Objective:To investigate the expression of Artemin (ARTN) in malignant peripheral nerve sheath tumor (MPNST), its effect on the malignant behavior of MPNST cells, and its signaling pathway.Methods:Fifty-one MPNST paraffin embedded tissues through surgical resection at Tianjin Medical University Cancer Hospital from January 1995 to November 2011 were collected, the expression of the ARTN protein was detected by immunohistochemistry, and the relationship between the ARTN protein expression and the clinical pathological characteristics and prognosis were analyzed. In human MPNST cell lines ST-8814 (NF-1) and STS26T(sporadic), ARTN overexpression and low expression cell lines were constructed by transfecting ARTN overexpression plasmids and ARTN small interfering RNA (siRNA), respectively. The expression of ARTN mRNA was detected by real time quantitative polymerase chain reaction (RT-qPCR), the expression of the ARTN protein and Phosphoinositide 3-kinase(PI3K)/Akt signaling pathway related proteins were detected by Western blot. CCK-8 assay was used to detect cell proliferation ability, and cell invasion assay was used to detect cell invasion ability. The pathway proteins that interacted with ARTN were searched in the STRING database, and the functional pathways were clarified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The PI3K/Akt pathway specific inhibitor LY294002 was used to block the PI3K/Akt pathway of ST-8814 and STS26T cells to observe the changes in cell proliferation and invasion.Results:Among the 51 MPNST tissue specimens, 22 cases showed a high expression of the ARTN protein and 29 cases showed a low expression of the protein. Higher expressions of the ARTN protein was associated with larger tumor diameters and disease progression (recurrence or metastasis) (both P＜0.05). The median disease-free survival (DFS) of patients with a low expression of the ARTN protein was 26.2 months, and the median overall survival (OS) was 66.9 months. The median DFS and median OS of patients with a high expression of the ARTN protein were 10.7 months and 53.8 months, respectively. The log rank test results showed that the progression free survival rate of patients with a high expression of the ARTN protein was worse than that of patients with a low expression ( P=0.027), but the difference in overall survival rate between the two groups was not statistically significant ( P=0.790), which was also confirmed by Cox regression analysis. The CCK-8 assay results showed that after 48 hours of transfection, the absorbance ( A) values of ST-8814 and STS26T cells in the ARTN overexpression group were 1.35±0.01 and 1.10±0.02, respectively, which were higher than those in the empty plasmid control group (1.05±0.01 and 0.78±0.01, both P＜0.01), while the A values of ST-8814 and STS26T cells in the ARTN siRNA group were 0.35±0.01 and 0.61±0.01, respectively, which were lower than those in the control siRNA group (0.74±0.01 and 1.10±0.04, both P＜0.01). The results of cell invasion assay showed that the number of transmembrane cells in ST-8814 and STS26T cells overexpressing ARTN was (29.67±2.08) and (31.67±2.08), respectively, which were higher than those in the empty plasmid control group [(20.00±1.00) and (24.33±1.15), both P＜0.01]. The number of transmembrane cells in ST-8814 and STS26T cells in the ARTN siRNA group were (14.00±2.00) and (19.33±1.53), respectively, which were lower than those in the control siRNA group [(19.33±2.52) and (23.33±0.58), both P＜0.05].The KEGG results showed that ARTN is associated with multiple tumor signaling pathways, especially the PI3K/Akt signaling pathway. Western blot results showed that overexpression of ARTN upregulated the expression of p-PI3K and p-Akt proteins in ST-8814 and STS26T cells (both P＜0.01).After knocking down ARTN expression, the expression of p-PI3K and p-Akt proteins was significantly down regulated (both P＜0.01). LY294002 could significantly inhibit the effect of ARTN overexpression on ST-8814 and STS26T cells after blocking the PI3K/Akt pathway. The A values of ST-8814 and STS26T cells in the ARTN overexpression+LY294002 group were 1.09±0.06 and 0.82±0.01, respectively, which were lower than those in the ARTN overexpression group (1.50±0.01 and 1.29±0.01, respectively, both P＜0.01). The numbers of transmembrane cells in the cell invasion assay were 16.67±3.21 and 19.67±2.31, respectively, which were also lower than those in the ARTN overexpression group (29.67±2.08 and 31.67±2.08, respectively, both P＜0.01). Conclusions:In MPNST, a high expression of the ARTN protein was associated with larger tumor size, disease progression, and worse DFS. ARTN promotes the proliferation and invasion of MPNST cells through the PI3K/Akt signaling pathway.

Objective:To investigate the clinical efficacy of liquid nitrogen cryopreservation and reimplantation versus allograft reconstruction in patients underwent resection of primary malignant bone tumors of the long bones of the extremities.Methods:A retrospective analysis was conducted on 144 patients who underwent resection of primary malignant bone tumors of the long bones of the extremities followed by either liquid nitrogen cryopreservation and reimplantation or massive allografts reconstruction at the Beijing Jishuitan Hospital affiliated with Capital Medical University from January 2012 to July 2023. The study included 82 males and 62 females, with an average age of 23.8±12.3 years (range, 6-64 years). Patients were divided into two groups based on the reconstruction method: the cryopreservation and reimplantation group (72 cases) and the allograft group (72 cases). The following outcomes were recorded during follow-up: local tumor recurrence, bone union, union time, graft survival, and reasons for graft removal. Graft-related complications were recorded using the modified Henderson classification system of the International Limb Salvage Association. Limb function was assessed at the last follow-up using the Musculoskeletal Tumor Society score (MSTS-93).Results:All patients completed surgery and were followed up for a mean of 60.2±32.1 months (range, 12-149 months). At the last follow-up, 24 patients were dead from the tumor, 16 patients survived with the tumor (2 cases of local recurrence and 14 cases of distant metastasis), and 104 patients survived without the tumor. The bone union rate and union time in cryopreservation and reimplantation group were 90% (65/72) and 9.6±4.8 months, respectively, which was significantly superior to those in allograft group [68% (49/72) and 15.9±6.7 months, P<0.05]. The 5-year overall graft survival rate was 86.8% [95% CI (80.1%, 95.7%)] in cryopreservation and reimplantation group, higher than 73.2% [95% CI(68.4%, 84.5%)] in allograft group significantly (χ 2=7.122, P=0.017). The rates of graft removal due to non-union and infection in the cryopreservation and reimplantation group were 0% (0/72) and 1.4% (1/72), respectively, which were significantly lower than those in the allograft group [5.6% (4/72) and 9.7% (7/72), P<0.05]. Overall, 48.6% (70/144) of patients experienced at least one graft-related complication, with a complication rate of 33.3% in cryopreservation and reimplantation group, lower than the 61.1% in allograft group significantly (χ 2=11.146, P<0.001). The complications with the highest incidence rate were nonunion (20.8%, 30/144), followed by structural failure (17.4%, 25/144), tumor progression (10.4%, 15/144), infection (10.4%, 15/144), and soft tissue failure (5.6%, 8/144). The incidence rates of the atrophic non-union and the structural failure of grafts were 9.7% (7/72) and 1.4% (1/72) respectively in the cryopreservation and reimplantation group, which were significantly lower compared to the allograft group [29.2% (21/72) and 13.9% (10/72), P<0.05]. At the last follow-up, the MSTS-93 score was 89.7%±8.3% in the cryopreservation and reimplantation group, and 87.6%±7.5% in the allograft group, with no statistically significant difference ( t=0.326, P=0.542). Conclusion:Compared with allograft reconstruction, autologous inactivated bone grafting demonstrated superior bone union efficiency and fewer complications, it may be considered for reconstruction in cases whose tumor bone is not severely osteolytic or pathologically fractured.

OBJECTIVE To compare the emergency specialist clinical pharmacist training programs between China and the United States， providing valuable insight for the development of specialized clinical pharmacist training in emergency departments within China. METHODS By reviewing the official website of the American Society of Health-System Pharmacists （ASHP）， the websites of some training institutions offering PGY2 emergency medicine （EM） residency programs in the United States， the official website of China’s National Health Commission， and the website of the Pharmaceutical Affairs Committee of the Chinese Hospital Association， relevant materials and data on the training of emergency medicine clinical pharmacists were collected. Microsoft Excel and NVivo software were utilized to analyze the implementation status of these training programs. Literature searches were conducted via Chinese （CNKI） and English （PubMed） databases， followed by screening， categorization， and thematic analysis aligned with research objectives. RESULTS As of now， there are 115 accredited PGY2 EM residency programs in the United States， which provide 120 specialized pharmacist training positions. These programs are distributed across 35 states and are hosted by a variety of institutions， including hospitals， medical centers， and universities. The predominant training model follows a hospital+acute care framework. Eligibility requirements for PGY2 EM residency programs include possession of a doctor of pharmacy （Pharm.D.） degree， pharmacist licensure， and completion of a PGY1 residency. The training standards are structured into three tiers： competency areas， competency goals， and learning objectives. The curriculum typically includes core rotations， elective rotations， and longitudinal training components. Assessment is conducted through a combination of formative and summative evaluations， with results categorized into four proficiency levels. In China， there is only one training base currently for emergency clinical pharmacist specialty training with an annual enrollment of three trainees. Applicant eligibility primarily involves requirements regarding academic degree， professional background， years of experience， and professional title. The training content covers four domains： general competency， clinical theoretical knowledge and skills， pharmacological knowledge and application， and clinical medication practice skills. The training process centers on rotations within emergency departments. Assessment methods include theoretical examinations， daily performance evaluations， and final completion assessments. CONCLUSIONS PGY2 EM residency programs in the United States emphasize inclusivity and professionalism in their implementation. Program admission involves a rigorous selection process， and they offer attractive incentive structures for trainees. The training content focuses on competency-based approaches and pragmatic applicability， while assessment methods are closely aligned with defined competence objectives. In contrast， specialist clinical pharmacist training in emergency medicine in China is currently in the exploratory and nascent stages. Admission criteria tend to be less stringent， and incentives for trainees are often insufficient. The training content appears relatively stereotyped and superficial， with assessment methods still primarily reliant on quantifiable metrics. In expanding and popularizing China’s emergency specialist clinical pharmacist training programs， it is essential to draw on advanced experiences from developed countries like the United States， particularly in areas such as training base distribution， application requirements， training content， and assessment methods. Aligned with the realities of emergency clinical practice in China， efforts should focus on enhancing program accessibility and training efficacy.

BACKGROUND:Currently,exercise therapy is an effective non-pharmacological treatment for low back pain,and exercise therapy can maintain lumbar spine stabilization through mechanical-chemical coupling between bones and muscles,but there is no clear description of the research progress and optimal treatment protocols for exercise therapy to relieve chronic non-specific lower back pain through mechanical-chemical coupling. OBJECTIVE:To review the research progress related to the influence of paravertebral muscles on lumbar spine stabilization during exercise therapy through mechanical-chemical coupling,which in turn relieves chronic non-specific lower back pain,as well as the current optimal treatment protocols of exercise therapy for chronic non-specific lower back pain. METHODS:Literature searches were performed in WanFang database,CNKI,VIP,Web of Science,and PubMed database,with search terms of"chronic non-specific low back pain,lumbar spine stabilization,paravertebral muscles,exercise therapy"in Chinese and English.Relevant literature published from database inception to January 2024 was searched and 93 articles were included for final summarization. RESULTS AND CONCLUSION:Exercise therapy can act on the paravertebral muscles and bones through appropriate mechanical stimulation and produce corresponding changes.Exercise therapy is an important intervention for chronic non-specific lower back pain as it improves the quality of the paravertebral muscles,primarily through mechanical-chemical coupling,and thus maintains lumbar spine stabilization for better relief of chronic non-specific lower back pain.However,there are no clear reports on the exact effective protocols for exercise therapy to treat chronic non-specific lower back pain through lumbar spine stabilization.The development of an individualized exercise program is particularly important for the treatment and prognosis of chronic non-specific low back pain.Muscle mass and bone mass of the same individual are closely related,and imaging assessment of paravertebral muscle mass and quantity is important for disease detection and intervention.

OBJECTIVE To explore the effect and potential mechanism of Chaihu longgu muli decoction on hippocampal neuronal damage in refractory epilepsy rats. METHODS The refractory epilepsy rat model was established by intracerebroventricular injection of kainic acid. The refractory epilepsy rats were randomly assigned into model group， celecoxib group （positive control）， Chaihu longgu muli decoction group， Chaihu longgu muli decoction+empty loading group， Chaihu longgu muli decoction+cyclooxygenase-2 （COX-2） overexpression group， with 12 rats in each group. Another 12 rats were selected as sham operation group （injected with normal saline into the lateral ventricle）. After 8 weeks of corresponding drug/normal saline intervention， Racine grading criteria were applied to evaluate the seizure behavior of rats； pathological changes and cell apoptosis in CA3 region of hippocampal tissue were observed； ELISA method was adopted to detect the levels of COX-2， prostaglandin E2 （PGE2）， and tumor necrosis factor-α （TNF-α） in hippocampal tissue. The colorimetric method was applied to detect the content of malondialdehyde （MDA） and the activity of superoxide dismutase （SOD） in hippocampal tissue. Real-time fluorescence quantitative PCR and Western blot were applied to detect the mRNA and protein expressions of COX-2 and P-glycoprotein （P-gp） in hippocampal tissue. RESULTS The rats in the sham operation group had no epileptic seizures. Compared with sham operation group， Racine grade of epilepsy， apoptotic rate of hippocampal tissue cells， COX-2， PGE2 and TNF-α levels， MDA contents， mRNA and protein levels of COX-2 and P-gp in the model group increased significantly， while SOD activity decreased significantly （P＜0.05）； prominent damage was observed in the hippocampal CA3 region. Compared with model group， Racine grade of epilepsy， apoptotic rate of hippocampal tissue cells， COX-2， PGE2 and TNF-α levels， MDA contents， mRNA and protein levels of COX-2 and P-gp in celecoxib group and Chaihu longgu muli decoction group decreased significantly， while SOD activity increased significantly（P＜0.05）； the neuronal damage in hippocampal CA3 region was alleviated. Compared with Chaihu longgu muli decoction group and Chaihu longgu muli decoction+ empty loading group， the improvement in the aforementioned indicators in rats from Chaihu longgu muli decoction+COX-2 overexpression group was significantly reversed （P＜0.05）， with exacerbated neuronal damage in the CA3 region of the hippocampal tissue. CONCLUSIONS Chaihu longgu muli decoction may alleviate hippocampal neuronal damage in refractory epilepsy rats by inhibiting the activation of COX-2/PGE2 signaling pathway， inhibiting inflammation and oxidative stress， thereby alleviating hippocampal neuronal damage in refractory epilepsy rats.

Objective:To investigate the clinical efficacy of liquid nitrogen cryopreservation and reimplantation versus allograft reconstruction in patients underwent resection of primary malignant bone tumors of the long bones of the extremities.Methods:A retrospective analysis was conducted on 144 patients who underwent resection of primary malignant bone tumors of the long bones of the extremities followed by either liquid nitrogen cryopreservation and reimplantation or massive allografts reconstruction at the Beijing Jishuitan Hospital affiliated with Capital Medical University from January 2012 to July 2023. The study included 82 males and 62 females, with an average age of 23.8±12.3 years (range, 6-64 years). Patients were divided into two groups based on the reconstruction method: the cryopreservation and reimplantation group (72 cases) and the allograft group (72 cases). The following outcomes were recorded during follow-up: local tumor recurrence, bone union, union time, graft survival, and reasons for graft removal. Graft-related complications were recorded using the modified Henderson classification system of the International Limb Salvage Association. Limb function was assessed at the last follow-up using the Musculoskeletal Tumor Society score (MSTS-93).Results:All patients completed surgery and were followed up for a mean of 60.2±32.1 months (range, 12-149 months). At the last follow-up, 24 patients were dead from the tumor, 16 patients survived with the tumor (2 cases of local recurrence and 14 cases of distant metastasis), and 104 patients survived without the tumor. The bone union rate and union time in cryopreservation and reimplantation group were 90% (65/72) and 9.6±4.8 months, respectively, which was significantly superior to those in allograft group [68% (49/72) and 15.9±6.7 months, P<0.05]. The 5-year overall graft survival rate was 86.8% [95% CI (80.1%, 95.7%)] in cryopreservation and reimplantation group, higher than 73.2% [95% CI(68.4%, 84.5%)] in allograft group significantly (χ 2=7.122, P=0.017). The rates of graft removal due to non-union and infection in the cryopreservation and reimplantation group were 0% (0/72) and 1.4% (1/72), respectively, which were significantly lower than those in the allograft group [5.6% (4/72) and 9.7% (7/72), P<0.05]. Overall, 48.6% (70/144) of patients experienced at least one graft-related complication, with a complication rate of 33.3% in cryopreservation and reimplantation group, lower than the 61.1% in allograft group significantly (χ 2=11.146, P<0.001). The complications with the highest incidence rate were nonunion (20.8%, 30/144), followed by structural failure (17.4%, 25/144), tumor progression (10.4%, 15/144), infection (10.4%, 15/144), and soft tissue failure (5.6%, 8/144). The incidence rates of the atrophic non-union and the structural failure of grafts were 9.7% (7/72) and 1.4% (1/72) respectively in the cryopreservation and reimplantation group, which were significantly lower compared to the allograft group [29.2% (21/72) and 13.9% (10/72), P<0.05]. At the last follow-up, the MSTS-93 score was 89.7%±8.3% in the cryopreservation and reimplantation group, and 87.6%±7.5% in the allograft group, with no statistically significant difference ( t=0.326, P=0.542). Conclusion:Compared with allograft reconstruction, autologous inactivated bone grafting demonstrated superior bone union efficiency and fewer complications, it may be considered for reconstruction in cases whose tumor bone is not severely osteolytic or pathologically fractured.

Objective:To investigate the expression of Artemin (ARTN) in malignant peripheral nerve sheath tumor (MPNST), its effect on the malignant behavior of MPNST cells, and its signaling pathway.Methods:Fifty-one MPNST paraffin embedded tissues through surgical resection at Tianjin Medical University Cancer Hospital from January 1995 to November 2011 were collected, the expression of the ARTN protein was detected by immunohistochemistry, and the relationship between the ARTN protein expression and the clinical pathological characteristics and prognosis were analyzed. In human MPNST cell lines ST-8814 (NF-1) and STS26T(sporadic), ARTN overexpression and low expression cell lines were constructed by transfecting ARTN overexpression plasmids and ARTN small interfering RNA (siRNA), respectively. The expression of ARTN mRNA was detected by real time quantitative polymerase chain reaction (RT-qPCR), the expression of the ARTN protein and Phosphoinositide 3-kinase(PI3K)/Akt signaling pathway related proteins were detected by Western blot. CCK-8 assay was used to detect cell proliferation ability, and cell invasion assay was used to detect cell invasion ability. The pathway proteins that interacted with ARTN were searched in the STRING database, and the functional pathways were clarified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The PI3K/Akt pathway specific inhibitor LY294002 was used to block the PI3K/Akt pathway of ST-8814 and STS26T cells to observe the changes in cell proliferation and invasion.Results:Among the 51 MPNST tissue specimens, 22 cases showed a high expression of the ARTN protein and 29 cases showed a low expression of the protein. Higher expressions of the ARTN protein was associated with larger tumor diameters and disease progression (recurrence or metastasis) (both P＜0.05). The median disease-free survival (DFS) of patients with a low expression of the ARTN protein was 26.2 months, and the median overall survival (OS) was 66.9 months. The median DFS and median OS of patients with a high expression of the ARTN protein were 10.7 months and 53.8 months, respectively. The log rank test results showed that the progression free survival rate of patients with a high expression of the ARTN protein was worse than that of patients with a low expression ( P=0.027), but the difference in overall survival rate between the two groups was not statistically significant ( P=0.790), which was also confirmed by Cox regression analysis. The CCK-8 assay results showed that after 48 hours of transfection, the absorbance ( A) values of ST-8814 and STS26T cells in the ARTN overexpression group were 1.35±0.01 and 1.10±0.02, respectively, which were higher than those in the empty plasmid control group (1.05±0.01 and 0.78±0.01, both P＜0.01), while the A values of ST-8814 and STS26T cells in the ARTN siRNA group were 0.35±0.01 and 0.61±0.01, respectively, which were lower than those in the control siRNA group (0.74±0.01 and 1.10±0.04, both P＜0.01). The results of cell invasion assay showed that the number of transmembrane cells in ST-8814 and STS26T cells overexpressing ARTN was (29.67±2.08) and (31.67±2.08), respectively, which were higher than those in the empty plasmid control group [(20.00±1.00) and (24.33±1.15), both P＜0.01]. The number of transmembrane cells in ST-8814 and STS26T cells in the ARTN siRNA group were (14.00±2.00) and (19.33±1.53), respectively, which were lower than those in the control siRNA group [(19.33±2.52) and (23.33±0.58), both P＜0.05].The KEGG results showed that ARTN is associated with multiple tumor signaling pathways, especially the PI3K/Akt signaling pathway. Western blot results showed that overexpression of ARTN upregulated the expression of p-PI3K and p-Akt proteins in ST-8814 and STS26T cells (both P＜0.01).After knocking down ARTN expression, the expression of p-PI3K and p-Akt proteins was significantly down regulated (both P＜0.01). LY294002 could significantly inhibit the effect of ARTN overexpression on ST-8814 and STS26T cells after blocking the PI3K/Akt pathway. The A values of ST-8814 and STS26T cells in the ARTN overexpression+LY294002 group were 1.09±0.06 and 0.82±0.01, respectively, which were lower than those in the ARTN overexpression group (1.50±0.01 and 1.29±0.01, respectively, both P＜0.01). The numbers of transmembrane cells in the cell invasion assay were 16.67±3.21 and 19.67±2.31, respectively, which were also lower than those in the ARTN overexpression group (29.67±2.08 and 31.67±2.08, respectively, both P＜0.01). Conclusions:In MPNST, a high expression of the ARTN protein was associated with larger tumor size, disease progression, and worse DFS. ARTN promotes the proliferation and invasion of MPNST cells through the PI3K/Akt signaling pathway.

Objective:To investigate the effect of Hong's One Stitch Method in pancreaticoduodenectomy(PD).Methods:A total of 40 patients who underwent PD in our hospital from Jan 2021 to Dec 2022 were divided into two groups according to random number table method,with 20 patients in each group.The control group was treated with end to end pancreatojejunal anastomosis,and the observation group was treated with"Hong's One Stitch Method".The perioperative indicators,complications,secondary surgery,mortality and quality of life were compared between the two groups.Results:The pancreatoenteroanastomosis time,operation time and hospitalization time in the observation group were shorter than those in the control group,and the incidence of pancreatic fistula was lower than that in the control group(P＜0.05).There were no significant differences in intraoperative blood loss,pancreatic biochemical leakage,bile fistula,hemorrhage,localized abdominal infection,gastric emptying obstruction,pulmonary infection,secondary surgery and mortality between the two groups(P＞0.05).The mental health score,emotional function score,social function score,energy score,general health status score,body pain score,and physiological function score in the observation group were higher than those in the control group(P＜0.05).Conclusion:In PD surgery,the application of"Hong's One Stitch Method"to perform pancreatoenterostomy is beneficial to shorten the pancreatoenterostomy time,operation time and hospitalization time,accelerate the postoperative recovery,reduce the incidence of pancreatic fistula,and improve the quality of life of patients.

OBJECTIVE To investigate the mechanism by which salvianolic acid A (Sal A) reduces the inflammatory response and oxidative stress of BV2 cells injured by oxygen and glucose deprivation/reperfusion (OGD/R).METHODS An OGD/R injury model of BV2 cells was established with sugar free Earle solution containing Na2S2O410 mmol·L-1.Na2S2O4 sugar free Earle solution was added and cultured in an incubator (37 ℃,5％CO2) for 1.5 h (oxygen glucose deprivation) before a normal medium was used for 24 h (reperfusion).Then,the cells were divided into the cell control group,OGD/R group,OGD/R+Sal A 1,5 and 10 μmol·L-1 group,OGD/R+ML385 group,OGD/R+ML385+Sal A 1,5 and 10μmol·L-1 group and OGD/R+edaravone (Eda,50μmol·L-1) group.After twenty-four hours of culture,the cell survival rate was measured by CCK8 kit.The contents of Interleukin-1β (IL-1β),IL-6,tumor necrosis factor-α(TNF-α),IL-10,IL-4 and transforming growth factor-β(TGF-β) in the cell supernatant were detected by ELISA.Reactive oxygen species (ROS) in cells was detected using the chemical fluo-rescence method.The contents of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD),glutathione peroxidase (GSH-PX) and chloramphenicol acetyltransferase (CAT) in cells were determined with the colorimetric method.Protein expressions of Kelch like ECH-associated protein 1 (Keap1),nuclear factor erythroid-2 related factor 2 (Nrf2),Heme oxygenase-1 (HO-1),NADPH:quinone oxidoreductase-1 (NQO1) and p-nuclear factor kappa-B p65 (p-NF-κB p65) were detected by Western blotting.RESULTS ①Compared with the cell control group,the cell survival rate of the OGD/R group was significantly decreased (P＜0.01).Compared with the OGD/R group,the survival rates of OGD/R+Sal A 1,5 and 10μmol·L-1 groups were significantly increased (P＜0.05,P＜0.01).②Compared with the cell control group,the contents of IL-1β,IL-6 and TNF-α were significantly increased,the contents of IL-10,IL-4 and TGF-β were significantly decreased,the contents of ROS and MDA were significantly increased,and the activities of SOD,CAT and GSH-Px were significantly decreased in the OGD/R group (P＜0.01).Compared with the OGD/R group,the content of IL-6 was significantly decreased,the contents of IL-10,IL-4 and TGF-β were significantly increased,the contents of ROS and MDA were significantly decreased,and the activities of SOD,CAT and GSH-Px were significantly increased in OGD/R+Sal A 1,5 and 10μmol·L-1 and OGD/R+Eda groups (P＜0.05,P＜0.01).③Compared with the cell control group,the protein expression of p-NF-κB P65 in the OGD/R group was significantly increased (P＜0.01).Compared with the OGD/R group,the protein expressions of Keap1 and cytoplasmic Nrf2 were significantly decreased,the expressions of nuclear Nrf2,HO-1 and NQO1 proteins were significantly increased,and the expression of p-NF-κB p65 protein was significantly decreased in OGD/R+Sal A 5 and 10 μmol·L-1 and OGD/R+Eda groups (P＜0.05,P＜0.01).In OGD/R+ML385,OGD/R+ML385+Sal A 1,5 and 10μmol·L-1 groups,the protein expression of Keap1 was significantly increased,the protein expressions of cytoplasmic Nrf2,nuclear Nrf2,HO-1 and NQO1 protein were significantly decreased,and the protein expression of p-NF-κB P65 was significantly increased (P＜0.01).CONCLU-SION Sal A reduces the inflammatory response and oxidative stress of OGD/R injured BV2 cells possi-bly by activating the Keap1/Nrf2 pathway and inhibiting the NF-κB pathway.

Objective:To construct a microfluidic organ-on-a-chip and evaluate its capability in simulating subchondral bone remodeling during the progression of osteoarthritis.Methods:The chip′s main body was designed based on the microfluidic technology and cell co-culture technique. MC3T3-E1 cells were cultured adherently within the cell seeding micro-chamber, with the culture medium perfused at a flow rate of 0.5 ml/min at the bottom of the micro-chamber. Evaluation metrics were as follows: (1) Assessment of the microfluidic organ-on-a-chip: The growth culture medium was perfused and simulation experiments were conducted to test the concentration differences and equilibrium times of the fluid inside and at the bottom of the cell seeding micro-chamber at various time points; live-dead staining was performed to observe the biocompatibility of cells cultured continuously for 3 days and 7 days at a set flow rate, which was divided into 3-day and 7-day groups. (2) Osteogenic potential of the microfluidic organ-on-a-chip: The osteogenic induction medium was perfused, and ALP staining and PCR were performed to compare the number of the black alkaline phosphatase (ALP)-positive cells and the expression levels of osteogenesis-related marker genes including osteoblast-specific transcription factor 2 (RUNX2), type I collagen (COL1A1), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN) under static, 3-day and 7-day perfusion conditions, which was divided into static non-induced, static-induced and perfusion-induced groups. (3) Characterization of morphology and size, and biocompatibility of extracellular vesicles (EVs) of three osteoblast subtypes: Three different subtypes of osteoblasts were obtained [endothelial-type osteoblasts (EnOB)-EVs, stromal-type osteoblasts (StOB)-EVs and mineralizing-type osteoblasts (MinOB)-EVs]. Their morphology and size were obtained through transmission electron microscopy and particle size analysis. Growth medium containing EVs of three different cell subtypes was perfused, and cell proliferation/apoptosis assay was performed to compare the biocompatibility of the addition of different EVs concentrations (1, 1.25, 2.5, and 5 μg/ml) for 24 hours, which was categorized into the EnOB-EVs group, StOB-EVs group and MinOB-EVs group. (4) Osteogenic effect of EVs from three subtypes of osteoblasts: Osteogenic induction media containing EVs from three different osteoblast subtypes were perfused for 3 days, and ALP staining and PCR were performed to compare the number of black ALP-positive cells and the expression levels of osteogenesis-related marker genes including RUNX2, COL1A1, BMP-2, and OCN, which was divided into non-EVs group, EnOB-EVs group, StOB-EVs group and MinOB-EVs group.Results:(1) Evaluation of the microfluidic organ-on-a-chip: Simulation results showed that the concentration in the top layer of the upper chamber reached more than 95% of that in the lower chamber and that the concentration in the bottom layer was about 96.5% of that in the lower chamber after 12 hours of continuous perfusion, reaching an equilibrium state of the concentration difference between the upper and lower chambers. The results of live-dead staining showed that the chip was biocompatible at a flow rate of 0.5 ml/min, and the cell survival rate at 3 and 7 days of perfusion was (99.48±0.12)% and (97.07±1.05)% ( P<0.01). (2) ALP staining results showed that at 3 days, the perfusion-induced group showed the highest number of black ALP-positive cells, followed by the static-induced group, and the least in the static non-induced group. At 7 days, the static-induced group had the highest number of black ALP-positive cells, followed by the perfusion-induced group, and the least in the static non-induced group. PCR results indicated that at 3 days, the expression levels of RUNX2, COL1A1, BMP-2, and OCN were 1.00±0.03, 1.00±0.12, 1.00±0.01, and 1.00±0.02 respectively in the static non-induced group; 1.80±0.04, 4.05±0.37, 9.80±1.94, and 4.38±0.89 respectively in the static-induced group, and 2.45±0.23, 5.48±0.42, 91.50±4.56, and 10.82±4.96 respectively in the perfusion-induced group ( P<0.01). At 7 days, the expression levels of RUNX2 was 1.00±0.01 in the static non-induced group, 1.46±0.46 in the static-induced group, and 1.11±0.08 in the perfusion-induced group ( P>0.05); the expression levels of COL1A1, BMP-2, and OCN were 1.00±0.03, 1.00±0.13, and 1.00±0.09 respectively in the static non-induced group, 9.38±0.25, 14.27±4.35, and 84.01±4.02 respectviely in the static-induced group, and 2.39±0.08, 133.64±8.87, and 86.64±8.36 respectively in the perfusion-induced group ( P<0.01). When comparing the static non-induced, static-induced, and perfusion-induced groups at both 3 and 7 days, the perfusion-induced group demonstrated the strongest osteogenic capability. (3) Characterization of morphology and size and biocompatibility of EVs from three osteoblast subtypes: Under the transmission electron microscope, EVs from EnOB-EVs, StOB-EVs, and MinOB-EVs all exhibited a typical saucer-shaped morphology. The particle sizes of EnOB-EVs, StOB-EVs, and MinOB-EVs were (91.3±14.7)nm, (106.0±16.0)nm, and (68.1±10.7)nm, respectively. Cell proliferation/apoptosis assay results indicated that the optimal administration concentration of EnOB-EVs, StOB-EVs, and MinOB-EVs was all 1.25 μg/mL. (4) Validation of osteogenic effect of the microfluidic organ-on-a-chip on three types of EVs: ALP staining results showed that the non-EVs group had the fewest black ALP-positive cells, followed by the EnOB-EVs group, then the StOB-EVs group, and the MinOB-EVs group had the most. PCR results showed that the expression levels of RUNX2, COL1A1, BMP-2, and OCN were 1.00±0.01, 1.00±0.03, 1.00±0.02, and 1.00±0.02 respectively in the non-EVs group, 1.95±0.11, 6.78±2.04, 7.99±0.57, and 6.93±3.83 repectively in the EnOB-EVs group, 0.79±0.12, 5.68±1.53, 12.59±3.15, and 25.59±0.95 respectively in the StOB-EVs group, and 0.68±0.10, 4.36±0.69, 18.75±3.21, and 34.74±3.98 repectively in the MinOB-EVs group ( P<0.01). Compared with the non-EVs group, EnOB-EVs group, StOB-EVs group, and MinOB-EVs group, the MinOB-EVs group showed the most significant osteogenic effect. Conclusions:The microfluidic organ-on-a-chip constructed using microfluidic technology and cell co-culture techniques is capable of maintaining the normal growth of MC3T3-E1 cells, enhancing their proliferation and osteogenic induction differentiation. EVs released by osteoblasts at different stages possess osteogenic effects and can accelerate the bone sclerosis in the remodeling of subchondral bone during the progression of osteoarthritis.