BACKGROUND:Formononetin demonstrates potent anti-inflammatory and antioxidant abilities.However,its protective effect on nucleus pulposus mesenchymal stem cells is not yet clear.OBJECTIVE:To investigate the effect and mechanism of formononetin on nucleus pulposus mesenchymal stem cells under an inflammatory microenvironment.METHODS:(1)Primary nucleus pulposus mesenchymal stem cells were isolated from the intervertebral discs of SD rats,and flow cytometry was performed to identify the surface markers of mesenchymal stem cells.(2)The CCK-8 assay was employed to evaluate the impact of lipopolysaccharide and formononetin on the proliferation viability of nucleus pulposus mesenchymal stem cells,aiming to determine the appropriate concentration of formononetin for subsequent cell treatments.(3)An inflammatory microenvironment was simulated by adding 5 μg/mL lipopolysaccharide to the DMEM/F-12 culture medium,and nucleus pulposus mesenchymal stem cells were treated with different concentrations of formononetin for 24 hours.Levels of inflammation markers were detected using western blot assay,real-time quantitative PCR,and immunofluorescence.Western blot assay was conducted to measure the protein levels of the nuclear factor kappa B signaling pathway.RESULTS AND CONCLUSION:(1)The nucleus pulposus mesenchymal stem cells cultured in adherent wall were shuttle-shaped with good growth status.The results of flow cytometry showed that the surface markers of mesenchymal stem cells were positive for CD29,CD44,and CD90,and the surface markers of hematopoietic stem cells were negative for CD34 and CD45.(2)The treatment with formononetin at 12.5,25,50,100,and 200 μmol/L concentrations for 24 hours had no significant proliferation inhibitory effect on nucleus pulposus mesenchymal stem cells.Compared with the lipopolysaccharide group,the cell viability of nucleus pulposus mesenchymal stem cells treated with 12.5,25,and 50 μmol/L formononetin for 24 hours was significantly increased,so formononetin at 12.5,25,and 50 μmol/L concentrations was subsequently selected as the low,medium,and high concentrations for treating nucleus pulposus mesenchymal stem cells.(3)Compared with the lipopolysaccharide group,the protein and mRNA expressions of matrix metalloproteinase-3,matrix metalloproteinase-13,and tumor necrosis factor-α in nucleus pulposus mesenchymal stem cells in the low,medium,and high concentrations of formononetin groups were significantly decreased(P＜0.05)in a dose-dependent manner.(4)Compared with the lipopolysaccharide group,the expressions of phosphorylated nuclear factor kappa B and phosphorylated nuclear factor kappa B inhibitor protein in nucleus pulposus mesenchymal stem cells in the low,medium,and high concentrations of formononetin groups were significantly decreased(P＜0.05)in a dose-dependent manner.The above results suggest that formononetin may attenuate lipopolysaccharide-induced inflammation in rat nucleus pulposus mesenchymal stem cells by inhibiting the activation of the nuclear factor kappa B signaling pathway.

Objective:To collect registered clinical research plans on TCM characteristic therapies for treating cervical spondylosis; To explore their research registration status; To provide references for future clinical trial registration and implementation.Methods:Clinical research on TCM characteristic therapies for treating cervical spondylosis was retrieved from ChiCTR, ITMCTR and Clinical Trials. gov from the establishment of the databases to July 1, 2024. Excel 2019 was used to conduct descriptive statistics on registration time, registration area and institution, funding source, research type and design scheme, research participation center and sample size, cervical spondylosis type, intervention measures, outcome indicators, reporting quality, research openness and methodological application.Results:A total of 138 clinical trials for the TCM treatment of cervical spondylosis were included, of which 136 were registered by domestic researchers in 22 provincial-level administrative regions. The top three in terms of registration numbers were Shanghai, Guangdong Province, and Beijing. Additionally, 2 were registered by foreign researchers in Egypt and Malaysia. The main sources of funding were 50 local finances, followed by 26 hospital subsidies and 18 national finances. The intervention research accounted for the largest proportion of research types, with 123 items (89.13%). The research center mainly focused on single center studies (98 projects). Most randomized controlled trials (115 trials) described randomization methods, while a small number of randomized controlled trials (50 trials) indicated blinding. The intervention measures were mostly combined with TCM therapy, and the outcome indicators were mainly efficacy indicators, with fewer safety indicators.Conclusions:At present, clinical trial registrations for TCM treatment of cervical spondylosis are increasing, but issues remain, such as poor study design, uneven distribution, and incomplete information. It is recommended to refine registration details, optimize study protocols, and promote high-quality clinical research.

BACKGROUND:Studies have shown that upregulation of heme oxygenase-1 expression enhances cellular antioxidant and anti-apoptotic abilities.However,the effects of upregulating heme oxygenase-1 expression on the proliferation and differentiation of neural stem cells under oxidative stress conditions remain unclear.OBJECTIVE:To investigate the influence of heme oxygenase-1 overexpression on the survival and differentiation capacity of neural stem cells under oxidative stress conditions.METHODS:(1)Mouse primary neural stem cells were isolated and cultured from newborn Balb/c mice.Immunofluorescence was used to detect the neural stem cell marker Nestin.(2)Lentivirus was used to infect neural stem cells to induce heme oxygenase-1 overexpression.Flow cytometry was used to assess green fluorescent protein fluorescence.Western blot assay was performed to detect the expression levels of heme oxygenase-1.(3)H2O2 was added to the lentivirus-infected neural stem cell culture medium to simulate the oxidative stress microenvironment after spinal cord injury.Effects of heme oxygenase-1 overexpression on neural stem cell proliferation and apoptosis levels were analyzed using cell proliferation assay kits,cell apoptosis assay kits,and TUNEL staining kits.(4)The levels of lipid oxidation markers malondialdehyde,catalase,superoxide dismutase,and glutathione peroxidase were detected using assay kits.(5)Flow cytometry was used to measure intracellular reactive oxygen species levels,and neural stem cell differentiation into astrocytes and neurons.(6)The effect of heme oxygenase-1 overexpression on neuronal axon growth during neural stem cell differentiation was observed under optical and fluorescence microscopes.RESULTS AND CONCLUSION:(1)Mouse neural stem cells exhibited stable morphology,good growth status,and high expression of Nestin as detected by immunofluorescence.(2)Western blot analysis showed that the overexpression of heme oxygenase-1 in the overexpression group was significantly higher than that in the empty carrier control group.Flow cytometry was used to detect the expression of green fluorescent protein in the neural stem cells of the heme oxygenase-1 overexpression group and empty vector control group.(3)Overexpression of heme oxygenase-1 maintained the proliferative activity of neural stem cells and significantly reduced the number of apoptotic cells under oxidative stress conditions.(4)Overexpression of heme oxygenase-1 inhibited lipid peroxidation of neural stem cells under oxidative stress microenvironment,enhanced the expression of enzymes related to maintaining the oxidative-reductive balance,and significantly reduced intracellular reactive oxygen species levels.(5)Overexpression of heme oxygenase-1 promoted the differentiation of neural stem cells into neurons and reduced differentiation into astrocytes.(6)The heme oxygenase-1 overexpression group exhibited longer axons,and more intercellular connections.The above results indicate that overexpression of heme oxygenase-1 can alleviate oxidative damage of H2O2-induced neural stem cells,reduce neural stem cell apoptosis,promote proliferation,and facilitate differentiation of neural stem cells into neurons.

Objective:To compare the advantages and disadvantages and training costs of different training methods for microsurgical vascular anastomosis, and to provide a basis for establishing a systematic training model and improving surgeons microsurgical skills and clinical competence.Methods:Doctors came from various primary hospitals and exchange groups from foreign hospitals to China, and several groups of data statistics from 2018-2023 were randomly selected for this study. The microsurgical vascular anastomosis training lasted 10 days, including 1 day of theoretical study and 9 days of practical training. A total of 48 doctors were equally divided into group A (one-person operation), group B (two-person cooperation), and group C (two-person cooperation in the first four days and one-person operation in the last five days). The differences in anastomosis time and number of anastomoses between the groups were analysed by one-way ANOVA using the software GraphPad Prism 8.3.0, with P<0.05 indicating that there were statistically significant differences in the variable data. The cost of the three training methods was investigated, and a questionnaire survey of the trainees was conducted. Results:For all the three groups, the speed of anastomosis and the number of anastomoses increased with the course of training. The duration of single-vessel anastomosis was significantly different between groups A and B as well as between groups A and C at all time points except on day 1 (A1 d vs. B1 d, P=0.335; A1 d vs. C1 d, P=0.064; P<0.05 for all the other time points); groups B and C showed no significant differences on day 1 ( P=0.196) and day 3 ( P=0.115) but had significant differences on days 5, 7, and 9 (all P<0.05) in the duration of anastomosis. The number of anastomoses was not significantly different between A1 d and B1 d ( P=0.081), between A3 d and B3 d ( P=0.160), between B1 d and C1 d ( P=0.695), between B3 d and C3 d ( P=0.520), and between A1 d and C1 d ( P=0.123), with significant differences at the other time points (all P<0.05). The training costs were group A > group C > group B. The training questionnaire showed that the proportion of trainees who applied this technique in their daily work was 100.00% (48/48), the proportion of those who wished to participate in the training of this technique occasionally was 100.00% (48/48), the proportion of participants whose institutions had no relevant training conditions was 37.50% (18/48), the proportion of those whose institutions lacked necessary instruments and equipment was 35.42% (17/48), the proportion of those who had difficulties in access to laboratory animals was 68.75% (33/48), and the proportion of inability to carry out relevant training due to the lack of animal experimentation techniques such as anesthesia, preservation, and euthanasia was 91.67% (44/48), indicating that there is a great need for microsurgical vascular anastomosis training. Conclusions:The three training modes have their own advantages and disadvantages. The A mode is suitable for small-scale training. The B mode is suitable for training with adequate funds, a large number of personnel, and a high use frequency. The C mode is the best choice for microsurgical vascular anastomosis training, in which trainees can not only practice the whole vascular anastomosis process but also cooperative skills for anastomosis.

BACKGROUND:Formononetin demonstrates potent anti-inflammatory and antioxidant abilities.However,its protective effect on nucleus pulposus mesenchymal stem cells is not yet clear.OBJECTIVE:To investigate the effect and mechanism of formononetin on nucleus pulposus mesenchymal stem cells under an inflammatory microenvironment.METHODS:(1)Primary nucleus pulposus mesenchymal stem cells were isolated from the intervertebral discs of SD rats,and flow cytometry was performed to identify the surface markers of mesenchymal stem cells.(2)The CCK-8 assay was employed to evaluate the impact of lipopolysaccharide and formononetin on the proliferation viability of nucleus pulposus mesenchymal stem cells,aiming to determine the appropriate concentration of formononetin for subsequent cell treatments.(3)An inflammatory microenvironment was simulated by adding 5 μg/mL lipopolysaccharide to the DMEM/F-12 culture medium,and nucleus pulposus mesenchymal stem cells were treated with different concentrations of formononetin for 24 hours.Levels of inflammation markers were detected using western blot assay,real-time quantitative PCR,and immunofluorescence.Western blot assay was conducted to measure the protein levels of the nuclear factor kappa B signaling pathway.RESULTS AND CONCLUSION:(1)The nucleus pulposus mesenchymal stem cells cultured in adherent wall were shuttle-shaped with good growth status.The results of flow cytometry showed that the surface markers of mesenchymal stem cells were positive for CD29,CD44,and CD90,and the surface markers of hematopoietic stem cells were negative for CD34 and CD45.(2)The treatment with formononetin at 12.5,25,50,100,and 200 μmol/L concentrations for 24 hours had no significant proliferation inhibitory effect on nucleus pulposus mesenchymal stem cells.Compared with the lipopolysaccharide group,the cell viability of nucleus pulposus mesenchymal stem cells treated with 12.5,25,and 50 μmol/L formononetin for 24 hours was significantly increased,so formononetin at 12.5,25,and 50 μmol/L concentrations was subsequently selected as the low,medium,and high concentrations for treating nucleus pulposus mesenchymal stem cells.(3)Compared with the lipopolysaccharide group,the protein and mRNA expressions of matrix metalloproteinase-3,matrix metalloproteinase-13,and tumor necrosis factor-α in nucleus pulposus mesenchymal stem cells in the low,medium,and high concentrations of formononetin groups were significantly decreased(P＜0.05)in a dose-dependent manner.(4)Compared with the lipopolysaccharide group,the expressions of phosphorylated nuclear factor kappa B and phosphorylated nuclear factor kappa B inhibitor protein in nucleus pulposus mesenchymal stem cells in the low,medium,and high concentrations of formononetin groups were significantly decreased(P＜0.05)in a dose-dependent manner.The above results suggest that formononetin may attenuate lipopolysaccharide-induced inflammation in rat nucleus pulposus mesenchymal stem cells by inhibiting the activation of the nuclear factor kappa B signaling pathway.

BACKGROUND:Studies have shown that upregulation of heme oxygenase-1 expression enhances cellular antioxidant and anti-apoptotic abilities.However,the effects of upregulating heme oxygenase-1 expression on the proliferation and differentiation of neural stem cells under oxidative stress conditions remain unclear.OBJECTIVE:To investigate the influence of heme oxygenase-1 overexpression on the survival and differentiation capacity of neural stem cells under oxidative stress conditions.METHODS:(1)Mouse primary neural stem cells were isolated and cultured from newborn Balb/c mice.Immunofluorescence was used to detect the neural stem cell marker Nestin.(2)Lentivirus was used to infect neural stem cells to induce heme oxygenase-1 overexpression.Flow cytometry was used to assess green fluorescent protein fluorescence.Western blot assay was performed to detect the expression levels of heme oxygenase-1.(3)H2O2 was added to the lentivirus-infected neural stem cell culture medium to simulate the oxidative stress microenvironment after spinal cord injury.Effects of heme oxygenase-1 overexpression on neural stem cell proliferation and apoptosis levels were analyzed using cell proliferation assay kits,cell apoptosis assay kits,and TUNEL staining kits.(4)The levels of lipid oxidation markers malondialdehyde,catalase,superoxide dismutase,and glutathione peroxidase were detected using assay kits.(5)Flow cytometry was used to measure intracellular reactive oxygen species levels,and neural stem cell differentiation into astrocytes and neurons.(6)The effect of heme oxygenase-1 overexpression on neuronal axon growth during neural stem cell differentiation was observed under optical and fluorescence microscopes.RESULTS AND CONCLUSION:(1)Mouse neural stem cells exhibited stable morphology,good growth status,and high expression of Nestin as detected by immunofluorescence.(2)Western blot analysis showed that the overexpression of heme oxygenase-1 in the overexpression group was significantly higher than that in the empty carrier control group.Flow cytometry was used to detect the expression of green fluorescent protein in the neural stem cells of the heme oxygenase-1 overexpression group and empty vector control group.(3)Overexpression of heme oxygenase-1 maintained the proliferative activity of neural stem cells and significantly reduced the number of apoptotic cells under oxidative stress conditions.(4)Overexpression of heme oxygenase-1 inhibited lipid peroxidation of neural stem cells under oxidative stress microenvironment,enhanced the expression of enzymes related to maintaining the oxidative-reductive balance,and significantly reduced intracellular reactive oxygen species levels.(5)Overexpression of heme oxygenase-1 promoted the differentiation of neural stem cells into neurons and reduced differentiation into astrocytes.(6)The heme oxygenase-1 overexpression group exhibited longer axons,and more intercellular connections.The above results indicate that overexpression of heme oxygenase-1 can alleviate oxidative damage of H2O2-induced neural stem cells,reduce neural stem cell apoptosis,promote proliferation,and facilitate differentiation of neural stem cells into neurons.

Objective:To compare the advantages and disadvantages and training costs of different training methods for microsurgical vascular anastomosis, and to provide a basis for establishing a systematic training model and improving surgeons microsurgical skills and clinical competence.Methods:Doctors came from various primary hospitals and exchange groups from foreign hospitals to China, and several groups of data statistics from 2018-2023 were randomly selected for this study. The microsurgical vascular anastomosis training lasted 10 days, including 1 day of theoretical study and 9 days of practical training. A total of 48 doctors were equally divided into group A (one-person operation), group B (two-person cooperation), and group C (two-person cooperation in the first four days and one-person operation in the last five days). The differences in anastomosis time and number of anastomoses between the groups were analysed by one-way ANOVA using the software GraphPad Prism 8.3.0, with P<0.05 indicating that there were statistically significant differences in the variable data. The cost of the three training methods was investigated, and a questionnaire survey of the trainees was conducted. Results:For all the three groups, the speed of anastomosis and the number of anastomoses increased with the course of training. The duration of single-vessel anastomosis was significantly different between groups A and B as well as between groups A and C at all time points except on day 1 (A1 d vs. B1 d, P=0.335; A1 d vs. C1 d, P=0.064; P<0.05 for all the other time points); groups B and C showed no significant differences on day 1 ( P=0.196) and day 3 ( P=0.115) but had significant differences on days 5, 7, and 9 (all P<0.05) in the duration of anastomosis. The number of anastomoses was not significantly different between A1 d and B1 d ( P=0.081), between A3 d and B3 d ( P=0.160), between B1 d and C1 d ( P=0.695), between B3 d and C3 d ( P=0.520), and between A1 d and C1 d ( P=0.123), with significant differences at the other time points (all P<0.05). The training costs were group A > group C > group B. The training questionnaire showed that the proportion of trainees who applied this technique in their daily work was 100.00% (48/48), the proportion of those who wished to participate in the training of this technique occasionally was 100.00% (48/48), the proportion of participants whose institutions had no relevant training conditions was 37.50% (18/48), the proportion of those whose institutions lacked necessary instruments and equipment was 35.42% (17/48), the proportion of those who had difficulties in access to laboratory animals was 68.75% (33/48), and the proportion of inability to carry out relevant training due to the lack of animal experimentation techniques such as anesthesia, preservation, and euthanasia was 91.67% (44/48), indicating that there is a great need for microsurgical vascular anastomosis training. Conclusions:The three training modes have their own advantages and disadvantages. The A mode is suitable for small-scale training. The B mode is suitable for training with adequate funds, a large number of personnel, and a high use frequency. The C mode is the best choice for microsurgical vascular anastomosis training, in which trainees can not only practice the whole vascular anastomosis process but also cooperative skills for anastomosis.

Objective:To compare dosimetric and radiobiological parameters between automatic and manual uARC plans in the treatment of esophageal cancer patients, aiming to provide reference for clinical application.Methods:High-quality uARC plans of 100 patients with esophageal cancer were selected, and the mean values of the dosimetric parameters in the target area and organs at risk (OAR) were counted, and the goal table of uRT-TPOIS intelligent plan was established. Automatic and manual uARC plans were generated with UIH (United Imaging) treatment planning system (TPS) for 21 esophageal cancer patients. The differences in mean dose (D mean), approximate minimum (D 98%) and maximum (D 2%) dose of planning target volume (PTV), homogeneity index (HI) and conformity index (CI), dose of OAR, mean planning time, monitor unit (MU), tumor control probability (TCP) and normal tissue complication probability (NTCP) were compared between automatic and manual uARC plans. Normally distributed data between two groups were compared by paired t-test, and non-normally distributed data were assessed by nonparametric Wilcoxon test. Results:The D 98% (PTV 60 Gy: P<0.001, PTV 54 Gy: P=0.001) , CI (PTV 60 Gy: P<0.001, PTV 54 Gy: P=0.002) and target volume of area covered by prescription dose (V 54 Gy: P<0.001) of the automatic uARC plans were better than those of manual uARC plans (all P<0.05). There was no significant difference in D mean or HI between the two plans [PTV 54 Gy (59.32±1.87) Gy vs. (59.13±1.64) Gy, (0.19±0.02) vs. (0.18±0.02), all P>0.05]. The D mean and D max of spinal cord of the automatic plan were better than those of the manual plan [(13.22±4.27) Gy vs. (13.75±4.44) Gy, P=0.020 and (36.99±1.67) Gy vs. (38.14±1.31) Gy, P=0.011]. There was no significant difference in the mean dose of V 20 Gy of the lung between two plans ( P>0.05), whereas the mean doses of V 5 Gy and V 10 Gy of the lung of the manual plan were less than those of the automatic plan ( both P<0. 001). Automatic uARC plan had a significantly shorter mean planning time than manual uARC plan [(11.79±1.71) min vs. (53.36±8.23) min, P<0.001]. MU did not significantly differ between two plans [(762.84±74.83) MU vs. (767.41±80.63) MU, P>0.05]. The TCP of the automatic plan was higher than that of the manual plan (PTV 60 Gy 89.15%±0.49% vs. 86.75%±6.46%, P=0.004 and PTV 54 Gy 79.79%±3.48% vs. 77.51%±5.04%, P=0.006). However, manual plan had a lower NTCP of the lung than automatic uARC plan (0.46%±0.40% vs. 0.35%±0.32%, P<0.001). There was no significant difference in NTCP of heart and spinal cord between two plans (all P>0.05). Conclusion:It is feasible to generate automatic uARC plan with uRT-TPOIS TPS for esophageal cancer patients, which can increase the target CI and shorten the plan design time.

ObjectiveAnalyze the current situation, hotspots and development trends of sarcopenia animal model to provide research direction and basic information for sarcopenia animal model research. MethodsEnglish literature of research on animal models of sarcopenia was retrieved from the Web of Science core data (WOS) set from 1900-01-01 to 2022-12-31. Chinese literature related to animal models of sarcopenia was retrieved from CNKI database between 1915 and 2022. The bibliometric analysis software VOSviewer was used to explore the countries, orgonizations, authors, hotspots and frontier directions in these studies. ResultsA total of 2 819 articles on animal models of sarcopenia were retrieved from WOS core database. The first paper was published in 1995. The United States has the largest number of animal model studies of sarcopenia with 1 105 articles. The institution with the most published articles is the University of Florida in the United States, with 69 articles. The University of Hong Kong has the highest number of publications in China, with 20 articles. American author Van Remmen H, with 50 publications, is the author of the most articles. The journal with the largest number of articles published on animal models of sarcopenia is the American journal called FASEB Journal, with 196 articles. In total, 423 articles on animal models of sarcopenia were retrieved from the CNKI database. Author LI Zhuyi has published 19 articles, and is the author of the most articles in China. The keyword co-occurrence clustering analysis of WOS literature search found that the research focus on animal model of sarcopenia can be summarized as the correlation between sarcopenia and metabolism, cytology and regenerative medicine of sarcopenia animal models, the study of sarcopenia animal models in bone, muscle, nerve and exercise therapy. The retrieval results of CNKI database revealed that the most extensive research was about on the model of denervated sarcopenia, and researches on the effects of Chinese medicine on sarcopenia were also widely reported. Through reading the full articles or abstracts of the literature, the animal models of sacopenia mainly include natural aging model, genetic modification model, high-fat diet induction model, disuse model, hormone induction model and complex sarcopenia models of other diseases. ConclusionIn recent years, the study on animal model of sarcopenia has become a hotspot at home and abroad.The bibliometric analysis provides a basis for the research of animal models of sarcopenia in terms of research direction, hotspots, model animal selection, animal model making, and domestic and international communication and cooperation.

An on-line HPLC-DPPH system was developed to determine the antioxidant activity of 16 batches of Polygoni Multiflori Radix Praeparata. By analyzing the chromatographic and biological activity fingerprints of 16 batches of Polygoni Multiflori Radix Praeparata, the dose-effect relationship was established and the total antioxidant activity was quantified by activity addition.The results suggested that the online HPLC-DPPH method can evaluate the antioxidant activity of different bathches of Polygoni Multiflori Radix Praeparata, with different processing methods, aiming to provide datasupport and scientific basis forquality evaluation of Polygoni Multiflori Radix Praeparata.