This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

This study aims to establish the S_(180) tumor-bearing mice model, and to investigate the influence of Shenqi Erpi Granules(SQEPG) on immune function, as well as the drug's tumor-suppressive effect and mechanism. SPF grade KM mice(half male and half female) were randomly divided into 6 groups: a control group, a model group, a cyclophosphamide group(50 mg·kg~(-1)), as well as SQEPG groups in low-, medium-, and high-dose(5.25, 10.5, 21 g·kg~(-1)). The control group and the model group were given distilled water, and the other 4 groups were given the corresponding drugs by gavage. The administration continued for 10 days before the mice were sacrificed. The antitumor and immune regulation effects of SQEPG were evaluated. The effect of SQEPG on delayed type hypersensitivity reaction(DTH), carbon clearance index, and serum hemolysin antibody level was observed to reflect the effect on the immune function of tumor-bearing mice. Tumor weight was recorded to calculate the tumor suppression rate and the immune organ index. Hematoxylin-eosin(HE) staining was used to detect morphological changes in tumor tissues. Flow cytometry was employed to detect the percentage of CD4~+ and CD8~+ T-cells in the spleen tissues and the tumor tissue apoptosis levels. Immunohistochemistry was conducted to detect the KI67 protein expression level of tumor tissues. ELISA resorted to the detection of the following expression levels in tumor tissues: tumor necrosis factor-α(TNF-α), interleukin-2(IL-2), interferon-γ(IFN-γ). Western blot was performed to detect the expression levels of caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cyclin-dependent kinases 4(CDK4), G_1/S-specific cyclin D1(cyclin D1), and vascular endothelial growth factor A(VEGFA). The results showed that, compared with the model group, the SQEPG could increase the swelling of the auricle of the tumor-bearing mice; significantly increase the phagocytic index of carbon granule contour(P<0.05 or P<0.01), and the middle dose of SQEPG could significantly increase the antibody level of hemolysin(P<0.05); different doses of SQEPG significantly inhibit the growth of the tumor, and decrease the mass of the tumor tissues(P<0.05 or P<0.01); the low dose of SQEPG significantly decreased spleen index(P<0.05), low and high doses of SQEPG increased thymus index, while medium doses of SQEPG decreased thymus index. High doses of SQEPG significantly elevated the levels of CD4~+ and CD8~+ T-cells in the spleens of the homozygous mice(P<0.01 or P<0.001), and increased the apoptosis rate of the cells of the tumor tissues(P<0.05); Meanwhile, high-dose SQEPG elevated the levels of immunity factors such as IL-2, IFN-γ and TNF-α in the serum of tumor-bearing mice(P<0.01); medium-and high-dose SQEPG significantly lowered the rate of positive expression of KI67 protein in tumor tissues(P<0.01). Compared with the model group, high-dose SQEPG significantly up-regulated the expression of caspase-3 and Bax proteins in tumor tissues(P<0.05), and significantly down-regulated the expression of CDK4, cyclin D1, and VEGFA proteins(P<0.05 or P<0.01). In conclusion, SQEPG has the effect of improving immune function and inhibiting tumor growth in tumor-bearing mice. Its mechanism of tumor-suppressive effects may be related to apoptosis promotion, cell cycle progression block, and tumor cell proliferation inhibition.
Animals ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Male ; Female ; Apoptosis/drug effects* ; Sarcoma 180/genetics* ; Humans

BACKGROUND: The association between uric acid-albumin ratio (UAR) with different diseases has been evaluated before. However, the association between UAR with spontaneous reperfusion (SR) in patients with ST-segment elevation myocardial infarction (STEMI) has not been explored. METHODS: STEMI patients admitted to our department and underwent primary coronary angiography between 1^st November 2018 and 31^st December 2020 were retrospectively enrolled. The patients were divided into the SR group and the non-SR group according to the index coronary angiography results. The association between UAR and SR was evaluated by uni-variable and multi-variable logistic analysis. Receiver operating characteristic curve analysis was used to determine the optimum cut-off level of UAR in predicting SR. RESULTS: Three hundred and fifty-seven patients were finally enrolled in our study, 55 patients were divided into the SR group and 302 patients were divided into the non-SR group. In uni-variable analysis, patients with SR were older (P = 0.032), with higher red blood cell distribution width (P < 0.001) and red blood cell distribution width-to-platelet ratio (P < 0.001), higher level of C-reactive protein (P = 0.046), higher level of uric acid (P < 0.001) compared with patients without SR. Patients with SR had a lower level of platelets (P = 0.008), lower level of on-admission B-type natriuretic peptide (P < 0.001). As for the level of UAR, STEMI patients with SR had significantly higher levels of UAR compared with STEMI patients without SR [11.1 (8.9-13.4) vs. 8.3 (6.6-10.0), P < 0.001]. Further multi-variable logistic analysis reveals that UAR was the independent risk factor of SR in different models after adjusting different variables. Receiver operating characteristic analysis showed that UAR had good predictive value in SR (AUC = 0.75, 95% CI: 0.702-0.794, P < 0.01). CONCLUSIONS Our study shows that UAR is an independent risk factor for predicting SR in STEMI patients.

The exoskeleton robot for lower limb medical rehabilitation in foreign countries and China was introduced in terms of the research status,structure and working principle,and analysis was carried out over its key technologies.It's pointed out the exoskeleton robot for lower limb medical rehabilitation would be enhanced in energy endurance,safety and comfort,individualized and intelligent control,modularity and lightweight design.[Chinese Medical Equipment Journal,2025,46(1):88-100]

To investigate the molecular mechanisms underlying the mechanosensitive ion channel PI-EZO2 in osteoarthritis(OA),we developed a lentiviral vector for endogenous PIEZO2 overexpression and established a stable PIEZO2-high-expressing immortalized human primary chondrocyte line.By map-ping the open reading frame of the PIEZO2 locus and designing sequence-specific sgRNA,we employed the CRISPR/Cas9 synergistic activation mediator(SAM)system to precisely integrate transcriptional ac-tivation elements into the PIEZO2 promoter region.Lentiviral-mediated targeted genomic integration en-sured endogenous PIEZO2 overexpression,confirmed by mCherry fluorescence tracing coupled with flow cytometric sorting,which revealed membrane-specific localization of PIEZO2 protein(localization effi-ciency:78.49％).Quantitative PCR demonstrated a 17-fold upregulation of PIEZO2 mRNA,while Western blotting validated enhanced membrane-localized protein expression.Strikingly,PIEZO2-overex-pressing chondrocytes exhibited hallmark OA metabolic phenotypes compared to wild-type controls:typeⅡ collagen mRNA expression decreased to 50％of baseline levels,whereas matrix metalloproteinase 13(MMP13)mRNA surged by 20-fold.These alterations recapitulated the pathological matrix metabolic phenotype observed in biomechanical OA models induced by cyclic mechanical stress(10％strain,0.5 Hz,8 h/day for 2 consecutive days).Collectively,we successfully generated a human chondrocyte model with stable PIEZO2 overexpression,which faithfully mirrors mechanotransduction-driven OA progression.This engineered cellular system provides a robust platform for dissecting PIEZO2-mediated mechanosig-naling networks and advancing targeted therapeutic discovery.

Background/Aims: Transmembrane 4 L six family member 1 (TM4SF1) is highly expressed and contributes to the progression of various malignancies. However, how it modulates hepatocellular carcinoma (HCC) progression and senescence remains to be elucidated. Methods: TM4SF1 expression in HCC samples was evaluated using immunohistochemistry and flow cytometry. Cellular senescence was assessed through SA-β-gal activity assays and Western blot analysis. TM4SF1-related protein interactions were investigated using immunoprecipitation-mass spectrometry, co-immunoprecipitation, bimolecular fluorescence complementation, and immunofluorescence. Tumor-infiltrating immune cells were analyzed by flow cytometry. The HCC mouse model was established via hydrodynamic tail vein injection. Results: TM4SF1 was highly expressed in human HCC samples and murine models. Knockdown of TM4SF1 suppressed HCC proliferation both in vitro and in vivo, inducing non-secretory senescence through upregulation of p16 and p21. TM4SF1 enhanced the interaction between AKT1 and PDPK1, thereby promoting AKT phosphorylation, which subsequently downregulated p16 and p21. Meanwhile, TM4SF1-mediated AKT phosphorylation enhanced PD-L1 expression while reducing major histocompatibility complex class I level on tumor cells, leading to impaired cytotoxic function of CD8+ T cells and an increased proportion of exhausted CD8+ T cells. In clinical HCC samples, elevated TM4SF1 expression was associated with resistance to anti-PD-1 immunotherapy. Targeting TM4SF1 via adeno-associated virus induced tumor senescence, reduced tumor burden and synergistically enhanced the efficacy of anti-PD-1 therapy. Conclusions Our results revealed that TM4SF1 regulated tumor cell senescence and immune evasion through the AKT pathway, highlighting its potential as a therapeutic target in HCC, particularly in combination with first-line immunotherapy.

Bacillus mycoides JA20-1 was screened and identified as a biocontrol bacterium with a high capacity for producing volatile organic compounds(VOCs) in the laboratory. This strain had significant inhibitory effects on various postharvest disease pathogens in crops, such as Botrytis cinerea, as well as soil-borne disease pathogens in ginseng, such as Sclerotinia ginseng. In order to accelerate its industrialization process, in this study, single-factor experiments and response surface optimization methods were used. The fermentation medium and fermentation conditions in the shake flask of strain JA20-1 were systematically optimized by using cell production volume as the response variable. Meanwhile, the biocontrol effect of JA20-1 on B. cinerea of ginseng during the storage period was evaluated by using the method of fumigation in a dry dish in vitro. The results indicated that the optimal fermentation medium formulation for strain JA20-1 was as follows: 1% yeast paste, 1% soluble starch, 0.25% K_2HPO_4·3H_2O, and 0.2% NaCl. The optimal fermentation conditions in the shake flask were vaccination size of 3%, culture volume of 50 mL in a 250 mL Erlenmeyer flask, pH of 6.2, fermentation temperature of 34 ℃, shaking speed of 180 r·min~(-1), and incubation time of 18 hours. The bacteria count in the fermentation broth under these conditions reached 2.17 × 10~8 CFU·mL~(-1), which was 6.58 times higher than before. The average control efficacy of the fermentation broth on Botrytis cinerea of ginseng under in vitro fumigation reached 61.70% and 84.04% respectively, when 20 mL and 30 mL per dish were used. The research provided theoretical support and technical foundation for the development and utilization of Bacillus mycoides JA20-1 and the biocontrol of soil-borne diseases in ginseng and postharvest diseases in crops.
Botrytis/drug effects* ; Fermentation ; Panax/microbiology* ; Plant Diseases/prevention & control* ; Volatile Organic Compounds/metabolism* ; Bacillus/physiology* ; Pest Control, Biological/methods* ; Biological Control Agents/metabolism* ; Culture Media/chemistry*

Objective To initially explore the possibility of applying the fluorescence micro-optical sectioning tomography(fMOST)high-resolution 3D reconstruction system to the morphological study of the intestinal nervous system and to preliminarily establish a method for studying the morphology of the intestinal nervous system using this system.Methods fMOST high-resolution 3D reconstruction system was used to study the intestinal nervous system of C57BL/6 mice in detail.Based on this method,a new morphological method of the visceral nervous system of small animal models was explored at the single-cell level.Results Compared with the large intestine,the small intestine lacked the typical myenteric plexus(Auerbach),deep mucosal plexus(Henley),and submucosal superficial plexus(Meissner).Conclusion The result of this paper provide a clearer and systematic display of the anatomical structure of the enteric nervous system in C57BL/6 mice,and further clarify the similarities and differences between the enteric nervous system of mice and human,and provide a theoretical basis for its rational application in the study of digestive system diseases.The morphological study of fMOST high-resolution 3D reconstruction system is not limited to the central nervous system,but can be extended to the morphological study of multiple visceral nervous systems.

Background/Aims: Transmembrane 4 L six family member 1 (TM4SF1) is highly expressed and contributes to the progression of various malignancies. However, how it modulates hepatocellular carcinoma (HCC) progression and senescence remains to be elucidated. Methods: TM4SF1 expression in HCC samples was evaluated using immunohistochemistry and flow cytometry. Cellular senescence was assessed through SA-β-gal activity assays and Western blot analysis. TM4SF1-related protein interactions were investigated using immunoprecipitation-mass spectrometry, co-immunoprecipitation, bimolecular fluorescence complementation, and immunofluorescence. Tumor-infiltrating immune cells were analyzed by flow cytometry. The HCC mouse model was established via hydrodynamic tail vein injection. Results: TM4SF1 was highly expressed in human HCC samples and murine models. Knockdown of TM4SF1 suppressed HCC proliferation both in vitro and in vivo, inducing non-secretory senescence through upregulation of p16 and p21. TM4SF1 enhanced the interaction between AKT1 and PDPK1, thereby promoting AKT phosphorylation, which subsequently downregulated p16 and p21. Meanwhile, TM4SF1-mediated AKT phosphorylation enhanced PD-L1 expression while reducing major histocompatibility complex class I level on tumor cells, leading to impaired cytotoxic function of CD8+ T cells and an increased proportion of exhausted CD8+ T cells. In clinical HCC samples, elevated TM4SF1 expression was associated with resistance to anti-PD-1 immunotherapy. Targeting TM4SF1 via adeno-associated virus induced tumor senescence, reduced tumor burden and synergistically enhanced the efficacy of anti-PD-1 therapy. Conclusions Our results revealed that TM4SF1 regulated tumor cell senescence and immune evasion through the AKT pathway, highlighting its potential as a therapeutic target in HCC, particularly in combination with first-line immunotherapy.