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 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 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 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.

Tuberculosis （TB） and human immunodeficiency virus infection / acquired immune deficiency syndrome （HIV/AIDS） are both serious global public health threats. Early detection of infected persons and/or patients through TB/HIV bi-directional screening is crucial for prevention and control strategy in China and globally. In recent years， with the promotion and application of new TB and HIV detection technologies worldwide， TB/HIV bi-directional screening technologies and strategies have made remarkable changes. This expert consensus introduces the significance and challenges of TB/HIV bi-directional screening， summarizes important progress of research and applications， and makes recommendations on screening measures and procedures to further strengthen TB/HIV bi-directional screening in China.

Targeted delivery of antibody bound to antigen is a precise drug delivery mode. It is regarded as one of the ideal targeted drug delivery modes due to its high specificity and affinity, which opens up a new way to successfully solve the problem of poor selectivity of chemotherapy drugs in antitumor therapy. Currently, the research on antibody drug conjugates(ADCs) that bind monoclonal antibodies to target antigens has become a research hotspot of molecular targeted therapy. This paper reviews the mechanism of action, targeting strategies and progress in the targeted delivery of ADCs, in order to provide reference for the clinical development of new ADCs.

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.

Objective To investigate the relationship between Kirsten rat sarcoma viral oncogene homolog(KRAS),neuroblastoma viral oncogene RAS homolog(NRAS),V-raf murine sarcoma viral oncogene homo-log B(BRAF),human epidermal growth factor receptor 2(HER2)gene mutations and microsatellite instabili-ty(MSI)status and clinicopathological features in patients with colorectal cancer.Methods The clinical data of 226 patients with colorectal cancer treated in the hospital from October 2019 to March 2022 were collected.Next-generation sequencing technology was used to detect KRAS,NRAS,BRAF,HER2 gene mutations and MSI status.Immunohistochemistry was used to evaluate the mismatch repair system(MMR)status.Multiva-riate Logistic regression analysis was used to analyze the relationship between KRAS,NRAS,BRAF,HER2 gene mutations and clinicopathological features.Results Among 226 colorectal cancer patients,the mutation frequencies of KRAS,NRAS,BRAF and HER2 were 54.89％,5.3％,8.4％and 1.8％,respectively.The fre-quency of KRAS mutation in mucinous adenocarcinoma was higher than that in common adenocarcinoma(P＜0.05).The risk of KRAS mutation in right colon cancer was increased(OR=2.145,P=0.012).NR AS gene mutation was more frequent in left colon and rectal cancer(P＜0.05).The frequency of BRAF gene mu-tation was higher in poorly differentiated and microsatellite instability-high(MSI-H)colorectal cancer(P＜0.05),and the risk of BRAF gene mutation in the right colon was increased(OR=2.844,P=0.042).HER2 gene amplification mutation showed distant metastasis(P＜0.05).KRAS mutations were mutually exclusive with NRAS,BRAF and HER2 amplification mutations(P＜0.05).MSI-H was more frequent in the right co-lon(P＜0.05).Of the 226 cases,10 cases were defective mismatch repair(dMMR)/MSI-H,8 cases were dM-MR/microsatellite stable,and 5 cases were proficient mismatch repair/MSI-H.There was a moderate agree-ment between dMMR and MSI-H(Kappa=0.575).Conclusion KRAS,NRAS,BRAF,HER2 and MSI sta-tus are associated with clinicopathological features in patients with colorectal cancer.Combined detection of KRAS,NRAS,BRAF,HER2 and MSI can provide more accurate and effective data to guide the treatment and prognosis of patients.