ObjectiveTo investigate the mechanism of action of Kaixinsan in the treatment of Alzheimer's disease （AD） based on network pharmacology， molecular docking， and animal experimental validation. MethodsThe Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP） and the Encyclopedia of Traditional Chinese Medicine（ETCM） databases were used to obtain the active ingredients and targets of Kaixinsan. GeneCards， Online Mendelian Inheritance in Man（OMIM）， TTD， PharmGKB， and DrugBank databases were used to obtain the relevant targets of AD. The intersection （common targets） of the active ingredient targets of Kaixinsan and the relevant targets of AD was taken， and the network interaction analysis of the common targets was carried out in the STRING database to construct a protein-protein interaction（PPI） network. The CytoNCA plugin within Cytoscape was used to screen out the core targets， and the Metascape platform was used to perform gene ontology（GO） functional enrichment analysis and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment analysis. The “drug-active ingredient-target” interaction network was constructed with the help of Cytoscape 3.8.2， and AutoDock Vina was used for molecular docking. Scopolamine （SCOP） was utilized for modeling and injected intraperitoneally once daily. Thirty-two male C57/BL6 mice were randomly divided into blank control （CON） group （0.9% NaCl， n=8）， model （SCOP） group （3 mg·kg^-1·d^-1， n=8）， positive control group （3 mg·kg^-1·d^-1 of SCOP+3 mg·kg^-1·d^-1 of Donepezil， n=8）， and Kaixinsan group （3 mg·kg^-1·d^-1 of SCOP+6.5 g·kg^-1·d^-1 of Kaixinsan， n=8）. Mice in each group were administered with 0.9% NaCl， Kaixinsan， or Donepezil by gavage twice a day for 14 days. Morris water maze experiment was used to observe the learning memory ability of mice. Hematoxylin-eosin （HE） staining method was used to observe the pathological changes in the CA1 area of the mouse hippocampus. Enzyme linked immunosorbent assay（ELISA） was used to determine the serum acetylcholine （ACh） and acetylcholinesterase （AChE） contents of mice. Western blot method was used to detect the protein expression levels of signal transducer and activator of transcription 3（STAT3） and nuclear transcription factor（NF）-κB p65 in the hippocampus of mice. ResultsA total of 73 active ingredients of Kaixinsan were obtained， and 578 potential targets （common targets） of Kaixinsan for the treatment of AD were screened out. Key active ingredients included kaempferol， gijugliflozin， etc.. Potential core targets were STAT3， NF-κB p65， et al. GO functional enrichment analysis obtained 3 124 biological functions， 254 cellular building blocks， and 461 molecular functions. KEGG pathway enrichment obtained 248 pathways， mainly involving cancer-related pathways， TRP pathway， cyclic adenosine monophosphate（cAMP） pathway， and NF-κB pathway. Molecular docking showed that the binding of the key active ingredients to the target targets was more stable. Morris water maze experiment indicated that Kaixinsan could improve the learning memory ability of SCOP-induced mice. HE staining and ELISA results showed that Kaixinsan had an ameliorating effect on central nerve injury in mice. Western blot test indicated that Kaixinsan had a down-regulating effect on the levels of NF-κB p65 phosphorylation and STAT3 phosphorylation in the hippocampal tissue of mice in the SCOP model. ConclusionKaixinsan can improve the cognitive impairment function in SCOP model mice and may reduce hippocampal neuronal damage and thus play a therapeutic role in the treatment of AD by regulating NF-κB p65， STAT3， and other targets involved in the NF-κB signaling pathway.

Objective: Based on spinopelvic parameters and biomechanical principles, the pedicle-facet joint (PFJ) morphological characteristics of isthmic and degenerative spondylolisthesis were analyzed, and the mechanism of their onset and progression was discussed. Methods: This retrospective cross-sectional study included 194 patients with L5 spondylolysis or L5–S1 low-grade isthmic spondylolisthesis (IS group), 172 patients with L4–5 degenerative spondylolisthesis (DS group), and 366 patients with nonlumbar spondylolysis (NL group). The spinopelvic parameters and PFJ morphological parameters of the patients were measured, the differences in these parameters among and within the 3 groups were compared, and the correlations were analyzed. Results: Sacral slope (SS) and lumbar lordosis (LL) were the highest in the IS group, the second highest in the DS group, and the lowest in the NL group. Among the 3 groups, the L4 facet joint angle (FJA) was the largest in the IS group, the second largest in the NL group, and the smallest in the DS group. The L4 pedicle-facet joint angle (PFA) was the largest in the DS group, the second largest in the IS group, and the smallest in the NL group. Pearson correlation analysis showed that within each group, SS and LL were negatively correlated with FJA and positively correlated with PFA. Conclusion This study found a correlation between the PFJ morphological characteristics of patients with lumbar spondylolisthesis and spinopelvic parameters, suggesting that the morphological characteristics of PFJs may be caused by varying stresses under different spinopelvic morphologies.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objective: Based on spinopelvic parameters and biomechanical principles, the pedicle-facet joint (PFJ) morphological characteristics of isthmic and degenerative spondylolisthesis were analyzed, and the mechanism of their onset and progression was discussed. Methods: This retrospective cross-sectional study included 194 patients with L5 spondylolysis or L5–S1 low-grade isthmic spondylolisthesis (IS group), 172 patients with L4–5 degenerative spondylolisthesis (DS group), and 366 patients with nonlumbar spondylolysis (NL group). The spinopelvic parameters and PFJ morphological parameters of the patients were measured, the differences in these parameters among and within the 3 groups were compared, and the correlations were analyzed. Results: Sacral slope (SS) and lumbar lordosis (LL) were the highest in the IS group, the second highest in the DS group, and the lowest in the NL group. Among the 3 groups, the L4 facet joint angle (FJA) was the largest in the IS group, the second largest in the NL group, and the smallest in the DS group. The L4 pedicle-facet joint angle (PFA) was the largest in the DS group, the second largest in the IS group, and the smallest in the NL group. Pearson correlation analysis showed that within each group, SS and LL were negatively correlated with FJA and positively correlated with PFA. Conclusion This study found a correlation between the PFJ morphological characteristics of patients with lumbar spondylolisthesis and spinopelvic parameters, suggesting that the morphological characteristics of PFJs may be caused by varying stresses under different spinopelvic morphologies.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

BACKGROUND:Human periodontal ligament stem cells are potential functional cells for periodontal tissue engineering.However,long-term in vitro culture may lead to reduced stemness and replicative senescence of periodontal ligament stem cells,which may impair the therapeutic effect of human periodontal ligament stem cells. OBJECTIVE:To investigate the effect of oxymatrine on the stemness maintenance and osteogenic differentiation of periodontal ligament stem cells in vitro,and to explore the potential mechanism. METHODS:Periodontal ligament stem cells were isolated from human periodontal ligament tissues by tissue explant enzyme digestion and cultured.The surface markers of mesenchymal cells were identified by flow cytometry.Periodontal ligament stem cells were incubated with 0,2.5,5,and 10 μg/mL oxymatrine.The effect of oxymatrine on the proliferation activity of periodontal ligament stem cells was detected by CCK8 assay.The appropriate drug concentration for subsequent experiments was screened.Western blot assay was used to detect the expression of stem cell non-specific proteins SOX2 and OCT4 in periodontal ligament stem cells.qRT-PCR and western blot assay were used to detect the expression levels of related osteogenic genes and proteins in periodontal ligament stem cells. RESULTS AND CONCLUSION:(1)The results of CCK8 assay showed that 2.5 μg/mL oxymatrine significantly enhanced the proliferative activity of periodontal stem cells,and the subsequent experiment selected 2.5 μg/mL oxymatrine to intervene.(2)Compared with the blank control group,the protein expression level of SOX2,a stem marker of periodontal ligament stem cells in the oxymatrine group did not change significantly(P>0.05),and the expression of OCT4 was significantly up-regulated(P<0.05).(3)Compared with the osteogenic induction group,the osteogenic genes ALP,RUNX2 mRNA expression and their osteogenic associated protein ALP protein expression of periodontal ligament stem cells were significantly down-regulated in the oxymatrine+osteogenic induction group(P<0.05).(4)The oxymatrine up-regulated the expression of stemness markers of periodontal ligament stem cells and inhibited the bone differentiation of periodontal ligament stem cells,and the results of high-throughput sequencing showed that it may be associated with WNT2,WNT16,COMP,and BMP6.

Objective To investigate the screening results and factors affecting abnormal detection rates among high-risk groups of esophageal cancer and to explore effective intervention measures. Methods We investigated and collected the information on gender, education level, age, marital status, symptoms of reflux esophagitis (heartburn, acid reflux, belching, hiccup, foreign body sensation in the pharynx, and difficulty swallowing), consumption of pickled vegetables, salt use, and esophageal cancer incidence of villagers in a natural village in Wenfeng District, Anyang City, Henan Province. Changes in reflux esophagitis symptoms in the high-incidence area of esophageal cancer before and after 16 years were observed, and the relationship of such changes with esophageal cancer was analyzed. Results In 2008, 711 cases were epidemiologically investigated, including 213 cases with one of the symptoms of reflux esophagitis such as heartburn, acid reflux, belching, hiccups, foreign body sensation in the pharynx, and difficulty swallowing (sorted in accordance with the abovementioned symptoms; if there are multiple symptoms, then the former is taken); 55 cases with at least two related symptoms, among which the most symptom was heartburn in 108 cases (15.1%); followed by acid reflux, belching, etc. In 2024, the same group of people was investigated, and eight people were missing because of relocation. A total of 703 people were successfully investigated, of which 189 cases (26.8%) had one of the symptoms of reflux esophagitis, such as heartburn (sorted in accordance with relevant symptoms, if there are multiple symptoms, then the former will be selected); 167 cases (23.7%) had at least two related symptoms, among which the most symptom was heartburn in 139 cases (19.7%); followed by acid reflux. Over 16 years, among the 703 people investigated, 45 cases had esophageal cancer and they had one or more of the abovementioned symptoms. No significant differences in the reflux esophagitis-like symptoms were found between 2008 and 2024 (P=0.26); no significant differences in the composition of reflux esophagitis-like symptoms were found between 2008 and 2024 (P=0.299). Conclusion The detection rate of reflux esophagitis-related symptoms in the population in a high-risk area of esophageal cancer has not decreased in the past 16 years, and the high-risk factors still exist. Esophageal cancer is closely related to reflux esophagitis, and clinical practice can provide early diagnosis and treatment for high-risk population.

Objective To investigate the expression of Cyclin F in clear cell renal cell carcinoma (ccRCC), its clinicopathological characteristics, and its effect on the biological behavior of renal cancer cell lines Methods RT-qPCR and Western blot were used to detect the mRNA and protein expression of Cyclin F in fresh ccRCC specimens. Immunohistochemistry assay was performed to detect the expression of Cyclin F protein in 80 paraffin samples. CCK-8 assay, scratch assay, and flow cytometry were conducted to determine the effects of Cyclin F overexpression on the proliferation, migration, and apoptosis of renal cancer cell lines. Results The expression of Cyclin F in cancer tissues was higher than that in adjacent tissues at the mRNA level (P<0.0285). The expression of Cyclin Fprotein in cancer tissues was lower than that in adjacent tissues (P<0.001). The analysis of clinicopathological parameters showed that the low expression of Cyclin F was correlated with WHO/ISUP grade, Ki67 index, and age (P=0.041, 0.047, 0.008,). In vitro experiments confirmed that overexpression of Cyclin F promoted the proliferation (P<0.001) and migratory ability (24 h P=0.003, 48 h P=0.0058) of the RCC 786-O cell line, while inhibiting apoptosis (P=0.0019). Conclusion The low expression of Cyclin F protein in ccRCC tissuesis associated with high ISUP grade, high Ki67 index, oldage, and prognosis of patients.

ObjectiveTo understand the whole genome characteristics and the information for genetic evolution in the two coxsackievirus A2 (CVA2) strains isolated from patients with herpangina in Shanghai, and to provide a scientific basis for the prevention and treatment of herpetic angina. MethodsTwo CAV2 strains isolated from patients with herpetic angina in Shanghai were performed whole genome sequencing and analysis for phylogenetics, nucleotide homology, and evolution. ResultsA phylogenetic analysis of the VP1 region revealed that the two Shanghai strains both belonged to CVA2 genotype D, with the highest homology to OL357660, a strain from Yunnan. The average nucleotide identity (ANI) of the whole genome between the two Shanghai strains was 98.88%, and the ANI of the whole genome comparisons to other CVA2 genotype D strains and CVA2 genotypes A-C strains ranged from 84.64% to 97.42% and from 79.21% to 84.20%, respectively. The two Shanghai strains had low homology in the 3D region compared to the existing CVA2 strains. The phylogenetic analysis and sliding window nucleotide similarity analysis indicated that the two Shanghai strains and the Yunnan OL357660 strain might constitute a new genetic lineage. ConclusionThe two CVA2 strains isolated for the first time in Shanghai are assigned to genotype D (GenBank: PQ130039 and PQ130040), which is identical to the existing subtype prevalent in China. As represented by the Shanghai strains, a new CVA2 genetic lineage is been identified. This study has enriched the data on genetic evolution and genetic variation of CVA2 in Shanghai, indicating the requirement to strengthen surveillance for the epidemiological pattern of CVA2.