Stem-leaf saponins from Panax notoginseng (SLSP) comprise numerous PPD-type saponins with diverse pharmacological properties; however, their role in Parkinson's disease (PD), characterized by microglia-mediated neuroinflammation, remains unclear. This study evaluated the effects of SLSP on suppressing microglia-driven neuroinflammation in experimental PD models, including the 1-methyl-4-phenylpyridinium (MPTP)-induced mouse model and lipopolysaccharide (LPS)-stimulated BV-2 microglia. Our findings revealed that SLSP mitigated behavioral impairments and excessive microglial activation in models of PD, including MPTP-treated mice. Additionally, SLSP inhibited the upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) and attenuated the phosphorylation of PI3K, protein kinase B (AKT), nuclear factor-κB (NFκB), and inhibitor of NFκB protein α (IκBα) both in vivo and in vitro. Moreover, SLSP suppressed the production of inflammatory markers such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) in LPS-stimulated BV-2 cells. Notably, the P2Y2R agonist partially reversed the inhibitory effects of SLSP in LPS-treated BV-2 cells. These results suggest that SLSP inhibit microglia-mediated neuroinflammation in experimental PD models, likely through the P2Y2R/PI3K/AKT/NFκB signaling pathway. These novel findings indicate that SLSP may offer therapeutic potential for PD by attenuating microglia-mediated neuroinflammation.
Animals ; Panax notoginseng/chemistry* ; Saponins/pharmacology* ; Microglia/immunology* ; Mice ; NF-kappa B/immunology* ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/immunology* ; Phosphatidylinositol 3-Kinases/genetics* ; Male ; Parkinson Disease/immunology* ; Mice, Inbred C57BL ; Disease Models, Animal ; Plant Leaves/chemistry* ; Neuroinflammatory Diseases/drug therapy* ; Humans

Background and purpose:Intracellular deubiquitylating enzymes,such as ubiquitin-specific peptidase 2(USP2),play a pivotal role in regulating protein degradation and cellular homeostasis by modulating protein ubiquitin deconjugation,which have been implicated in the proliferation and survival of multiple myeloma(MM)cells.Targeting the inhibition of USP2 activity in MM cells might modulate their biological behavior.This study aimed to investigate regulatory effects of the leukotriene receptor antagonist montelukast sodium on USP2 in MM cells and its subsequent biological effects.Methods:An in vitro deubiquitination reaction system was established using purified USP2 protein and its substrate,the glutathione S-transferase(GST)tagged ubiquitin A-52 residue ribosomal protein fusion product(UbA52),known as GST-UbA52 protein.This system was used to characterize inhibitory effects of montelukast sodium on USP2 deubiquitinase activity.The MM cell lines MM1.S and H929 were used as in vitro models.Cellular thermal shift assay(CETSA)was subsequently employed to test interaction mode between montelukast sodium and USP2 in MM cells.Western blot assay was applied to detect expression levels of USP2 and its targeting regulators,including cell cycle supervisors cyclin D1(CCND1)and cyclin A1(CCNA1),classical signaling transducer KRAS and glucose regulated protein 78kD(GRP78),as well as apoptotic molecule C/EBP-homologous protein(CHOP)in MM1.S and H929 cells before and after the treatment with different concentrations of montelukast sodium.MM cells with either overexpression(H929-OE,MM1.S-OE)or knockdown(H929-LE,MM1.S-LE)of USP2 were generated using a lentiviral vector.Cell counting kit-8(CCK-8)and flow cytometry were utilized to detect the proliferation and apoptotic rates of H929-OE,MM1.S-OE,H929-LE and MM1.S-LE cells treated with montelukast sodium.Results:Montelukast sodium was found to inhibit USP2 mediated degradation of GST-UbA52 protein in a concentration-dependent manner,with a half inhibitory concentration(IC50)of 3.814 μmol/L.Additionally,montelukast sodium significantly enhanced the thermal stability of USP2 at temperatures of 49.1,53.2 and 56.4℃.It was also shown that montelukast sodium could down-regulate expressions of CCND1,CCNA1 and KRAS,while increase levels of GRP78 and CHOP in MM1.S and H929 cells.Furthermore,after treating with 40 μmol/L montelukast sodium for 24 h,the proliferation inhibition and apoptotic rate of H929-OE cells reached to(37.68±1.10)%and(18.99±0.26)%,while the proliferation inhibition and apoptotic rate of MM1.S-OE cells reached to(24.48±0.49)%and(33.29±0.75)%,which were significantly lower than those in H929 and MM1.S cells[H929:(57.19±1.93)%and(45.65±0.24)%;MM1.S:(50.04±0.53)%and(40.25±0.91)%;P＜0.05,n=3].Conversely,the proliferation inhibition and apoptotic rates of H929-LE and MM1.S-LE cells were significantly higher[H929-LE-1#:(80.70±1.60)%and(89.08±0.49)%;H929-LE-2#:(75.30±3.80)%and(82.41±1.07)%;MM1.S-LE-1#:(70.64±0.84)%and(67.63±0.21)%;MM1.S-LE-2#:(68.47±1.32)%and(85.90±0.18)%;P＜0.05,n=3].Conclusion:Montelukast sodium can target ubiquitin proteasome regulator USP2 and inhibit its deubiquitylating activity,which may modulate USP2 directing protein and trigger endoplasmic reticulum stress to induce cell cycle arrest and apoptosis in MM cells.

Objective To explore the mechanism of TPT1-AS1 targeting miR-324/TWIST1 axis to regulate the proliferation, invasion, migration and angiogenesis of epithelial ovarian cancer (EOC) cells, thereby affecting ovarian cancer (OC) progression. Methods RT-qPCR was used to detect the expression of TPT1-AS1 and miR-324 in 29 OC lesions and adjacent tissue samples. The two OC cell models of TPT1-AS1 overexpression and miRNA324 knockdown were constructed, and the cell proliferation, invasion and migration abilities were detected by CCK-8, Transwell^TM and scratch test. Western blot analysis was used to detect the protein expression levels of TWIST1, epithelial cadherin (E-cadherin), Vimentin, and vascular endothelial growth factor A (VEGF-A) in OC cells. Fluorescence in situ hybridization (FISH) and RNA pull-down experiments were used to verify the interaction between TPT1-AS1 and miR-324. Immunohistochemistry and Targetscan bioinformatics analysis were used to verify the negative regulatory role of miR-324 in the epithelial-mesenchymal transition (EMT) process. Results The TPT1-AS1 expression was significantly higher in OC tissues than that in para-cancerous tissues, while the miR-324 expression was significantly lower. In SKOV3 cells with TPT1-AS1 overexpression, the miR-324 expression decreased significantly, and TPT1-AS1 was negatively correlated with miR-324. It was also found that TPT1-AS1 and miR-324 were co-expressed in OC cells, and there was a direct binding relationship between them. Down-regulation of miR-324 significantly promoted the proliferation, invasion and migration of SKOV3 cells. Further studies revealed that miR-324 had a binding site at the 3'-UTR end of the TWIST1, a key transcription factor for EMT. Inhibiting miR-324 expression increased the transcription level of TWIST1, leading to a decrease in E-cadherin protein expression and an increase in Vimentin protein expression. Additionally, the downregulation of miR-324 resulted in an increased expression level of VEGF-A protein, which in turn enhanced angiogenesis of OC. Conclusion TPT1-AS1 promotes EOC cell proliferation, invasion, migration and angiogenesis by negatively regulating the miR-324/TWIST1 axis, thus promoting the development of OC. These findings provide new potential targets for the diagnosis and treatment of OC.
Humans ; MicroRNAs/metabolism* ; Female ; Cell Movement/genetics* ; Ovarian Neoplasms/blood supply* ; Twist-Related Protein 1/metabolism* ; Cell Line, Tumor ; Neovascularization, Pathologic/genetics* ; Neoplasm Invasiveness ; Carcinoma, Ovarian Epithelial/metabolism* ; Nuclear Proteins/metabolism* ; Cell Proliferation/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic ; RNA, Long Noncoding/metabolism* ; Cadherins/genetics* ; Vascular Endothelial Growth Factor A/genetics* ; Vimentin/genetics* ; Angiogenesis

Background and purpose:Intracellular deubiquitylating enzymes,such as ubiquitin-specific peptidase 2(USP2),play a pivotal role in regulating protein degradation and cellular homeostasis by modulating protein ubiquitin deconjugation,which have been implicated in the proliferation and survival of multiple myeloma(MM)cells.Targeting the inhibition of USP2 activity in MM cells might modulate their biological behavior.This study aimed to investigate regulatory effects of the leukotriene receptor antagonist montelukast sodium on USP2 in MM cells and its subsequent biological effects.Methods:An in vitro deubiquitination reaction system was established using purified USP2 protein and its substrate,the glutathione S-transferase(GST)tagged ubiquitin A-52 residue ribosomal protein fusion product(UbA52),known as GST-UbA52 protein.This system was used to characterize inhibitory effects of montelukast sodium on USP2 deubiquitinase activity.The MM cell lines MM1.S and H929 were used as in vitro models.Cellular thermal shift assay(CETSA)was subsequently employed to test interaction mode between montelukast sodium and USP2 in MM cells.Western blot assay was applied to detect expression levels of USP2 and its targeting regulators,including cell cycle supervisors cyclin D1(CCND1)and cyclin A1(CCNA1),classical signaling transducer KRAS and glucose regulated protein 78kD(GRP78),as well as apoptotic molecule C/EBP-homologous protein(CHOP)in MM1.S and H929 cells before and after the treatment with different concentrations of montelukast sodium.MM cells with either overexpression(H929-OE,MM1.S-OE)or knockdown(H929-LE,MM1.S-LE)of USP2 were generated using a lentiviral vector.Cell counting kit-8(CCK-8)and flow cytometry were utilized to detect the proliferation and apoptotic rates of H929-OE,MM1.S-OE,H929-LE and MM1.S-LE cells treated with montelukast sodium.Results:Montelukast sodium was found to inhibit USP2 mediated degradation of GST-UbA52 protein in a concentration-dependent manner,with a half inhibitory concentration(IC50)of 3.814 μmol/L.Additionally,montelukast sodium significantly enhanced the thermal stability of USP2 at temperatures of 49.1,53.2 and 56.4℃.It was also shown that montelukast sodium could down-regulate expressions of CCND1,CCNA1 and KRAS,while increase levels of GRP78 and CHOP in MM1.S and H929 cells.Furthermore,after treating with 40 μmol/L montelukast sodium for 24 h,the proliferation inhibition and apoptotic rate of H929-OE cells reached to(37.68±1.10)%and(18.99±0.26)%,while the proliferation inhibition and apoptotic rate of MM1.S-OE cells reached to(24.48±0.49)%and(33.29±0.75)%,which were significantly lower than those in H929 and MM1.S cells[H929:(57.19±1.93)%and(45.65±0.24)%;MM1.S:(50.04±0.53)%and(40.25±0.91)%;P＜0.05,n=3].Conversely,the proliferation inhibition and apoptotic rates of H929-LE and MM1.S-LE cells were significantly higher[H929-LE-1#:(80.70±1.60)%and(89.08±0.49)%;H929-LE-2#:(75.30±3.80)%and(82.41±1.07)%;MM1.S-LE-1#:(70.64±0.84)%and(67.63±0.21)%;MM1.S-LE-2#:(68.47±1.32)%and(85.90±0.18)%;P＜0.05,n=3].Conclusion:Montelukast sodium can target ubiquitin proteasome regulator USP2 and inhibit its deubiquitylating activity,which may modulate USP2 directing protein and trigger endoplasmic reticulum stress to induce cell cycle arrest and apoptosis in MM cells.

Lean six sigma (LSS) emphasizes patient demand-oriented, and continuously optimizing prolesses to achieve efficiency and standardization in medical services. Starting in 2021, a specialized children′s hospital (comprising one main campus and three branch campuses) introduced the LSS management method. Through define, measure, analyze, improve, and control, the hospital utilized tools such as questionnaire surveys, SIPOC models, and fishbone diagrams to identify pain points in the building of multi-campus child-friendly hospital and develop improvement measures.To address the main issues of insufficient cross-campus collaboration, significant disparities in service quality among different campuses, unclear functional positioning of each campus, and inadequate integration with social security mechanisms, the hospital implemented a " homogeneity-differentiation-coordination" management mode. The hospital implemented unified diagnosis and treatment standards and clinical pathways, carried out remote consultations and expert rotations, completed mutual recognition of examination and testing results, optimized child friendly labeling and child friendly environment, and built a " hospital-to-hospitals, hospital to medical schools, and hospital to community health centers" linkage platform, etc., to promote the standardization of diagnosis and treatment processes, clear functional positioning of the hospital area, and efficient resource allocation. Through practice, the outpatient appointment rate and patient satisfaction rate in the main hospital had increased from 86.72% and 98.64% in January December 2021 to 91.87% and 99.72% in January December 2024, respectively; The patient waiting time had been shortened from 26.54 minutes to 21.94 minutes, and the efficiency of medical treatment and service experience had been significantly improved. As of 2024, mutual recognition of 214 inspection and testing items had been achieved cross hospital campuses, forming a collaborative pattern of " main hospital leading, campuses support, and resource complementarity", and significantly improving the level of collaboration and child friendly connotation among multi-campus. This practice explored the integration path of multi-campus collaborative governance and child-friendly services, which could provide reference and inspiration for the similar hospitals.

Objective:To explore the clinicopathological features of rectal neuroendocrine tumor (R-NET) G2, identify prognostic factors, and summarize treatment experience.Methods:The clinical data of patients diagnosed with R-NET G2 by pathological diagnosis admitted to Cancer Hospital of the Chinese Academy of Medical Sciences from January 2003 to September 2023 were retrospectively analyzed. The Fisher's exact test and Kaplan-Meier curves were performed to analyze the association between pathological features and prognosis.Results:A total of 22 patients were enrolled in this study and 21 patients were followed up for a period of 6-98 months with a median follow-up time of 42 months. 5 patients died due to tumor progression during the follow-up period. The 1-, 3-, and 5-year cancer-specific survival (CSS) of the whole group were 100.0%, 92.9%, and 69.6%, respectively. Of the 22 patients, 20 underwent surgical treatment, of which 15 underwent postoperative adjuvant therapy; 2 underwent medical treatment for liver and bone multiple metastases. The 5-year survival rates of patients with tumours ≥2 cm in length, T2-3 stage, lymph node metastasis, and distant metastasis (57.1%, 68.8%, 66.7%, and 63.6%, respectively) were shorter than those of patients with tumours <2 cm in length, T1 stage, no lymph node metastasis, and no distant metastasis (all 100.0%, P＜0.001). In addition, patients with liver metastases had larger primary tumor diameters and higher T-stages compared with those without distant metastasis ( P＜0.05). Conclusions:R-NET G2 has a high degree of malignancy compared with G1 and a high propensity for metastasis. Clinicians should formulate appropriate diagnostic and treatment strategies based on factors such as tumor size, depth of invasion, lymph node status, presence of distant metastasis, and the location and extent of distant metastasis.

Objective:To explore the clinicopathological features of rectal neuroendocrine tumor (R-NET) G2, identify prognostic factors, and summarize treatment experience.Methods:The clinical data of patients diagnosed with R-NET G2 by pathological diagnosis admitted to Cancer Hospital of the Chinese Academy of Medical Sciences from January 2003 to September 2023 were retrospectively analyzed. The Fisher's exact test and Kaplan-Meier curves were performed to analyze the association between pathological features and prognosis.Results:A total of 22 patients were enrolled in this study and 21 patients were followed up for a period of 6-98 months with a median follow-up time of 42 months. 5 patients died due to tumor progression during the follow-up period. The 1-, 3-, and 5-year cancer-specific survival (CSS) of the whole group were 100.0%, 92.9%, and 69.6%, respectively. Of the 22 patients, 20 underwent surgical treatment, of which 15 underwent postoperative adjuvant therapy; 2 underwent medical treatment for liver and bone multiple metastases. The 5-year survival rates of patients with tumours ≥2 cm in length, T2-3 stage, lymph node metastasis, and distant metastasis (57.1%, 68.8%, 66.7%, and 63.6%, respectively) were shorter than those of patients with tumours <2 cm in length, T1 stage, no lymph node metastasis, and no distant metastasis (all 100.0%, P＜0.001). In addition, patients with liver metastases had larger primary tumor diameters and higher T-stages compared with those without distant metastasis ( P＜0.05). Conclusions:R-NET G2 has a high degree of malignancy compared with G1 and a high propensity for metastasis. Clinicians should formulate appropriate diagnostic and treatment strategies based on factors such as tumor size, depth of invasion, lymph node status, presence of distant metastasis, and the location and extent of distant metastasis.

Lean six sigma (LSS) emphasizes patient demand-oriented, and continuously optimizing prolesses to achieve efficiency and standardization in medical services. Starting in 2021, a specialized children′s hospital (comprising one main campus and three branch campuses) introduced the LSS management method. Through define, measure, analyze, improve, and control, the hospital utilized tools such as questionnaire surveys, SIPOC models, and fishbone diagrams to identify pain points in the building of multi-campus child-friendly hospital and develop improvement measures.To address the main issues of insufficient cross-campus collaboration, significant disparities in service quality among different campuses, unclear functional positioning of each campus, and inadequate integration with social security mechanisms, the hospital implemented a " homogeneity-differentiation-coordination" management mode. The hospital implemented unified diagnosis and treatment standards and clinical pathways, carried out remote consultations and expert rotations, completed mutual recognition of examination and testing results, optimized child friendly labeling and child friendly environment, and built a " hospital-to-hospitals, hospital to medical schools, and hospital to community health centers" linkage platform, etc., to promote the standardization of diagnosis and treatment processes, clear functional positioning of the hospital area, and efficient resource allocation. Through practice, the outpatient appointment rate and patient satisfaction rate in the main hospital had increased from 86.72% and 98.64% in January December 2021 to 91.87% and 99.72% in January December 2024, respectively; The patient waiting time had been shortened from 26.54 minutes to 21.94 minutes, and the efficiency of medical treatment and service experience had been significantly improved. As of 2024, mutual recognition of 214 inspection and testing items had been achieved cross hospital campuses, forming a collaborative pattern of " main hospital leading, campuses support, and resource complementarity", and significantly improving the level of collaboration and child friendly connotation among multi-campus. This practice explored the integration path of multi-campus collaborative governance and child-friendly services, which could provide reference and inspiration for the similar hospitals.

ObjectiveTo investigate the effect of Gualou Xiebai Banxiatang on cardiac function and myocardial histopathological changes in rats with ischemic myocardial injury， and to observe the effect of myocardial microvascular density （MVD）， phosphatidylinositol 3-kinase （PI3K）， mammalian target of rapamycin （mTOR）， hypoxia-inducible factor-1 alpha （HIF-1α）， and vascular endothelial growth factor （VEGF） signaling pathways on myocardial microangiogenesis. MethodSeventy male SD rats were randomly selected， with six rats in the normal group. The remaining rats were fed a high-fat diet and injected with isoproterenol hydrochloride （ISO，80 mg·kg^-1·d^-1， 2 d） to induce a hyperlipidemia-based ischemic heart disease model. After successful modeling， the rats were randomly divided into the model group， high， medium， and low dose groups of Gualou Xiebai Banxiatang， and the metoprolol group. The high， medium， and low dose groups of Gualou Xiebai Banxiatang were given Gualou Xiebai Banxiatang at 10.42， 5.21， 2.61 g·kg^-1·d^-1， respectively， while the metoprolol group was given metoprolol at 2.6 mg·kg^-1·d^-1. Both the normal and model groups were given an equivalent volume of physiological saline for 28 days. After the intervention， relevant tests were conducted， and serum was collected to measure heart function-related indicators. Hematoxylin-eosin （HE） and Masson staining were performed on ventricular tissue to observe pathological changes under a light microscope. Immunohistochemistry （IHC） was used to detect the positive expression of platelet endothelial cell adhesion molecule （CD31）. Enzyme-linked immunosorbent assay （ELISA） was used to detect the expression of N-terminal pro-brain natriuretic peptide （NT-proBNP） and VEGF. Western blot was used to detect the protein expression levels of PI3K/mTOR/HIF-1α/VEGF. ResultCompared with the normal group， the model group showed significantly increased serum levels of LDH， CK， CK-MB， NT-proBNP， and VEGF （P<0.01）， significantly increased collagen volume fraction （CVF） （P<0.01）， significantly decreased MVD （P<0.01）， and elevated protein expression levels of PI3K， mTOR， HIF-1α， and VEGF （P<0.05， P<0.01）. Compared with the model group， the metoprolol group had significantly lower serum levels of LDH， CK， CK-MB， and NT-proBNP （P<0.01）， significantly higher VEGF levels （P<0.01）， significantly decreased CVF （P<0.01）， significantly increased MVD （P<0.01）， and significantly increased protein expression levels of PI3K， mTOR， and VEGF （P<0.01）， with no statistically significant change in HIF-1α protein expression. Compared with the model group， the high and medium dose groups of Gualou Xiebai Banxiatang had decreased serum levels of LDH， CK， CK-MB， and NT-proBNP （P<0.05， P<0.01）， increased VEGF levels （P<0.05， P<0.01）， significantly reduced CVF （P<0.01）， increased MVD （P<0.05， P<0.01）， and significantly increased protein levels of PI3K， mTOR， HIF-1α， and VEGF （P<0.01）. In the low dose group of Gualou Xiebai Banxiatang， compared with the model group， serum levels of LDH and NT-proBNP were decreased （P<0.05）， VEGF was increased （P<0.05）. Moreover， CVF was decreased （P<0.05）， and the protein expression levels of PI3K， mTOR， HIF-1α， and VEGF were significantly increased （P<0.01）. ConclusionGualou Xiebai Banxiatang can improve cardiac function， reduce myocardial pathological damage， enhance endothelial cell function， promote myocardial microvascular formation， and upregulate the expression of PI3K， mTOR， HIF-1α， and VEGF proteins in myocardial tissue in rats with ischemic myocardial injury.

Inducing the degradation of KRAS represents a novel strategy to combat cancers with KRAS mutation. In this study, we identify ubiquitin-specific protease 2 (USP2) as a novel deubiquitinating enzyme of KRAS in multiple myeloma (MM). Specifically, we demonstrate that gambogic acid (GA) forms a covalent bond with the cysteine 284 residue of USP2 through an allosteric pocket, inhibiting its deubiquitinating activity. Inactivation or knockdown of USP2 leads to the degradation of KRAS, resulting in the suppression of MM cell proliferation in vitro and in vivo. Conversely, overexpressing USP2 stabilizes KRAS and partially abrogates GA-induced apoptosis in MM cells. Furthermore, elevated USP2 levels may be associated with poorer prognoses in MM patients. These findings highlight the potential of the USP2/KRAS axis as a therapeutic target in MM, suggesting that strategically inducing KRAS degradation via USP2 inhibition could be a promising approach for treating cancers with KRAS mutations.