ObjectiveTo investigate the feasibility， safety， and efficacy of surgery-assisted transjugular intrahepatic portosystemic shunt （SA-TIPS） in the treatment of portal hypertension comorbid with complex portal vein thrombosis， including cavernous transformation of the portal vein （CTPV）. MethodsAn analysis was performed for the data of 36 patients with portal hypertension and complex portal vein thrombosis who underwent SA-TIPS in Beijing Shijitan Hospital， Capital Medical University， from November 2023 to January 2025， including general status， technical data of the surgical process （surgical success rate， puncture times， time of operation， the number of stents used， and the length of shunt）， perioperative complications， and surgical recovery. The change in portal pressure gradient （PPG） after shunt was compared， and the rate of reaching the standard for PPG reduction was calculated， as well as stent patency rate within 1 week after surgery. The paired samples t-test was used for comparison of continuous data between two groups. ResultsAmong the 36 patients， 34 （94.4%） underwent SA-TIPS successfully. The incidence rate of perioperative complications was 16.7% （6/36）， including 3 cases of thoraco-abdominal hemorrhage， 2 cases of intraoperative arrhythmia， and 1 case of incision infection. There was a significant reduction in PPG after SA-TIPS （t=19.85， P<0.01）， and the patients achieving a ≥50% reduction in PPG accounted for 76.5% （26/34）. Imaging reexamination within 1 week showed a shunt patency rate of 100%. ConclusionSA-TIPS has a high technical success rate， a favorable safety profile， and good efficacy in the treatment of portal hypertension comorbid with complex portal vein thrombosis （including CTPV）， and therefore， it holds promise for clinical application.

ObjectiveThis paper aims to observe the syndrome improvement and negative antigen conversion rate of Qingxuan Daozhi formula in the treatment of influenza in children （heat accumulation in the lung and stomach syndrome）. MethodsThrough a multi-center randomized controlled methodology design，confirmed influenza cases were collected from October 2022 to April 2023 in the pediatrics department of eight hospitals，such as Dongfang Hospital of Beijing University of Chinese Medicine. A total of 180 children with influenza and heat accumulation in the lung and stomach syndrome conforming to the standard were recruited through the clinic. The sick children meeting the inclusion criteria were randomly divided into groups by a block-randomized method. The children in the experimental group were treated with Qingxuan Daozhi formula for five days，and those in the control group were treated with Oseltamivir Phosphate Granules for five days. The primary efficacy indicator was the negative conversion rate of influenza antigen detection. Secondary efficacy indicators were the Canadian acute respiratory illness and flu scale （CARIFS） and the incidence of complications，severe cases， and critical cases. Follow-up observation was conducted on the day of enrollment，48 hours after medication，72 hours after medication， and （6+1） d after medication. ResultsOne hundred and eighty participants were randomly assigned to the experimental group （90 cases） or the control group （90 cases）. All participants were followed up during the study. Comparison of influenza antigen detection results in the primary efficacy indicators showed that the average time of negative influenza antigen conversion in the experimental group was （5.29±1.25） d，and that in the control group was （5.40±1.68） d，without a statistically significant difference. After five days of intervention，52 cases in the experimental group and 51 cases in the control group converted to negative，without a statistically significant difference. CARIFS score results in the secondary efficacy indicators showed that during 72 hours after intervention，there were statistically significant differences between the experimental group and the control group in three dimensions， including headache，muscle soreness， and the need for extra care （P<0.05）. On the （6+1） days after the intervention，the differences in both the experimental group and the control group were statistically significant in 10 dimensions， including sore throat，bad sleep，uncomfortable feeling，poor spirit and fatigue，crying more than usual，the need for extra care，symptom，function，influence on parents，and total score （P<0.05）. The comparison results within the group in the dimensional scores of symptom， function， and influence on parents，as well as the CARIFS total score showed that with the delay of follow-up time，scores of both groups decreased significantly，with a statistically significant difference （P<0.01）. Inter-group comparison results showed that the mean score of the experimental group was higher than that of the control group at the time of enrollment. With the progress of intervention，the score of the experimental group was significantly decreased compared with that of the control group. At the end of follow-up，the mean score of the experimental group was lower than that of the control group，with no statistically significant difference. In terms of the incidence of complications，severe cases， and critical cases， there were no complications，severe cases， and critical cases in the two groups，without a statistically significant difference. ConclusionThe symptom improvement effect and negative antigen conversion rate of Qingxuan Daozhi formula in the treatment of influenza in children （heat accumulation in the lung and stomach syndrome） are not inferior to Oseltamivir Phosphate granules， and children's acceptance is better. It can be more widely used in clinical treatment of influenza in children （heat accumulation in the lung and stomach syndrome）.

Objective·To construct an interpenetrating network hydrogel(HMGL)that can induce hypoxia in situ,investigate its effects on promoting the secretion of extracellular matrix in nucleus pulposus cells(NPCs),and evaluate its potential in the treatment of intervertebral disc degeneration.Methods·HMGLs capable of long-term in situ hypoxia induction were prepared by a two-step polymerization method.Hypoxic hydrogels with laccase concentrations of 5 U/mL and 10 U/mL,namely HMGL-5 and HMGL-10,were constructed,whereas a hyaluronic acid methacryloyl(HAMA)hydrogel served as the control.The molecular structures of HAMA and gelatin grafted with vanillin(GelVA)were verified by 1H nuclear magnetic resonance spectra(1H NMR)and Fourier transform infrared spectroscopy(FTIR).The microstructures of the hydrogels were observed by scanning electron microscope(SEM),and the mechanical properties of the hydrogels were tested by a rotational rheometer and a dynamic mechanical analyzer(DMA).The induced hypoxic behaviors of the hydrogels were detected by an oxygen consumption fluorescent probe.The biocompatibility of the hydrogels was tested by live/dead staining and the cell counting kit 8(CCK-8)assay,and the expression of hypoxia-inducible factor-1α(HIF-1α)and type Ⅱ collagen(Col Ⅱ)in NPCs was detected by immunofluorescence staining.A rat model of intervertebral disc degeneration was established,and hydrogels loaded with NPCs were injected into degenerated intervertebral discs to evaluate their repair effects.Disc height and disc water content changes were detected by X-ray imaging and magnetic resonance imaging(MRI),and the integrity of disc structure and proteoglycan levels were detected by histological staining.Results·Structural characterization demonstrated that the materials had been successfully prepared.SEM showed that the three hydrogels all had a loose and porous structure,and their elastic moduli increased with the decrease of pore size.Hypoxia test results indicated that the hypoxia-inducing ability of HMGL-10 hydrogel was the strongest.In vitro experimental results showed that the three hydrogels all had good biocompatibility.Compared with the HAMA hydrogel,the HIF-1α of NPCs was significantly activated in the HMGL-10 hydrogel.The expression levels of HIF-1α and Col Ⅱ in the HMGL-10 hydrogel group were 3.38 and 4.15 times higher than those in the HAMA hydrogel group.In vivo experimental results showed that the integrity,height,and water content of the intervertebral discs in the hypoxia hydrogel group were all superior to those in the other treatment groups.Conclusion·The in situ hypoxia-inducing hydrogel effectively activates HIF-1α,promotes extracellular matrix secretion,and demonstrates superior regenerative potential for intervertebral disc repair.

Objective·To construct an interpenetrating network hydrogel(HMGL)that can induce hypoxia in situ,investigate its effects on promoting the secretion of extracellular matrix in nucleus pulposus cells(NPCs),and evaluate its potential in the treatment of intervertebral disc degeneration.Methods·HMGLs capable of long-term in situ hypoxia induction were prepared by a two-step polymerization method.Hypoxic hydrogels with laccase concentrations of 5 U/mL and 10 U/mL,namely HMGL-5 and HMGL-10,were constructed,whereas a hyaluronic acid methacryloyl(HAMA)hydrogel served as the control.The molecular structures of HAMA and gelatin grafted with vanillin(GelVA)were verified by 1H nuclear magnetic resonance spectra(1H NMR)and Fourier transform infrared spectroscopy(FTIR).The microstructures of the hydrogels were observed by scanning electron microscope(SEM),and the mechanical properties of the hydrogels were tested by a rotational rheometer and a dynamic mechanical analyzer(DMA).The induced hypoxic behaviors of the hydrogels were detected by an oxygen consumption fluorescent probe.The biocompatibility of the hydrogels was tested by live/dead staining and the cell counting kit 8(CCK-8)assay,and the expression of hypoxia-inducible factor-1α(HIF-1α)and type Ⅱ collagen(Col Ⅱ)in NPCs was detected by immunofluorescence staining.A rat model of intervertebral disc degeneration was established,and hydrogels loaded with NPCs were injected into degenerated intervertebral discs to evaluate their repair effects.Disc height and disc water content changes were detected by X-ray imaging and magnetic resonance imaging(MRI),and the integrity of disc structure and proteoglycan levels were detected by histological staining.Results·Structural characterization demonstrated that the materials had been successfully prepared.SEM showed that the three hydrogels all had a loose and porous structure,and their elastic moduli increased with the decrease of pore size.Hypoxia test results indicated that the hypoxia-inducing ability of HMGL-10 hydrogel was the strongest.In vitro experimental results showed that the three hydrogels all had good biocompatibility.Compared with the HAMA hydrogel,the HIF-1α of NPCs was significantly activated in the HMGL-10 hydrogel.The expression levels of HIF-1α and Col Ⅱ in the HMGL-10 hydrogel group were 3.38 and 4.15 times higher than those in the HAMA hydrogel group.In vivo experimental results showed that the integrity,height,and water content of the intervertebral discs in the hypoxia hydrogel group were all superior to those in the other treatment groups.Conclusion·The in situ hypoxia-inducing hydrogel effectively activates HIF-1α,promotes extracellular matrix secretion,and demonstrates superior regenerative potential for intervertebral disc repair.

Purpose To investigate the role of ultrasound-guided fine needle aspiration cytology(US-FNAC)in the diagnosis of breast cancer.Methods Fine needle aspiration cytology(FNAC)samples of 203 patients with breast cancer were selected and prepared by liquid-based cytology.The results of histopathology after neoadjuvant therapy were used as the gold standard to compare with the diagnostic results of FNAC samples,and then evaluated the diagnos-tic coincidence rate of FNAC samples.Results Of 203 axillary lymph node FNAC samples,111 cases were diagnosed as positive,38 cases were diagnosed as suspicious for positivity,and 54 cases were diagnosed as negative.The diag-nostic accuracy of FNAC was 85.2％,the sensitivity of FNAC was 98.0％,and the specificity of FNAC was 90.0％.Conclusion FNAC has high sensitivity and accuracy in the diagnosis of axillary lymph node metastasis.Sentinel lymph node biopsy is recommended for patients with negative FNAC but clinically and radiographically suspected lymph node metastasis.

Objective:To construct a calcium alginate (ALG-Ca 2+) composite hydrogel loaded with chlorella protein (Cp) (ALG-Ca 2+@Cp) and investigate its combined effect with microwave hyperthermia on the proliferation, apoptosis, and immune activation of mouse pancreatic cancer cells. Methods:ALG-Ca 2+@Cp was prepared using a physical cross-linking method and its physiochemical properties was characterized via scanning electron microscopy, rheological analysis, Ca 2+ release experiments, and microwave thermal conversion tests. The BCA protein quantification assay was used to evaluate the adsorption capacity of ALG-Ca 2+@Cp for pancreatic cancer cell antigens. The effects of ALG-Ca 2+@Cp extract combined with microwave intervention on pancreatic cancer cell proliferation, apoptosis protein expression, and cell viability were assessed using CCK-8 assays, ELISA, and Calcein-AM/PI double fluorescence staining. Flow cytometry was performed to determine the maturation-promoting ability of ALG-Ca 2+@Cp on immature mouse bone marrow-derived dendritic cells (BMDCs). Results:ALG-Ca 2+@Cp exhibited a three-dimensional network structure with a storage modulus (G') greater than the loss modulus (G''), demonstrating typical hydrogel properties. The hydrogel loaded with 0.5 mol/ml Ca 2+ reached 48°C after 5 minutes of microwave irradiation at 5.0 W/cm 2, and Ca 2+ release plateaued within 5 minutes. ALG-Ca 2+@Cp effectively adsorbed pancreatic cancer cell antigens. Combined with microwave treatment, it significantly reduced pancreatic cancer cell proliferation ( A450 value 0.39±0.07 vs 2.78±0.15) and increased apoptosis markers calreticulin (CRT) and high mobility group box-1 protein (HMGB1) [(557.09±37.84) pg/ml vs (135.14±11.84) pg/ml, (4.77±0.18) ng/ml vs (1.6±0.16) ng/ml], leading to decreased cell viability; and all the differences were statistically significant (all P value <0.05). ALG-Ca 2+@Cp synergistically promoted the maturation of immature BMDCs in the presence of pancreatic cancer antigens, with a CD 80+ positivity rate of (75.67±6.53)%. Conclusions:ALG-Ca 2+@Cp is successfully constructed. Its combination with microwave hyperthermia can significantly enhance the cytotoxicity and immune activation against mouse pancreatic cancer cells by targeting intracellular antigens and inducing immunogenic cell death.

Objective:To elucidate the molecular mechanisms through which high-dose dexamethasone exerts long-term effects on bone marrow mesenchymal stem cells (BMSCs), specifically its role in suppressing osteogenic differentiation, accelerating cellular senescence, triggering the senescence-associated secretory phenotype (SASP), and inducing apoptosis.Methods:Primary rat BMSCs were isolated and treated with high-dose dexamethasone (1×10 -4 mol/L) to establish the experimental group, while untreated cells served as the control. The gene and protein expression levels of osteogenic markers, bone alkaline phosphatase (bALP) and Runt-related transcription factor 2 (Runx2), were analyzed in both groups. Cellular senescence was evaluated using senescence-associated β-galactosidase (SA-β-gal) staining. The expression of senescence-related markers (P16 and P21), components of the senescence-associated secretory phenotype (SASP), including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-γ, as well as apoptosis-related proteins (Bcl-2, Bax, and Cleaved-Caspase-3), and key factors of the Nrf2/HO-1 signaling pathway were assessed at both transcriptional and protein levels using qRT-PCR, immunofluorescence, and Western-blot analyses. These comprehensive evaluations aimed to determine the senescent state, apoptotic features, and alterations in osteogenic differentiation of BMSCs. Results:Following treatment with dexamethasone and subsequent withdrawal, both qRT-PCR and Western blot analyses indicated a significant reduction in the expression of the osteogenic markers bALP and Runx2 at both mRNA and protein levels. The proportion of SA-β-gal positive cells was markedly higher in the dexamethasone group (74.33%±6.89%) than in the control group (20.30%±1.57%, t=17.300, P<0.001). qRT-PCR analysis revealed upregulated mRNA expression of the senescence-related genes P16 and P21 after dexamethasone treatment, which was further supported at the protein level by immunofluorescence showing increased P21 expression. Western-blot results confirmed that protein expression levels of P16 and P21 were significantly elevated in the dexamethasone group (7.025±0.255 and 6.362±0.456, respectively) compared with the control group (1.016±0.115 and 0.816±0.172; both P<0.05). Furthermore, gene expression levels of the senescence-associated secretory phenotype (SASP) factors TNF-α and IL-1β were significantly increased (TNF-α: 3.539±0.599 vs. 0.742±0.095; IL-1β: 4.469±0.331 vs. 0.799±0.175; both P<0.05), and their protein expression was consistently upregulated as validated by Western-blot. Additionally, protein expression levels of TNF-α, IL-1β, and IFN-γ were significantly higher in the dexamethasone-treated group (3.476±0.932 vs. 0.945±0.095; 4.111±0.220 vs. 0.762±0.105; 2.155±0.240 vs. 0.656±0.104; all P<0.05).Western-blot analysis also demonstrated that protein expression of Nrf2 and HO-1 was significantly suppressed in the dexamethasone group (0.21±0.07 and 0.19±0.06, respectively) compared with the control group (1.13±0.15 and 0.92±0.21; P<0.05). Moreover, Western-blot analysis revealed that the expression levels of the pro-apoptotic proteins Bax and Cleaved-Caspase-3 were significantly up, regulated in the dexamethasone, treated BMSCs (Bax: 3.673±0.397 vs. 0.453±0.111; Cleaved-Caspase-3: 3.863±0.399 vs. 0.465±0.057), while the expression of the anti-apoptotic protein Bcl-2 was markedly down, regulated (0.959±0.073 vs. 2.126±0.195), with all differences being statistically significant ( P<0.05). Conclusions:High-dose dexamethasone treatment of BMSCs, followed by withdrawal of dexamethasone, induces cellular senescence and enhances the expression of the senescence-associated secretory phenotype (SASP) through suppression of the Nrf2/HO-1 signaling pathway. Concurrently, it promotes apoptosis by activating the mitochondrial apoptotic pathway, collectively leading to impaired osteogenic differentiation of BMSCs.

ObjectiveTo investigate the effect and underlying mechanism of the Wulao Qisun prescription on pathological new bone formation in ankylosing spondylitis （AS）. MethodsSynovial fibroblasts were isolated from the hip joints of AS patients and observed under a microscope to assess cell morphology. The cells were identified using immunofluorescence staining. The isolated AS fibroblasts were divided into blank group， low drug-containing serum group， medium drug-containing serum group， high drug-containing serum group， and positive drug group. After drug intervention， cell proliferation was measured using the cell counting kit-8 （CCK-8） assay to observe fibroblast growth and determine the optimal intervention time. Alkaline phosphatase （ALP） activity was measured using the alkaline phosphatase assay. Protein expression of osteocalcin （OCN）， osteopontin （OPN）， and runt-related transcription factor 2 （Runx2） was detected by Western blot. The mRNA expression levels of Wnt5a， β-catenin， and Dickkopf-1 （DKK-1） were measured by real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsCompared with the blank group， each drug-containing serum group of Wulao Qisun prescription and the positive drug group inhibited the proliferation of AS fibroblasts and reduced ALP expression （P<0.01）. Compared with the blank group， the low drug-containing serum group of Wulao Qisun prescription downregulated β-catenin mRNA expression （P<0.05）. The medium and high drug-containing serum groups and the positive drug group significantly downregulated Wnt5a and β-catenin mRNA expression （P<0.05， P<0.01）， with the positive drug group showing the most pronounced effect （P<0.01）. The high drug-containing serum group and the positive drug group significantly upregulated DKK-1 mRNA expression （P<0.01）. Compared with the blank group， the low drug-containing serum group of Wulao Qisun prescription inhibited the expression of OPN and Runx2 proteins （P<0.05， P<0.01）， while the medium and high drug-containing serum groups and the positive drug group inhibited the expression of OCN， OPN， and Runx2 proteins （P<0.05， P<0.01）. ConclusionThe Wulao Qisun prescription can inhibit the proliferation and osteogenic differentiation of AS fibroblasts， thereby delaying the formation of pathological new bone in AS. The possible mechanism involves the regulation of Wnt/β-catenin-related gene expression， further inhibiting the transcription of downstream target genes.

BACKGROUND:Solute carrier family 1 member 5(SLC1A5)plays a potential role in a variety of diseases,but the exact mechanism of action is unclear.The construction of stable SLC1A5 overexpression and knockdown cell models can provide a powerful experimental tool for in-depth study of the exact role and mechanism of SLC1A5 in diseases and the discovery of potential therapeutic targets. OBJECTIVE:To construct lentiviral vectors for overexpression and knockdown of mouse SLC1A5 and establish stable transfected RAW264.7 cell lines,so as to provide an experimental foundation for further investigation of the role of SLC1A5 in inflammation. METHODS:Primers were designed and synthesized based on the SLC1A5 gene sequence,and the gene segment was amplified using polymerase chain reaction.Subsequently,the target gene segment was directionally inserted into the GV492 vector plasmid,which had been digested with AgeI/NheI enzymes,to construct recombinant lentiviral plasmids.Positive clones were further selected,and their sequences were confirmed.The pHelper1.0 plasmid vector and pHelper2.0 plasmid vector,along with the target plasmid vector,was co-cultured with 293T cells for transfection,resulting in the production and titration of lentiviral stocks.Furthermore,RAW264.7 cells were cultured in vitro,and the working concentration of puromycin was determined.Lentiviruses were separately co-cultured with RAW264.7 cells,and transfection efficiency was determined by measuring fluorescence intensity.Stable transfected cells were selected using puromycin,and real-time fluorescence quantitative PCR and western blot assay were employed to assess the gene and protein expression levels of SLC1A5 in stably transfected cell lines. RESULTS AND CONCLUSION:(1)Sequencing results indicated a perfect match between the sequencing and target sequences,confirming the successful construction of recombinant lentiviral vectors.(2)The titer for the overexpression SLC1A5 lentivirus was 1×109 TU/mL,while the titer for the knockdown SLC1A5 lentivirus was 3×109 TU/mL.(3)The working concentration of puromycin for RAW264.7 cells was determined to be 3 μg/mL.(4)The optimal conditions for transfecting RAW264.7 cells with overexpression/knockdown expression of SLC1A5 lentivirus involved the use of HiTransG P transfection enhancer with a multiplicity of infection value of 50.(5)A significant upregulation of the gene and protein expression levels of SLC1A5 was detected in cell lines stably overexpressing SLC1A5,while gene and protein expression levels of SLC1A5 were significantly decreased in the knockdown stable cell lines.These findings indicate that lentiviral vectors for mouse SLC1A5 overexpression and knockdown have been successfully constructed and a stably transfected RAW264.7 cell line has been obtained.

Purpose To investigate the role of ultrasound-guided fine needle aspiration cytology(US-FNAC)in the diagnosis of breast cancer.Methods Fine needle aspiration cytology(FNAC)samples of 203 patients with breast cancer were selected and prepared by liquid-based cytology.The results of histopathology after neoadjuvant therapy were used as the gold standard to compare with the diagnostic results of FNAC samples,and then evaluated the diagnos-tic coincidence rate of FNAC samples.Results Of 203 axillary lymph node FNAC samples,111 cases were diagnosed as positive,38 cases were diagnosed as suspicious for positivity,and 54 cases were diagnosed as negative.The diag-nostic accuracy of FNAC was 85.2％,the sensitivity of FNAC was 98.0％,and the specificity of FNAC was 90.0％.Conclusion FNAC has high sensitivity and accuracy in the diagnosis of axillary lymph node metastasis.Sentinel lymph node biopsy is recommended for patients with negative FNAC but clinically and radiographically suspected lymph node metastasis.