)patients.Sixty(93.75%)patients presented adenocarcinoma,and 4(6.25%)patients presented other types of tumors.The patients in T1/T2 and T3/T4 phases accounted for 17.19%(n =11)and 82.81%(n =53),respectively.Lymph node metastasis occurred in 24(37.50%)patients.The immunohistochemical staining results showed partially down-regulated or absent ex-pression of SMARCA4 in 1(1.56%)patient,positive BRAF expression in 4(6.25%)patients,and mutant expression of P53 in 35(54.69%)patients.The PD-1-expressing tumor associated immune cell was proportion score<10%in 45(70.31%)patients and≥10%in 19(29.69%)patients.The PD-L1 combined positive score was<10 in 52(81.25%)patients and≥10 in 12(18.75%)patients.The gene fusion of NTRK1,NTRK2,and NTRK3 was negative in all the patients,and BRAF V600E gene mutation was positive in 4(6.25%)patients.The SMARCA4 gene alteration was not detected in the patient with partial expression missing of SMARCA4.The PD-L1 combine positive score was correlated with the deficient mismatch repair(dMMR)/mic-rosatellite instability-high(MSI-H)and the PD-1 expression(χ2 = 10.223,P = 0.001;χ2 = 11.979,P = 0.001).Conclusions The down-regulated or absent SMARCA4 expression and NTRK gene fusion are rare in the patients with colorectal cancer in Tibet.A few patients present BRAF V600E gene mutations,and Pan-TRK and BRAF expression can be used for the primary screening of NTRK gene fusion and BRAF gene mutation.The patients with dMMR/MSI-H are prone to high expression of PD-L1 and expected to benefit from immunothera-py.No significant correlation exists between P53 mutation and PD-L1 expression.The high expression of PD-1 is positively correlated with the high expression of PD-L1.

Objective:To investigate the efficacy of the intelligent version of the Montreal Cognitive Assessment Scale(MoCA)in identifying mild cognitive impairment(MCI)in elderly inpatients.Methods:Seventy-five patients diagnosed with mild cognitive impairment(MCI group)from the Neurology and Geriatrics Departments of a tertiary hospital in Changsha City, along with 195 patients with normal cognitive function(normal control group), were selected between July 2020 and December 2022.Both groups underwent evaluations using the intelligent version of the Montreal Cognitive Assessment(MoCA)and the Mini Mental State Examination(MMSE), and the results were analyzed.Results:The MCI group showed significantly lower total MoCA scores and MMSE scores[(19.73±2.42) vs.(24.8±2.33)]compared to the normal control group[(24.47±2.02) vs.(26.50±1.65)], with a statistically significant difference( P<0.01).The optimal cut-off score for the MoCA was found to be 23.5 points in diagnosing MCI, showing higher sensitivity, specificity, and kappa value(96.0%, 82.1%, and 0.690)compared to the MMSE scale(90.0%, 62.6%, and 0.428). Conclusions:The intelligent MoCA has a high screening accuracy for identifying MCI in this population, demonstrating superior sensitivity and specificity compared to the MMSE.

Nuclear receptor corepressor (NCoR1) interacts with various nuclear receptors and regulates the anabolism and catabolism of lipids. An imbalance in lipid/energy homeostasis is also an important factor in obesity and metabolic syndrome development. In this study, we found that the deletion of NCoR1 in intestinal epithelial cells (IECs) mainly activated the nuclear receptor PPARα and attenuated metabolic syndrome by stimulating thermogenesis. The increase in brown adipose tissue thermogenesis was mediated by gut-derived tricarboxylic acid cycle intermediate succinate, whose production was significantly enhanced by PPARα activation in the fed state. Additionally, NCoR1 deletion derepressed intestinal LXR, increased cholesterol excretion, and impaired duodenal lipid absorption by decreasing bile acid hydrophobicity, thereby reversing the possible negative effects of intestinal PPARα activation. Therefore, the simultaneous regulatory effect of intestinal NCoR1 on both lipid intake and energy expenditure strongly suggests that it is a promising target for developing metabolic syndrome treatment.

Objective: To investigate the distribution of various types of screen time and examine the association of screen time with psychological and behavioral development problems in children aged 3-6 years, so as to provide scientific basis for children s screen use and mental health promotion. Methods: A total of 3 875 mother child dyads who completed the follow up in Shanghai Maternal-Child Pairs Cohort were included. The daily usage time of children s tablet, mobile phone, TV, projectors, and other types of screens were obtained in questionnaire survey. Children s psychological and behavioral development problems were evaluated by Age-Stage Questionnaire, Third Edition (ASQ-3) and Strengths and Difficulties Questionnaire (Parent version) (SDQ). The Chi-square test, Mann Whitney U and Kruskal Wallis rank sum test were used to compare the detection rate of psychological and behavioral development problems and screen time in children with different characteristics. Multivariate binary Logistic regression was used to analyze the association of screen time with psychological and behavioral development problems. Results: There were 49.91% of children having screen time more than 1 h/d. Children s TV, tablet and mobile phone screen time were 0.39(0.25, 0.96 ), 0.25(0,0.61) and 0.18(0,0.25) h/d. The detection rates of suspected developmental delay in fine motor, problem solving and personal-social domains and pro social behavior deficiency and externalizing behaviors in boys (8.54%, 6.77%, 5.46%, 30.07 %, 27.39%) were higher than that in girls (4.64%, 4.85%, 2.48%, 22.10%, 22.36%) ( χ 2=23.76, 6.49, 22.37, 31.81, 13.06, P <0.05). There were statistically significant differences in the detection rates of suspected developmental delay in communication, fine motor and problem solving, as well as internalizing behavior and externalizing behavior of children with different parents educational levels ( χ 2=14.37, 15.18, 21.10, 11.66, 9.27; 16.34, 26.75, 32.89, 16.97, 6.37, P <0.05). There were significant differences in the detection rates of suspected developmental delay in problem solving, prosocial behavior deficiency, internalizing behavior and externalizing behavior of children whose mothers had anxiety/depression symptoms during pregnancy ( χ 2= 5.61 , 9.05, 21.90, 7.17; 8.75, 6.06, 12.76 , 5.55, P <0.05). The average total screen time of boys was longer than that of girls (1.07, 1.00 h/d, Z=-2.08, P =0.04). Compared with children with other educational levels of their parents, the total screen time, mobile phone and TV screen time of children whose parents had college education or above were short (father: H =42.01, 44.49 , 21.24, mother: H =42.31, 39.21 , 26.47, P <0.01). Among all types of screen time, mobile phone screen time had the most impact on psychological and behavioral development. More mobile phone screen time increased the risk of suspected developmental delay and abnormal emotional behavior ( P < 0.05). Screen time of tablet, mobile phone and TV were positively correlated with externalizing behavior ( OR=1.36, 1.57, 1.27 , P <0.05). Conclusions Screen time is related to children s psychological and behavioral development problems and mobile phones affect the most. Parents should limit their children s screen time to avoid excessive screen time affecting their psychological and behavioral development.

Objective:To understand the current situation of early mobilization in elder patients with acute ischemic stroke, and to analyze the influencing factors of early mobilization in patients with different degrees of neurological deficits, aiming to provide evidence for promoting early mobilization in patients with stroke and for formulating intervention strategies.Methods:Patients with acute ischemic stroke admitted to the stroke centers of two class A tertiary hospitals in Hunan Province between January and December 2021 were enrolled.The degree of neurological impairment, activities of daily living and muscle strength were evaluated within 24 hours after admission, and patients' performance of early mobilization was recorded.Based on whether patients performed out-of-bed activities such as sitting at the bedside, standing or walking, transferring from bed to chair within 48 hours after admission, they were divided into an early ambulation group and a non-early ambulation group.Univariate analysis and Logistic regression analysis were then used to examine the influencing factors of early ambulation in patients with mild to moderate neurological deficits.Results:Among 257 patients with acute ischemic stroke, 60.7%(156 cases)conducted early mobilization.The rate of early mobilization for patients with moderate neurological deficits was only 12.9%(12/193), significantly lower than that for patients with mild neurological deficits(87.8%, 144/164)( χ2=139.571, P<0.001).Univariate analysis showed that there were significantly differences in the level of activities of daily living, stroke history, strength of the upper and lower limbs of the affected side between the early and non-early ambulation groups with moderate neurological impairment(all P<0.05), and there were significant differences in the level of activities of daily living between the early ambulation group and the non-early ambulation group in patients with mild neurological impairment( χ2=11.907, P<0.001).Logistic regression analysis showed that requiring assistance in daily life( OR=4.189, 95% CI: 1.724-10.668, P=0.002), muscle strength of the affected lower extremity ≤ grade 3( OR=9.605, 95% CI: 2.627-35.113, P=0.001)and moderate neurological deficits( OR=8.585, 95% CI: 3.216-22.913, P<0.001)were the risk factors of early mobilization in stroke patients. Conclusions:The rate of stroke patients with moderate neurological impairment performing early mobilization is low within 48 hours after admission.Activities of daily living ability, limb muscle strength and degrees of neurological deficits are influencing factors of early mobilization in stroke patients.

Objective:To evaluate the role of extracellular signal-regulated kinase (ERK)1/2 in glutamate-induced ferroptosis in PC12 cells.Methods:PC12 cells were divided into 6 groups ( n=21 each) using a random number table method: control group (C group), glutamategroup (Glu group), glutamate+ ERK1/2 over-expression group (Glu+ ERK1/2-OE group), glutamate+ ERK1/2 plasmid empty vector group (Glu+ Vec group), glutamate+ ERK1/2 knockdown group (Glu+ si-ERK1/2 group)and glutamate+ ERK1/2 SiRNA negative control group (Glu+ si-NC group). Cells were treated with glutamate at a final concentration of 6 mmol/L for 72 h in Glu group and with the equal volume of PBS buffer for 72 h in C group. Glu+ ERK1/2-OE group was transfected with ERK1/2 overexpression plasmid, Glu+ Vec group was transfected with plasmid empty vector, and Glu+ si-ERK1/2 group was transfected with ERK1/2 siRNA, Glu+ si-NC group was transfected with siRNA negative control for 48 h, and then glutamate at a final concentration of 6 mmol/L was added and cells were treated for 72 h. The cell viability, lactic dehydrogenase (LDH)activity and contents of glutathione (GSH), ferrous ions and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay. Mitochondrial membrane potential (MMP) and lipid reactive oxygen species (Lip-ROS) were measured by flow cytometry. Results:Compared with C group, the cell viability, GSH content and MMP were significantly decreased, and the LDH activity, ferrous ions content, MDA content and Lip-ROS levels were increased in Glu group ( P<0.05). Compared with Glu+ Vec group, the cell viability, GSH content and MMP were significantly increased, and the activity of LDH, contents of ferrous ions and MDA, and Lip-ROS levels were decreased in Glu+ ERK1/2-OE group( P<0.05). Compared with Glu+ si-NC group, the cell viability, GSH content and MMP were significantly decreased, and the LDH activity, contents of ferrous ions and MDA, and Lip-ROS level were increased in Glu+ si-ERK1/2 group ( P<0.05). Conclusions:ERK1/2 is involved in glutamate-induced ferroptosis in PC12 cells.

Objective:To investigate the clinical and genetic features of patients with ACTL6B gene variations, and to report novel pathogenic variations of the ACTL6B gene, summarize the clinical phenotypes and genotypes of the gene. Methods:The clinical phenotypes and genotypes of a infant with developmental epileptic encephalopathy carrying the ACTL6B gene variations, who visited the Department of Neurology, Capital Institute of Pediatrics-Peking University Teaching Hospital on March 12, 2021 were analyzed. The phenotypes and genotypes of patients carrying the ACTL6B gene variations reported in the literature were also summarized and analyzed. Results:The proband was a 2-month-old male presented with convulsive seizures, development delay, dystonia, and cherry erythema in the fundus. The whole exome sequencing of his family showed that he carried compound heterozygous variation c.937-2A>G(p.?), c.11delG(p.G4Afs *86) which derived from his parents respectively. These 2 genotypes had not been reported. A total of 42 cases with ACTL6B gene variation were reported in the literature and in this study. There were 11 de novo heterozygous variations and 31 bi-allelic variations inherited from the parents (24 homozygous and 7 compound heterozygous). Individuals with variations tended to have epilepsy, development delay, ambulation disability, speech disability and dystonia. Minor facial dysmorphisms and autism spectrum disorder also can be seen. Conclusion:This paper summarizes the clinical and genetic features of patients with ACTL6B gene variations, reports 2 novel variations and a novel combination of this gene with cherry erythema in the fundus.