ObjectiveTo investigate the mechanism of modified Si Junzitang and Shashen Maidong Tang ［Yiqi Yangyin Jiedu prescription （YQYYJD）］ in enhancing the sensitivity of cisplatin in epidermal growth factor receptor tyrosine kinase inhibitor （EGFR-TKI）-resistant lung adenocarcinoma cells based on aerobic glycolysis. MethodsThe effects of different concentrations of YQYYJD （0， 2， 3， 4， 5， 6， 7， 8 g·L^-1） and cisplatin （0， 3， 6， 9， 12， 15， 18， 21， 24， 27 mg·L^-1） on the proliferation and activity of PC9/GR cells were detected by the cell counting kit-8 （CCK-8） assay after 24 hours of intervention. The half-maximal inhibitory concentration （IC₅₀） for PC9/GR cells was calculated to determine the concentrations used in subsequent experiments. PC9/GR cells were divided into blank group （complete medium）， YQYYJD group （5 g·L^-1）， cisplatin group （12 mg·L^-1）， and combined group （YQYYJD 5 g·L^-1 + cisplatin 12 mg·L^-1）. After 24 hours of intervention， cell viability was measured using CCK-8 assay. Cell proliferation was assessed by colony formation assay， and cell migration was evaluated by scratch and Transwell assays. Glucose consumption， lactate production， and adenosine triphosphate （ATP） levels were measured by colorimetric assays. The expression levels of glycolysis-related proteins， including hexokinase 2 （HK2）， phosphofructokinase P （PFKP）， pyruvate kinase M2 （PKM2）， lactate dehydrogenase A （LDHA）， glucose transporter 1 （GLUT1）， and monocarboxylate transporter 4 （MCT4）， were determined by Western blot. ResultsBoth YQYYJD and cisplatin inhibited the viability of PC9/GR cells in a concentration-dependent manner. The IC₅₀ of PC9/GR cells for YQYYJD and cisplatin were 5.15 g·L^-1 and 12.91 mg·L^-1， respectively. In terms of cell proliferation， compared with the blank group， the cell survival rate and the number of colonies formed in the YQYYJD group， cisplatin group， and combined group were significantly decreased （P<0.01）. Compared with the YQYYJD and cisplatin groups， the combined group showed a further significant reduction in cell survival rate and colony formation （P<0.01）. In terms of cell migration， compared with the blank group， the cell migration rate and the number of cells passing through the Transwell membrane in the YQYYJD group， cisplatin group， and combined group were significantly decreased （P<0.01）. Compared with the YQYYJD and cisplatin groups， the combined group exhibited a further significant reduction in cell migration rate and the number of cells passing through the Transwell membrane （P<0.01）. In terms of glycolysis， compared with the blank group， glucose consumption， lactate production， and ATP levels in the YQYYJD group， cisplatin group， and combined group were significantly decreased （P<0.01）. Compared with the YQYYJD and cisplatin groups， the combined group showed a further significant reduction in glucose consumption， lactate production， and ATP levels （P<0.05）. Compared with the blank group， the protein expression levels of HK2， PFKP， PKM2， and LDHA in the YQYYJD， cisplatin， and combined groups were significantly decreased （P<0.01）. The combined group showed a further significant reduction in the expression levels of these proteins compared with the YQYYJD and cisplatin groups （P<0.01）. No significant differences were observed in the protein expression levels of GLUT1 and MCT4 among the groups. ConclusionYQYYJD can synergistically inhibit the proliferation and migration of PC9/GR cells and enhance their sensitivity to cisplatin. The mechanism may be related to the downregulation of the expression of glycolysis-related rate-limiting enzymes， including HK2， PFKP， PKM2， and LDHA， thereby inhibiting glycolysis.

ObjectiveTo investigate the possible mechanism by which Zhiqiao Gancao Decoction （枳壳甘草汤， ZGD） delays intervertebral disc degeneration （IDD） based on the Hippo-yes-associated protein （YAP）/transcriptional co-activator with PDZ-binding motif （TAZ） signaling pathway. MethodsA total of 50 SD rats were randomly divided into sham surgery group， model group， low-dose ZGD group， high-dose ZGD group， and high-dose ZGD + inhibitor group， with 10 rats in each group. In the sham surgery group， the rats were pierced in the skin and muscle at the Co6/7/8 segments of the tail with a 21G needle （depth approximately 2 mm） without damaging the intervertebral disc. In the other groups， rats were injected with a 21G needle at the Co6/7/8 segments of the tail to establish an IDD model by piercing the tail intervertebral disc 5 mm. One week after modeling， rats in the low-dose and high-dose ZGD groups were given 6.24 and 12.24 g/（kg·d） of the decoction via gastric gavage， respectively. The high-dose ZGD + inhibitor group was given 12.24 g/（kg·d） of the decoction and an intraperitoneal injection of YAP/TAZ inhibitor Verteporfin 10 mg/kg. The sham surgery and model groups were given 5 ml/（kg·d） of normal saline via gavage. The gavage was given once a day， and the intraperitoneal injection was given every other day. After 4 weeks of continuous intervention， the pathological changes of the tail intervertebral discs were observed using HE staining， Oil Red O-Green staining， and Toluidine Blue staining. Immunohistochemistry was used to detect the expression of aggrecan and MMP3 in the nucleus pulposus. TUNEL fluorescence staining was performed to detect apoptosis in the nucleus pulposus， and the apoptosis rate was calculated. Western blot was used to detect the Hippo-YAP/TAZ signaling pathway， including YAP， phosphorylated YAP （p-YAP）， phosphorylated MST1/2 （p-MST1/2）， phosphorylated TAZ （p-TAZ） and apoptosis-related proteins， such as Cleaved Caspase 3， P53， Bcl-2 and Bax. ResultsCompared with sham surgery group， the rats in the model group showed significant degenerative changes in the intervertebral disc. The levels of aggrecan， Bcl-2， and YAP proteins in the nucleus pulposus decreased， while the levels of p-MST1/2， p-YAP， p-TAZ， P53， Bax， Cleaved Caspase 3， MMP3 proteins， and the apoptosis rate increased （P < 0.01）. Compared with the model group， the drug intervention groups showed partial recovery in intervertebral disc degeneration. The levels of aggrecan， Bcl-2， and YAP proteins increased， while the levels of p-MST1/2， p-YAP， p-TAZ， P53， Bax， Cleaved Caspase 3， MMP3 proteins， and the apoptosis rate decreased （P＜0.05 or P＜0.01）. The high-dose ZGD group showed more significant recovery in intervertebral disc degeneration compared to the low-dose ZGD group， with a decrease in the levels of p-MST1/2， p-YAP， p-TAZ， P53， Bax， Cleaved Caspase 3， MMP3 proteins， and apoptosis rate， and an increase in the levels of aggrecan， Bcl-2， and YAP proteins （P＜0.05 or P＜0.01）. Compared with the high-dose ZGD group， the high-dose ZGD + inhibitor group showed a reduced recovery in intervertebral disc degeneration， with an increase in the levels of p-MST1/2， p-YAP， p-TAZ， P53， Bax， Cleaved Caspase 3， MMP3 proteins， and apoptosis rate， and a decrease in the levels of aggrecan， Bcl-2， and YAP proteins （P＜0.05 or P＜0.01）. ConclusionZGD may delay intervertebral disc degeneration by inhibiting the phosphorylation of YAP in the nucleus pulposus， maintaining the function of the Hippo-YAP/TAZ signaling pathway， and reducing apoptosis of nucleus pulposus cells.

ObjectiveTo explore the effect of timosaponin BⅡ （TBⅡ） combined with icariin （ICA） on osteoclast （OC）-osteoblast （OB） coupling and decipher the mechanism from the cellular level. MethodsThe cell counting kit-8 （CCK-8） was used to assess the effects of different concentrations of TBⅡ and different concentrations of TBⅡ+ICA on the growth of RAW264.7 cells. Soluble receptor activator of nuclear factor-κB ligand （sRANKL） was used to induce the differentiation of RAW264.7 pre-osteoclasts into osteoclasts. The cells were allocated into sRANKL， TBⅡ （1， 5， 10 μmol·L^-1）， and TBⅡ+ICA groups. Tartrate-resistant acid phosphatase staining was performed to assess the effects of TBⅡ and TBⅡ+ICA on osteoclast differentiation. Real-time quantitative polymerase chain reaction （Real-time PCR） was conducted to examine the effects of TBⅡ+ICA on the expression of key genes involved in osteoclast differentiation and osteoclast-derived coupling factors. The osteogenic differentiation conditioned medium mixed with osteoclast supernatant was used to induce osteogenic differentiation of MC3T3-E1 cells. Alkaline phosphatase staining and alizarin red S staining were employed to determine the effect of TBⅡ+ICA on osteogenic differentiation. Real-time PCR was employed to evaluate the effects of conditioned medium on key genes involved in osteogenic differentiation. ResultsTBⅡ at 1， 5， 10 μmol·L^-1 had no significant effect on the cell survival rate. Compared with the sRANKL group， TBⅡ inhibited osteoclast differentiation in a dose-dependent manner and achieved the best effect at 10 μmol·L^-1 （P<0.01）. Compared with the sRANKL group， different concentrations of TBⅡ down-regulated the mRNA levels of osteoclast differentiation-related genes c-Fos， RANK， and RANKL （P<0.05）. None of 10 μmol·L^-1 TBⅡ， 10 μmol·L^-1 TBⅡ+10^-4 μmol·L^-1 ICA， or 10 μmol·L^-1 TBⅡ+10^-3 μmol·L^-1 ICA affected the viability of RAW264.7 cells. TBⅡ and/or ICA inhibited osteoclast differentiation （P<0.01）， and TBⅡ + ICA had the best effect （P<0.01）. Compared with the sRANKL group， TBⅡ and/or ICA down-regulated the mRNA levels of c-Fos， RANK， and RANKL （P<0.05）. The single application of TBⅡ and ICA had no significant effect on the mRNA levels of Wnt10b， Cthrc1， and C3a， while TBⅡ+ICA exerted up-regulating effects （P<0.05）. Compared with those in the blank group， the bone differentiation and mineralization abilities of the normal osteogenic induction group and each osteogenic induction + osteoclast supernatant group were improved （P<0.01）. Compared with the blank group， the normal osteogenic induction group and the osteogenic induction + osteoclast supernatant group showed up-regulated mRNA levels of Runx2 and OCN （P<0.01）. ConclusionTBⅡ+ICA can inhibit osteoclast differentiation， maintain the normal osteoclast-osteoblast coupling， and promote osteogenic differentiation.

Objective Zinc finger protein 335 (Zfp335) plays a crucial role in the early development of thymic T cells and the differentiation of peripheral T cell subpopulations. The objective of this study is to investigate the role and underlying mechanisms of Zfp335 in the regulation of regulatory T cell (Treg) within tumor immunity. Methods The Zfp335 gene was specifically knocked out in Treg using tamoxifen (Zfp335^fl/fl FOXP3^creERT2), and the MC38 tumor model was established. On the 7th day after tumor inoculation, tumor size was observed and measured. Tumor size was monitored and recorded daily starting from day 7 post-inoculation. On day 12, tumors were harvested, and the proportions of CD4⁺ T cells, CD8⁺ T cells, and Treg were analyzed by flow cytometry. Additionally, the mitochondrial function of effector regulatory T cell (eTreg) was assessed. Results From day 10 post-tumor inoculation, tumor volume in the Zfp335^CKO group was significantly reduced compared to that of the wild-type (WT) group. Furthermore, the infiltration of CD4⁺ and CD8⁺ T cells, along with their respective effector cells, was significantly higher in the Zfp335^CKO group than in the WT group. The proportions of CD4⁺ and CD8⁺ T cells producing interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) were also significantly increased in the Zfp335^CKO group compared to that of the WT group. In addition, the percentage of CD8⁺ T cells secreting granzyme B (GzmB) was significantly higher in the Zfp335^CKO group than that in the WT group. In contrast, the proportion of Treg and inducible T cell co-stimulator (ICOS)⁺ Treg in the Zfp335^CKO group was significantly lower than that in the WT group. Finally, the expression level of Mitotracker Deep Red in eTreg from the Zfp335^CKO group was significantly reduced compared to that in the WT group. Conclusion During tumorigenesis, the specific deletion of Zfp335 impairs Treg activation, which is related to decreased mitochondrial function in eTreg. In Zfp335^CKO mice. Tumors exhibit increased infiltration of effector T cells, accompanied by elevated levels of cytotoxic cytokines, ultimately enhancing resistance to tumor progression.
Animals ; T-Lymphocytes, Regulatory/metabolism* ; Mice ; CD8-Positive T-Lymphocytes/immunology* ; Neoplasms/genetics* ; Cell Line, Tumor ; Mice, Inbred C57BL ; Mice, Knockout ; DNA-Binding Proteins/genetics* ; Female

OBJECTIVE: To explore the function of LIM and calponin homology domains 1 (LIMCH1) in the development and progression of oral squamous cell carcinoma (OSCC), along with their potential clinical applications. METHODS: By utilizing transcriptome sequencing data from two groups of oral squamous cell carcinoma patients, along with bioinformatics analytical techniques such as Gene Ontology (GO) and gene co-expression networks, we identified genes that might play a pivotal role in the pathogenesis of oral squamous cell carcinoma. We employed real-time quantitative PCR and Western blotting to validate the expression patterns of these genes across twelve patient tissue samples. Furthermore, we conducted CCK-8 assays, flow cytometry analyses, and scratch wound healing assays to assess the impact of key genes on the biological behaviors of both the Cal27 oral squamous cell carcinoma cell line and the potentially malignant DOK oral lesion cell line. Additionally, we examined correlations between these key genes and clinical disease parameters in 214 oral squamous cell carcinoma patients using The Cancer Genome Atlas (TCGA) data; gene set enrichment analysis (GSEA) analysis results were also incorporated to enhance our findings from real-time quantitative PCR and Western blotting regarding potential mechanisms underlying the action of these key genes. RESULTS: The integrated analysis of sequencing data and bioinformatics revealed that LIMCH1 exhibited significantly reduced mRNA (P < 0.001) and protein levels (P < 0.01) in the oral squamous cell carcinoma tissues compared with normal control tissues. In the Cal27 cells, the low LIMCH1 level group demonstrated a larger wound healing area within 24 hours than the control group (P < 0.01), enhanced proliferation capacity over 72 hours relative to the control group (P < 0.01), and an increased apoptosis rate within 24 hours compared with the high expression group (P < 0.05). However, no significant differences were observed between the low and high level groups in DOK cells. Furthermore, it was determined that low LIMCH1 level correlated with poor prognosis in the patients (P=0.013) and a higher lymph node metastasis rate (P < 0.05). Investigations into the potential mechanisms of action indicated that LIMCH1 did not influence the onset or progression of oral squamous cell carcinoma via the epithelial-mesenchymal transition pathway. CONCLUSION LIMCH1 level may function as a promising biomarker to aid in the prognostic assessment of oral squamous cell carcinoma; however, its precise mechanistic role requires further investigation.
Humans ; Mouth Neoplasms/metabolism* ; Prognosis ; Carcinoma, Squamous Cell/metabolism* ; Biomarkers, Tumor/metabolism* ; LIM Domain Proteins/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Male ; Female

Medical equipment, as an important indicator of smart hospital evaluation, plays a vital role in hospital operations. To ensure the safe and efficient operation of medical equipment, a reasonable performance evaluation system is indispensable. This study introduces a platform based on Internet of Things (IoT) technology that connects medical devices and collects data, achieving standardized and structured data processing, and supporting online operational supervision. Through the Delphi method, a performance evaluation system for large medical equipment is constructed, including 4 primary indicators and 22 secondary indicators. DICOM data acquisition devices are used to achieve functions such as efficiency analysis, benefit analysis, usage evaluation, and decision-making support for medical equipment. The study is still in its early stages, and in the future, it is expected to integrate more types of equipment, achieve rational resource allocation, and significantly impact decision-making for the development of public hospitals.
Internet of Things ; Delphi Technique

Objective To investigate the expression of microRNA(miR)-15b and miR-503 in peripheral blood in patients with depression and their relationship with cognitive function.Methods A total of 103 patients with depression admitted and diagnosed in Shaoxing Seventh People's Hospital from January 2023 to June 2024 were set as study group,and 94 healthy participants were selected as control group.The cognitive function of patients with depression was evaluated by Montreal cognitive assessment(MoCA),and the patients in study group were divided into normal cognitive function group(n=68)and cognitive dysfunction group(n=35).Pearson method was used to analyze the correlation between the expression level of miR-15b,miR-503 and cognitive function;The diagnostic value of miR-15b and miR-503 for cognitive dysfunction in patients with depression was analyzed by using receiver operating characteristic(ROC)curve.Results miR-15b and miR-503 in the peripheral blood of study group were lower than those of control group(P＜0.05),miR-15b and miR-503 in the peripheral blood of cognitive dysfunction group were lower than those of normal cognitive function group(P＜0.05),the scores of MoCA and mini-mental state examination in normal cognitive function group were higher than those in cognitive dysfunction group(P＜0.05);The results of Pearson analysis showed that the levels of miR-15b and miR-503 in peripheral blood of patients with depression were significantly positively correlated with MoCA scores(r=0.635,0.668,P＜0.05);The area under the curve(AUC)of miR-15b level alone in diagnosing cognitive dysfunction in patients with depression was 0.836(95％CI:0.750-0.902),the sensitivity and specificity were 77.14％and 89.71％respectively;The AUC of miR-503 level alone in diagnosing cognitive dysfunction in patients with depression was 0.892(95％CI:0.816-0.945),the sensitivity and specificity were 80.00％and 88.24％respectively;The AUC for the combined diagnosis of miR-15b and miR-503 was significantly greater than the AUC for miR-15b and miR-503 alone.Conclusion The expression of miR-15b and miR-503 is decreased in patients with depression,and they are of high diagnostic value for cognitive dysfunction in patients with depression.

Objective To investigate the effects of Aikeqing(AKQ),a compound of Chinese medicine,on drug-metabolizing activity and on the pharmacokinetic parameters of HIV-1 protease inhibitors lopinavir(LPV)and ritonavir(RTV)in Kaletra tablets.Methods Human liver microsomes were co-incubated with mixed probes and AKQ.HPLC-MS was employed to measure the content of probe.Half-inhibitory concentration(IC50)of AKQ on different subtypes of cytochrome P450 enzymes involved in drug metabolism of liver microsomes was calculated.The P450 subtype,whose activity was significantly inhibited by AKQ was then identified.HPLC-MS analytical method for simultaneous determination of LPV and RTV in rat plasma was established.SD rats were orally given AKQ or vehicle once a day for 7 consecutive days.After half an hour of the last gavage of AKQ,the rats were given Kaletra by intragastric administration.Then,the blood concentration of LPV and RTV were measured and the effect of AKQ on pharmacokinetic parameters of LPV and RTV were analyzed.Results The methanol extract of AKQ at the concentrations of 0～500 μg·mL-1 showed inhibitory effects on the metabolic activity of CYP2D6,CYP2C8、CYP2E1,CYP2C19,CYP1A2,CYP2B6,CYP2C9 and CYP3A4,with IC50 values of 7.7,75.3,144.0,99.5,43.5,104.5,49.3 and 204.9 μg·mL-1,respectively.An HPLC-MS/MS method was established for simultaneous quantification of LPV and RTV in blood samples.LPV and RTV showed linear relationships in the ranges of 30～10 000 ng·mL-1 and 3～1 000 ng·mL-1,respectively.The lowest limits of quantification were 30 ng·mL-1 and 3 ng·mL-1.Intra-day and inter-day precision were all less than 5%,and the accuracy of LPV and RTV was in the range of 96.3%～109%.The extraction recovery rates were not less than 88.7%,the matrix effects were 93.8%～105.0%,and the plasma samples were stable.Compared with Kaletra group,there was no significant changes in non-compartmental model parameters including AUC0-t,AUC0-∞,MRT0-t,t1/2z,tmax,Vz/F,Clz/F and Cmax of LPV in AKQ+Kaletra group(P＞0.05).But MRT0-∞ was found to be obviously affected by AKQ(P＜0.05).All pharmacokinetic parameters of RTV showed no significant change in AKQ+Kaletra group(P＞0.05).Conclusion Aikeqing exhibited varying degrees of inhibitory effects on 8 human drug-metabolizing cytochromes P450,especially CYP2D6.A human-equivalent dose of Aikeqing does not affect the pharmacokinetic parameters of lopinavir and ritonavir in rats.

Objective:To explore the role of metagenomic next-generation sequencing (mNGS) in the diagnosis of pathogens in primary infectious diseases of the spine (IDS) and to reveal its pathogen spectrum.Methods:This is a retrospective multi-center case series study. Clinical data of 380 patients with primary IDS who were treated at four medical centers in China from December 2019 to April 2024 were retrospectively analyzed. Among them, 82 cases were from the Department of Spine Surgery at the Affiliated Hospital of Qingdao University, 129 cases were from the Orthopedics Section Ⅱ (Bone Infection), Public Health Clinical Center Affiliated to Shandong University, 112 cases were from the Department of Spine Surgery, Fuzhou Second General Hospital, and 57 cases were from the Department of Orthopedic Surgery, Shanghai Sixth People′s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. There were 238 males and 242 females, with an age of (61.4±13.1) years (range: 10 to 91 years). Specimens from the site of spinal infection were obtained for pathogen culture, pathological examination, and mNGS detection preoperatively or intraoperatively in all patients. The number, types, and positive rates of pathogens detected by the two methods were analyzed and compared using the Chi-square test.Results:Among the 380 patients, 320 had confirmed pathogenic bacteria, with the highest proportion being pyogenic bacterial infections, accounting for 76.9% (246/320). The most common pathogen was Staphylococcus aureus, accounting for 22.8% (73/320). Brucella accounted for 13.8% (44/320); Mycobacterium tuberculosis accounted for 6.3% (20/320). Fungal infections accounted for 3.4% (11/320), mainly Aspergillus and Candida. In addition, Mycoplasma was detected in 3 cases (0.9%) and Benacox body in 4 cases (1.2%). The pathogen spectrum constructed by mNGS covered 46 types of pathogens, higher than the 22 types detected by traditional methods. The positive rate of mNGS was 80.8% (308/381), significantly higher than the 27.9% (106/381) of traditional methods ( χ2=182.53, P<0.01). Conclusions:mNGS improves the positive rate of pathogen diagnosis in IDS, detecting a broader spectrum of pathogens, and serves as a valuable complement to traditional diagnostic methods. Combining both methods in the diagnosis of IDS can maximize detection rates, providing robust evidence for precise anti-infective treatment.

The utilization of optimal orthodontic force is crucial to prevent undesirable side effects and ensure efficient tooth movement during orthodontic treatment.However,the sensitivity of existing detection techniques is not sufficient,and the criteria for evaluating optimal force have not been yet established.Here,by employing 3D finite element analysis methodology,we found that the apical distal region(A-D region)of mesial roots is particularly sensitive to orthodontic force in rats.Tartrate-resistant acidic phosphatase(TRAP)-positive osteoclasts began accumulating in the A-D region under the force of 40 grams(g),leading to alveolar bone resorption and tooth movement.When the force reached 80 g,TRAP-positive osteoclasts started appearing on the root surface in the A-D region.Additionally,micro-computed tomography revealed a significant root resorption at 80 g.Notably,the A-D region was identified as a major contributor to whole root resorption.It was determined that 40 g is the minimum effective force for tooth movement with minimal side effects according to the analysis of tooth movement,inclination,and hyalinization.These findings suggest that the A-D region with its changes on the root surface is an important consideration and sensitive indicator when evaluating orthodontic forces for a rat model.Collectively,our investigations into this region would aid in offering valuable implications for preventing and minimizing root resorption during patients'orthodontic treatment.