ObjectiveTo establish an in vitro culture model of rat corpora cavernous smooth muscle cells （CCSMCs） using a modified tissue block adherence method. MethodsCorpus cavernosum smooth muscle tissue was digested with collagenase type I and subsequently cultured using an adherent method. Cells were purified via differential adhesion and identified through immunofluorescence and Western blotting. ResultsCCSMCs began to emerge from the tissue block after 3 days， increased significantly by day 7， and converged by day 12. Post-passage， CCSMCs exhibited strong proliferation and a “peak-to-valley” phenomenon. After purification， the cells tested positive for α-smooth muscle actin （α-SMA）， confirming the successful establishment of the in vitro culture model. ConclusionThe modified tissue block adherence method is a cost-effective and efficient way to obtain high-purity CCSMCs.

A comprehensive phytochemical investigation of the leaves and twigs of Physalis angulata. var. villosa resulted in the isolation of 23 withanolide derivatives, including one novel 13,20-γ-lactone withanolide derivative (1) and three new withanolide derivatives (2-4). Architecturally, physalinin A (1) represents the first identified type B withanolide featuring a 13,20-γ-lactone moiety. The molecular structures of all isolates were elucidated using an integrated approach combining nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), infrared (IR) spectroscopy, and quantum chemical calculations to confirm structural assignments. The antiproliferative activities of all isolated withanolides were evaluated against four human cancer cell lines (HEL, HCT-116, Colo320DM, and MDA-MB-231). Among them, eight derivatives (2, 5-8, 14, 15, and 23) exhibited significant inhibitory effects, with half-maximal inhibitory concentration (IC₅₀) values of 0.18 ± 0.03 to 17.02 ± 0.21 μmol·L^-1. Structure-activity relationship (SAR) analysis suggested that the presence of an epoxide ring enhances anticancer activity, potentially through increased reactivity or specific interactions with molecular targets involved in cancer progression. These findings underscore the pharmacological potential of withanolides as promising lead compounds for the development of novel anticancer therapeutics.
Withanolides/isolation & purification* ; Physalis/chemistry* ; Humans ; Molecular Structure ; Cell Line, Tumor ; Antineoplastic Agents, Phytogenic/isolation & purification* ; Cell Proliferation/drug effects* ; Plant Leaves/chemistry* ; Plant Extracts/pharmacology*

The key pathogenesis of coronary heart disease （CHD） is spleen deficiency and phlegm stasis， and dysfunctional high-density lipoprotein （dys-HDL） may be the biological basis for the occurrence of CHD due to spleen deficiency and phlegm stasis. Considering the biological properties and effects of high-density lipoprotein （HDL）， it is believed that the structure and components of HDL are abnormal in the state of spleen deficiency which led to dys-HDL； and dys-HDL contributes to the formation of atherosclerotic plaques through two major pathways， namely， mediating the dysfunction of endothelial cells and mediating the foaminess of macrophages and smooth muscle cells， thus triggering the development of CHD. It is also believed that dys-HDL is a microcosmic manifestation and a pathological product of spleen deficiency， and spleen deficiency makes foundation for the production of dys-HDL； dys-HDL is also an important biological basis for the phlegm-stasis interactions in CHD. The method of fortifying spleen， resolving phlegm， and dispelling stasis， is proposed as an important principle in the treatment of CHD by traditional Chinese medicine， which can achieve the therapeutic purpose by affecting the changes in the structure and components of dys-HDL， thus revealing the scientific connotation of this method， and providing ideas for the diagnosis and treatment of CHD by traditional Chinese medicine.

Objective:To explore the clinical efficacy and safety analysis of a novel laser localization technology assisted percutaneous puncture of trigeminal nerve microsphere capsule compression surgery for the treatment of primary trigeminal neuralgia.Methods:A retrospective selection was conducted on 63 patients with primary trigeminal neuralgia who underwent percutaneous puncture of the trigeminal nerve microsphere capsule compression surgery at the First Hospital of Hunan University of Chinese Medicine from January 2020 to December 2021. According to different surgical methods, they were divided into a new laser localization assisted puncture group (observation group) of 32 cases and a traditional barehanded localization puncture group (control group) of 31 cases. An analysis was conducted on the surgical time, puncture time, puncture frequency, intraoperative exposure to radiation, number of cases of poor balloon formation, and clinical efficacy within 6 months after surgery for two groups of patients. The prognosis of the patients was followed up at 6 months after surgery.Results:The surgical time, puncture time, puncture frequency, and intraoperative exposure of the observation group were all less than those of the control group, and the differences were statistically significant (all P<0.05). There was no statistically significant difference ( P>0.05) in the number of cases of poor balloon angioplasty between the observation group and the control group, as well as the pain score grading of the Barlow Neurological Institute (BNI) on the first day after surgery. Within 6 months after surgery, there was no statistically significant difference in the incidence of facial numbness, diplopia, masseter weakness, perilabial herpes, and recurrent pain between the two groups of patients (all P>0.05). Conclusions:Laser positioning technology can assist in precise puncture of the foramen ovale and accurate placement of balloons based on surgical experience, which helps to improve surgical safety, reduce postoperative complications and intraoperative radiation dose, and achieve satisfactory short-term follow-up results.

Objective To explore the clinical efficacy and safety of donepezil(DNPQ)combined with butylphthalide sequential therapy(BST)in the treatment of Parkinson's syndrome(PS).Methods In this study,104 patients with Parkinson's disease(PD)who were diagnosed and treated in the Department of Neurology of The Fourth Hospital of Changsha from January 2020 to November 2023 were randomly divided into a control group(butylphthalide softcapsule combined with DNPQ)and an observation group(BST combined with DNPQ).The main observation indicators of this study were the clinical efficacy and drug-related adverse reactions after 3 months of treatment.The secondary observation indicators were the cognitive function[Montreal Cognitive Assessment(MoCA)and Mini-Mental State Examination(MMSE)],overall condition[Unified Parkinson's Disease Rating Scale(UPDRS)],activity of daily living(ADL),and oxidative stress-related cytokines[recombinant human Parkinson's disease protein 7(PARK7),neurotrophic factor 3(NT3),and C-reactive protein(CRP)]improvement after treatment.Results There were 52 patients in the experimental group and 52 patients in the control group.The treatment efficacy rate in the experimental group was significantly higher than that in the control group(P<0.05),while the incidence of adverse reactions was significantly lower than that in the control group(P<0.05).Before treatment,there were no significant differences in MoCA scores,MMSE scores,UPDRS scores,ADL scores,serum NT3,CRP,and PARK7 levels between the two groups(P>0.05).After treatment,the MoCA score,MMSE score,and ADL score in the experimental group were higher than those in the control group(P<0.05),while the UPDRS score was lower than that in the control group(P<0.05).After treatment,the serum NT3 level in the experimental group was higher than that in the control group(P<0.05),while the serum CRP and PARK7 levels were lower than those in the control group(P<0.05).Conclusion The combination of DNPQ and BST has better clinical efficacy and safety,which can improve cognitive function,ADL and oxidative stress-related cytokine content in patients with PS.

OBJECTIVES: To investigate the genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine (C4). METHODS: One hundred and twenty neonates with increased C4 levels detected by tandem mass spectrometry in the neonatal screening at Children's Hospital, Zhejiang University School of Medicine from January 2018 to June 2023 were included. The initial screening data and recalled data of C4 and C4/C3 were collected and converted into multiples of C4 reference range. Next generation sequencing was performed and the exons with adjacent 50 bp regions of ACAD8 and ACADS genes were captured by liquid phase capture technique. Variant information was obtained by bioinformatic analysis and the pathogenicity were classified according to the American College of Medical Genetics and Genomics criteria. The Wilcoxon rank sum test was used to analyze the differences in C4 levels among neonates with different variation types. RESULTS: In total, 32 variants in ACAD8 gene were detected, of which 7 variants were reported for the first time; while 41 variants of ACADS gene were detected, of which 17 variants have not been previously reported. There were 39 cases with ACAD8 biallelic variations and 3 cases with ACAD8 monoallelic variations; 34 cases with ACADS biallelic variations and 36 cases with ACADS monoallelic variations. Furthermore, 5 cases were detected with both ACAD8 and ACADS gene variations. Inter group comparison showed that the multiples of C4 reference range in initial screening and re-examination of the ACAD8 biallelic variations and ACADS biallelic variations groups were significantly higher than those of the ACADS monoallelic variations group (all P<0.01), while the multiples in the ACAD8 biallelic variations group were significantly higher than those in the ACADS biallelic variations group (all P<0.01). The multiples of C4 reference range in the initial screening greater than 1.5 times were observed in all neonates carrying ACAD8 or ACADS biallelic variations, while only 25% (9/36) in neonates carrying ACADS monoallelic variations. CONCLUSIONS ACAD8 and/or ACADS gene variants are the main genetic causes for elevated C4 in newborns in Zhejiang region with high genotypic heterogeneity. The C4 levels of neonates with biallelic variations are significantly higher than those of neonates with monoallelic variations. The cut-off value for C4 level could be modestly elevated, which could reduce the false positive rate in tandem mass spectrometry neonatal screening.
Child ; Humans ; Infant, Newborn ; Acyl-CoA Dehydrogenase/genetics* ; Genotype ; Phenotype ; Carnitine/metabolism* ; Mutation

Objective:To explore the expression of type 2 complement receptor (CR2) in mesangial cells of the renal tissue in IgA nephropathy (IgAN) and its possible mechanism involved in complement C3 deposition.Methods:The demographic data, samples of plasma and renal tissues of primary IgAN patients diagnosed by renal biopsy in the Guangdong Provincial People's Hospital from August 2021 to May 2022 were collected. According to the fluorescent intensity of mesangial complement C3 deposition, the patients were divided into complement C3 deposition ≥2+ group and complement C3 deposition <2+ group. The circulating IgA and complement C3 levels were detected by enzyme linked immunosorbent assay (ELISA). The influencing factors of kidney prognosis, plasma IgA and complement C3 levels were compared between the two groups. Immunofluorescence was used to detect the expression of IgA, complement C3 and CR2 in the renal mesangial cells of IgAN patients and normal renal tissues around renal carcinoma. Human mesangial cells were cultured in vitro and randomly divided into control group and experimental group. The experimental group was incubated with IgA protein (2 g/L) for 8 hours. The expressions of CR2 protein and mRNA were measured by Western blotting and real-time fluorescence quantitative PCR. The biological function of differential genes was analyzed by gene ontology (GO) and Kyto encyclopedia of genes and genomes (KEGG) enrichment analysis. Results:A total of 75 patients with IgAN were included in this study, including 50 patients in the complement C3 deposition ≥2+ group and 25 patients in the complement C3 deposition <2+ group. The proportions of patients with urine red blood cell count negative, 1+, 2+ and 3+-4+ in the complement C3 deposition ≥2+ group were 2.0%, 8.0%, 18.0% 72.0%, respectively, which were more serious than those in the complement C3 deposition <2+ group (4.0%, 4.0%, 52.0%, 40.0%) ( Z=-2.320, P=0.020). Meanwhile, the proportion of S1 in Oxford pathological classification in the complement C3 deposition ≥2+ group was higher than that in the complement C3 deposition <2+ group (68.0% vs. 40.0%, χ2=5.389, P=0.020), and there were no statistically significant differences in gender, age, 24-hour urinary protein, serum creatinine, other indicators of Oxford pathological classification between the two groups. ELISA results showed that plasma IgA concentration in the complement C3 deposition ≥2+ group was higher than that in the complement C3 deposition <2+ group [3.62 (2.95, 5.53) g/L vs. 2.72 (2.15, 4.24) g/L, Z=2.405, P=0.016], and the plasma complement C3 concentration was lower than that in the complement C3 deposition <2+ group [199.6 (116.0, 328.0) mg/L vs. 319.2 (158.3, 454.5) mg/L, Z=-2.383, P=0.017]. Spearman correlation analysis showed that the complement C3 deposition intensity was positively correlated with IgA deposition intensity in mesangial area ( rs=0.441, P<0.001). Immunofluorescence results showed that there was colocalization of IgA and complement C3 in the glomeruli of IgAN patients. The expression of CR2 in the kidney was consistent with complement C3 deposition, and CR2 was colocalization with complement C3. In vitro experiments, the expression of CR2 in IgA protein group was higher than that in the control group ( P<0.05). GO and KEGG enrichment analysis found that IgA protein induced active changes in various pathways of mesangial cells. Conclusion:IgA protein induces mesangial cells to express CR2 and participates in complement C3 deposition, which may be an important mechanism of complement C3 activation in IgAN.

Objective:To study and verify the molecular mechanism of Duzhong Pills for osteoporosis (OP) by means of network pharmacology and molecular docking.Methods:The main chemical components of Duzhong Pills were mined by TCMSP database and the related targets were predicted. The potential targets of osteoporosis in GeneCards, DisGeNET and OMIM databases were searched and the common targets of both were obtained. The STRING platform was used for protein interaction analysis and PPI network diagram was made. The common targets were imported into the David database for enrichment analysis of GO and KEGG pathways, and molecular docking of the main components and core targets was performed. Eighteen Sprague-Dawley rats were divided into control group, model group and Duzhong Pills group according to random number table method, with 6 rats in each group. Ovariectomy was used to make osteoporosis model in model group and Duzhong Pills group. Duzhong Pills group was intragaxed with Duzhong Pills extract of 5 g/kg, and control group and model group were intragaxed with normal saline of the same volume, once a day for 8 weeks. Serum levels of TNF-α, IL-6 and IL-1β were detected by ELISA, and femur PI3K and Akt were detected by Western blot.Results:34 active components were obtained from Duzhong Pills, corresponding to 243 targets, and 1 059 targets for osteoporosis. The core targets included TNF-α, IL-6, AKT1, TP53, IL-1β and others regulated osteoporosis through PI3K-Akt and TNF pathway. The experimental results indicated that compared with model group, the levels of serum TNF-α, IL-6 and IL-1β in Duzhong Pills group decreased ( P<0.05), and the expressions of PI3K and Akt in femur decreased ( P<0.05). Conclusion:Through β-sitosterol, quercetin, kaempferol and other active components, Duzhong Pills can act on TNF, IL-6, AKT1, TP53, IL-1β and other targets, regulating PI3K-Akt signaling pathway, TNF signaling pathway and other signaling pathways to play a role in the treatment of osteoporosis.

Objective:To establish and validate a predictive model for treatment failure of peritoneal dialysis-related peritonitis(PDAP)in elderly patients.Methods:Clinical data of peritoneal dialysis(PD)patients who were followed up from January 1, 2013 to December 31, 2019 at four Grade A tertiary hospitals in Jilin Province were collected.A total of 362 elderly patients with PDAP were eventually included as study subjects.Subjects recruited from 2013 to 2017 were used for model construction and the logistic regression model was used to screen risk factors for treatment failure of PDAP in elderly patients.A nomogarm was constructed to predict treatment failure of secondary PDAP using R language.The receiver operating curve(ROC)and calibration curve were used to evaluate discrimination accuracy of the model.Subjects from 2018 to 2019 were used as the cohort for validation of discrimination accuracy of the model.Results:Of 258 PDAP patients in the modeling cohort, 29 experienced treatment failure, including 15 PDAP-related deaths and 14 cases requiring catheter removal.The multivariate logistic regression model showed that types of pathogens( OR=8.849, 95% CI: 1.656-47.269, P=0.011), long dialysis age( OR=1.023, 95% CI: 1.005-1.042, P=0.013), pre-hospitalization antibiotic treatment( OR=5.123, 95% CI: 1.338-19.610, P=0.017), and dialysate white blood cell count on day 5>100×10 6/L( OR=7.085, 95% CI: 2.162-23.217, P=0.001)were independent risk factors for treatment failure of PDAP in elderly patients.For the nomogarm predictive model, the areas under the ROC curve(AUC)in the modeling cohort and the validation cohort were 0.818(95% CI: 0.735-0.902)and 0.762(95% CI: 0.656-0.889), respectively, and the calibration curves were close to a straight line with a slope of 1. Conclusions:Our nomogram predictive model based on types of pathogens, months of dialysis, pre-hospital admission antibiotic treatment, and dialysate white blood cell count on day 5 has demonstrated satisfactory discrimination accuracy for treatment failure of PDAP in elderly patients.

Objective:To fabricate biogenic gas vesicles (GVs)- polyethyleneimine (PEI)-marrow mesenchymal stem cells (BMSCs) and evaluate its potential on stem cell tracking with ultrasound imaging.Methods:GVs were cationized by PEI to fabricate GVs-PEI. The diameter and zeta potential of GVs-PEI were determined. GVs-PEI were co-incubated with BMSCs to obtain GVs-PEI-BMSCs and stem cell uptake was observed by fluorescence microscope. The cell viability of GVs-PEI-BMSCs was verified by cell counting kit-8 (CCK-8) assay were set. Ultrasound imaging was performed on 0, 2, 4 and 6 d in agarose phantom to evaluate ultrasound imaging capability of GVs-PEI-BMSCs group and BMSCs group in vitro. GVs-PEI-BMSCs and BMSCs were injected into quadriceps femoris of SD rats, and ultrasound imaging was performed on 0, 2, 4 and 6 d to evaluate the ultrasound imaging capability in vivo. One-way analysis of variance and independent-sample t test were used to analyze the data. Results:The diameter and zeta potential of GVs-PEI were (383.63±11.55) nm and (18.48±2.20) mV. Plenty of GVs-PEI were observed in GVs-PEI-BMSCs through microscope. When BMSCs were incubated with GVs-PEI in absorbance ( A) 500 nm of 0.5 and 1.0, there were no significant changes in the cell viability of GVs-PEI-BMSCs at 24, 48 and 72 h ( F values: 7.078-11.982, all P>0.05). Compared with BMSCs, GVs-PEI-BMSCs showed better ultrasound imaging capability in vitro in all time points with still significantly different signal at 6 d (634.29±10.78 vs 2 864.51±100.86; t=-121.86, P<0.001). The ultrasound imaging capability of GVs-PEI-BMSCs in vivo was much better than that of BMSCs at each time point with still significantly different signal at 6 d (2 108.02±217.96 vs 267.71±7.87; t=-121.39, P<0.001). Conclusion:GVs-PEI-BMSCs are successfully fabricated with the advantages of significant ultrasound imaging capability, long duration and safety, which provide a brand-new means for stem cells tracking in vivo.