Guided by the theory of "the kidney storing essence, governing the bones, and producing marrow", this study explored the mechanism of icariin(ICA) in regulating the osteogenic differentiation of rat bone mesenchymal stem cells(BMSCs) through caveolin-1(Cav1) via in vitro and in vivo experiments, aiming to provide a theoretical basis for the prevention and treatment of postmenopausal osteoporosis with traditional Chinese medicine(TCM). Primary cells were obtained from 4-week-old female SD rats using the whole bone marrow adherent method. Flow cytometry was used to detect the expression of surface markers CD29, CD90, CD11b, and CD45. The potential for osteogenic and adipogenic differentiation was assessed. The effect of ICA on cell viability was determined using the CCK-8 assay, and the impact of ICA on the formation of mineralized nodules was verified by alizarin red staining. A stable Cav1-silenced cell line was constructed using lentivirus. The effect of Cav1 silencing on osteogenic differentiation was observed via alizarin red staining. Western blot analysis was conducted to detect the expression of Cav1, Hippo/TAZ, and osteogenic markers such as Runt-related transcription factor 2(RUNX2) and alkaline phosphatase(ALP). The results showed that primary cells were successfully obtained using the whole bone marrow adherent method, positively expressing surface markers of rat BMSCs and possessing the potential for both osteogenic and adipogenic differentiation. The CCK-8 assay and alizarin red staining results indicated that 1×10~(-7) mol·L~(-1) was the optimal concentration of ICA for intervention in this experiment(P<0.05). During osteogenic induction, ICA inhibited Cav1 expression(P<0.05) while promoting TAZ expression(P<0.05). Alizarin red staining demonstrated that Cav1 silencing significantly promoted the osteogenic differentiation of BMSCs. After ICA intervention, TAZ expression was activated, and the expression of osteogenic markers ALP and RUNX2 was increased. In conclusion, Cav1 silencing significantly promotes the osteogenic differentiation of BMSCs, and ICA promotes this differentiation by inhibiting Cav1 and regulating the Hippo/TAZ signaling pathway.
Animals ; Mesenchymal Stem Cells/metabolism* ; Caveolin 1/genetics* ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; Cell Differentiation/drug effects* ; Female ; Signal Transduction/drug effects* ; Flavonoids/administration & dosage* ; Protein Serine-Threonine Kinases/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Cells, Cultured ; Humans

Ulcerative colitis(UC) is a recurrent, chronic, nonspecific inflammatory bowel disease. The longer the course of the disease, the higher the risk of cancerization. In recent years, the incidence and mortality rates of colon cancer in China have been increasing year by year, seriously threatening the life and health of patients. Therefore, studying the mechanism of "inflammation cancer transformation" in UC and conducting early intervention is crucial. The "Kenang" theory is an important component of traditional Chinese medicine(TCM) theory of phlegm and blood stasis. It is based on the coexistence of phlegm and blood stasis in the body and deeply explores the pathogenic syndromes and characteristics of phlegm and blood stasis. Kenang is a pathological product formed when long-term Qi stagnation leads to the internal formation of phlegm and blood stasis, which is hidden deep within the body. It is characterized by being hidden, progressive, and difficult to treat. The etiology and pathogenesis of "inflammation cancer transformation" in UC are consistent with the connotation of the "Kenang" theory. The internal condition for the development of UC "inflammation cancer transformation" is the deficiency of healthy Qi, with Qi stagnation being the key pathological mechanism. Phlegm and blood stasis are the main pathogenic factors. Phlegm and blood stasis accumulate in the body over time and can produce cancer toxins. Due to the depletion of healthy Qi and a weakened constitution, the body is unable to limit the proliferation and invasion of cancer toxins, eventually leading to cancer transformation in UC. In clinical treatment, the focus should be on removing phlegm and blood stasis, with syndrome differentiation and treatment based on three basic principles: supporting healthy Qi to strengthen the body's foundation, resolving phlegm and blood stasis to break up the Kenang, and regulating Qi and blood to smooth the flow of energy and resolve stagnation. This approach helps to dismantle the Kenang, delay, block, or even reverse the cancerization process of UC, reduce the risk of "inflammation cancer transformation", improve the patient's quality of life, and provide new perspectives and strategies for early intervention in the development of colon cancer.
Humans ; Colitis, Ulcerative/immunology* ; Medicine, Chinese Traditional ; Drugs, Chinese Herbal/therapeutic use* ; Cell Transformation, Neoplastic

OBJECTIVE: To explore the safety and efficacy of high-dose etoposide (VP-16) combined with recombinant human granulocyte colony-stimulating factor (rhG-CSF) as salvage mobilization for peripheral blood stem cells (PBSC) in newly diagnosed multiple myeloma (NDMM) patients. METHODS: From April 2021 to May 2023, eight NDMM patients who had failed to yield sufficient PBSC during initial mobilization with high-dose cyclophosphamide (CTX) combined with rhG-CSF underwent salvage mobilization with 1.2 g/m2 etoposide combined with rhG-CSF 10 μg/(kg·d). The effects and adverse reactions of initial mobilization and salvage mobilization were analyzed. RESULTS: For salvage mobilization and initial mobilization, the numbers of PBSC collections were 16 and 18, respectively. The mean value of total collected CD34⁺ cells were (11.90±5.75)×10⁶/kg and (1.67±0.75)×10⁶/kg (P =0.0010) in salvage mobilization group and initial mobilization group, respectively. The proportion of patients with a total collection of CD34⁺ cell count≥2×10⁶/kg were 100% and 37.5% (P =0.0625), and the proportion of patients with a total collection of CD34⁺ cell count≥5×10⁶/kg were 87.5% and 0% (P =0.0156) in salvage mobilization group and initial mobilization group, respectively. For five patients who underwent high-dose CTX initial mobilization but had a total CD34⁺ cell count < 2×10⁶/kg, successful collection was achieved through salvage mobilization with high-dose VP-16. Salvage mobilization with high-dose VP-16 was scheduled 2-3 weeks after failure of CTX mobilization. Adverse reactions of high-dose VP-16 mobilization did not increase compared to the initial mobilization with high-dose CTX. CONCLUSION As a salvage mobilization regimen, VP-16 1.2 g/m² combined with rhG-CSF is safe and highly effective in NDMM patients who failed to initial mobilization with high-dose CTX combined with rhG-CSF.
Humans ; Multiple Myeloma/therapy* ; Etoposide/therapeutic use* ; Hematopoietic Stem Cell Mobilization/methods* ; Cyclophosphamide/therapeutic use* ; Granulocyte Colony-Stimulating Factor ; Salvage Therapy ; Peripheral Blood Stem Cells ; Male ; Middle Aged ; Female ; Peripheral Blood Stem Cell Transplantation

The ventral anterior (VA) nucleus of the thalamus is a major target of the basal ganglia and is closely associated with the pathogenesis of Parkinson's disease (PD). Notably, the VA receives direct innervation from the hypothalamic histaminergic system. However, its role in PD remains unknown. Here, we assessed the contribution of histamine to VA neuronal activity and PD motor deficits. Functional magnetic resonance imaging showed reduced VA activity in PD patients. Optogenetic activation of VA neurons or histaminergic afferents significantly alleviated motor deficits in 6-OHDA-induced PD rats. Furthermore, histamine excited VA neurons via H1 and H2 receptors and their coupled hyperpolarization-activated cyclic nucleotide-gated channels, inward-rectifier K⁺ channels, or Ca²⁺-activated K⁺ channels. These results demonstrate that histaminergic afferents actively compensate for Parkinsonian motor deficits by biasing VA activity. These findings suggest that targeting VA histamine receptors and downstream ion channels may be a potential therapeutic strategy for PD motor dysfunction.
Animals ; Histamine/metabolism* ; Male ; Oxidopamine/toxicity* ; Rats ; Ventral Thalamic Nuclei/physiopathology* ; Rats, Sprague-Dawley ; Disease Models, Animal ; Parkinson Disease/metabolism* ; Neurons/physiology* ; Humans ; Optogenetics

Heterotypic cell-in-cell structures(heCICs)mediate unique non-autonomous cell death,which are widely involved in a variety of important pathological processes,such as tumorigenesis,pro-gression and clinical prognosis.Methylenetetrahydrofolata dehydrogenase 2(MTHFD2),one of the key enzymes of one-carbon metabolism,is highly expressed in a variety of tumor cells.In this study,in order to investigate the effect of MTHFD2 on the formation of heCICs,liver cancer cells and immune cells were first labeled separately by live cell dyes,and the heCIC model was established by using fluorescence mi-croscopy for cell imaging and analysis.After transiently knocking down MTHFD2 in cells by RNAi,we found that the ability of PLC/PRF/5 and Hep3B to form heCICs with immune cells was significantly in-creased(all P＜0.01).MTHFD2 recombinant expression plasmid was constructed by the homologous re-combination method,and MTHFD2 overexpression cell lines were further constructed.Then,the effect of MTHFD2 overexpression on the ability to form heCICs was detected by co-culturing the overexpression cell lines with immune cells.The results showed that the rate of heCIC formation was significantly re-duced after overexpression of MTHFD2(all P＜0.001).In conclusion,this study demonstrated that MTHFD2 is a negative regulator of heCIC formation,providing a research basis for targeting MTHFD2 to promote heCIC formation and enhance the in-cell killing of immune cells.

Objective To develop a matrix metalloproteinase(MMP)-responsive hyaluronic acid(HA)-based controlled-release material for brain-derived neurotrophic factor(BDNF)to provide a novel therapeutic strategy for intervention and repair of traumatic brain injury(TBI).Methods HA was modified with amination,followed by condensation with Suflo-SMCC carboxyl group to form amide,and then linked with glutathione(GSH)to synthesize HA-GSH.The recombinant glutathione S-transferase(GST)-tissue inhibitor of metalloproteinase(TIMP)-BDNF(GST-TIMP-BDNF)expression plasmid was constructed using molecular cloning technique with double enzyme digestion by Bam H Ⅰ and Eco R Ⅰ.The recombinant GST-TIMP-BDNF protein was expressed in the Escherichia coli prokaryotic expression system,and purified by ion exchange chromatography,confirmed by Western blotting.MMP diluents were supplemented with PBS,MMP inhibitor marimastat,and varing concentrations(0.4,0.6,0.8 mg/ml)of GST-TIMP-BDNF or GST-BDNF.MMP-2 activity was analyzed using an MMP activity detection kit to evaluate the inhibitory effect of the recombinant protein on MMP.Primary rat neurons were extracted and cultured to establish an iron death model induced by RSL3.The effect of recombinant protein GST-TIMP-BDNF on neuronal injury was detected by immunofluorescence staining.Results MRI hydrogen spectrum identification confirmed the successful synthesis of HA-GSH.Western blotting results showed the successful expression of the recombinant protein GST-TIMP-BDNF containing the GST tag using the E.coli prokaryotic expression system.MMP activity detection results indicated that the recombinant protein GST-TIMP-BDNF had a superior inhibitory effect on MMP-2 activity compared to GST-BDNF(P<0.05).Immunofluorescence staining results showed a significant increase in fluorescence intensity in rat neurons treated with GST-TIMP-BDNF after RSL3 induction(P<0.05).Conclusion A MMP-responsive HA-based BDNF controlled-release material has been successfully developed,exhibiting a protective effect on neuron damage.

AIM To study the chemical constituents from the large polar fraction of the roots of Lindera reflexa Hemsl.and their antitumor activities.METHODS The large polar fraction from the roots of L.reflexa was isolated and purified by silica gel column,Sephadex LH-20 gel column,semi-preparative HPLC and ODS medium pressure column,then the structures of obtained compounds were identified by physicochemical properties and spectral data.The antitumor activities were determined by MTT method.RESULTS Thirteen compounds were isolated and identified as 2,6-dimethoxy-4-hydroxyphenyl-1-O-β-D-glucopyranoside(1),3-hydroxy-4,5-dimethoxyphenol-β-D-glucopyranoside(2),syringin(3),1-O-3,4-dimethoxy-5-hydroxyphenyl-(6-O-3,5-dimethoxygalloyl)-β-D-glucopyranoside(4),p-cymen-7-yl β-D-glucopyranoside(5),pisumionoside(6),staphylionoside D(7),dendranthemoside B(8),lynoiside(9),nudiposide(10),icariside B1(11),(2S)-pinocembrin-7-O-(6-O-α-L-rhamnopyranosyl-β-D-glucopyranoside)(12),(+)-N-(methoxycarbonyl)-N-norboldine(13).Compounds 3 and 13 showed obvious cytotoxicity against human lung cancer cells(A549)and human gastric cancer cells(MGC80-3).CONCLUSION Compounds 1-13 are isolated from the roots of L.reflexa for the first time.Compounds 3 and 13 have good anti-tumor activities.

OBJECTIVE Cannabinoids modulate do-pamine(DA)transmission and DA-related behavior,which has been thought to be mediated initially by acti-vation of cannabinoid CB1 receptors(CB1Rs)on GABA neurons.However,the cellular and receptor mechanisms underlying cannabinoids' psychoactive effects are not fully understood.The present study is to explore the pos-sible expression character of CB1Rs and elucidated the underlying mechanism of them.METHODS We took advantage of RNAscope in situ hybridization(ISH)assays and triple-staining assays to detect the CB1R-expressing neurons.We established an optical intracranial self-stimulation(OICSS)behavioral model by using opto-genetics to study dopaminergic reinforcement function.Natural and synthetic cannabinoids were used to study the function of CB1Rs.Conditional genetic depletion of CB1Rs and behavioral assay were performed to study the modulatory role of CB1Rs in DA-related behaviors.RESULTS We found that CB1Rs are also expressed in a subset of DA neurons and functionally underlie cannabi-noid action in male and female mice.ISH assays demon-strated CB1 mRNA in tyrosine hydroxylase(TH)-posi-tive DA neurons in the ventral tegmental area(VTA)and glutamate decarboxylase 1(GAD1)-positive GABA neu-rons.The CB1R expressing DA neurons were located mainly in the middle portion of the VTA with the number of CB1-TH colocalization progressively decreasing from the medial to the lateral VTA.Triple-staining assays indi-cated CB1R mRNA colocalization with both TH and vesicular glutamate transporter 2(VgluT2,a glutamate neuronal marker)in the medial VTA close to the midline of the brain.Optogenetic activation of this population of DA neurons was rewarding as assessed by OICSS.D9-tetrahydrocannabinol(D9-THC)or ACEA(a selective CB1R agonist)dose-dependently inhibited optical intra-cranial self-stimulation in DAT-Cre control mice,but not in conditional knockout mice with the CB1R gene absent in DA neurons.In addition,deletion of CB1Rs from DA neurons attenuated D9-THC-induced reduction in DA release in the NAc,locomotion,and anxiety.CONCLU-SION Our results indicated that CB1Rs are expressed in a subset of DA neurons that corelease DA and gluta-mate,and functionally underlie cannabinoid modulation of DA release and DA-related behavior.

Objective:To explore the risk factors for lower extremity deep vein thrombosis (DVT) in patients with a spinal cord injury (SCI).Methods:The medical records of 276 hospitalized SCI patients were analyzed retrospectively. They were divided into a DVT group ( n=63) and a no-DVT group ( n=213). Gender, age, blood type, smoking history, surgical history, the time from SCI to admission, cause of SCI, fracture, SCI segments, American Spinal Cord Injury Association grade and complications were compared between the two groups. Binomial logistic regression was used to isolate the risk factors for lower extremity DVT among such patients. Results:Among 84% of the 63 with a lower extremity DVT, it was a calf muscle venous thrombosis. Anemia, hyponatremia and time from SCI to admission (which ranged from 74 to 195 days) were the most serious DVT risk factors.Conclusions:SCI patients are of high risk for DVT, with anemia and hyponatremia being independent risk factors.

Thirteen isoflavones were separated and purified from an ethanol extract of the rhizome of Dalbergia benthamii Prain by using silica gel, Sephadex LH-20, recrystallization et al. Their structures were identified by physicochemical properties and spectral analysis such as MS, 1D/2D-NMR as dalbergibenthamin (1), butesuperin A (2), xanthocercin A (3), butesuperin B (4), di-O-methylalpinum isoflavone (5), 2′-deoxgisoaunculutin (6), robustone (7), 4′-hydroxy-5,7-dimethoxy-6-(3-methyl-2-butenyl)-isoflavone (8), formononetin (9), 6″-O-rhamnosyldaidzin (10), 3′,4′-di-O-methylene-5-hydroxy-7-methoxy-6-isopentenyl isoflavone (11), derrubone dimethyl enter (12), and derrubone (13). Compound 1 is a pair of new isoflavonoid enantiomers, compound 12 is a new natural product and compounds 1-7 and 10-13 were obtained from D. benthamii Prain for the first time. In vitro cytotoxic activities of the compounds were explored by MTS testing with HL-60, A-549, SMMC-7721, MCF-7 and SW480 cell lines. Results show that compound 8 significantly inhibited cellular proliferation. The IC₅₀ of compound 8 in A-549 and SW480 cells was 16.68 ± 0.19 and 15.21 ± 0.60 μmol·L^-1.