ObjectiveThe paper aims to investigate the mechanism by which Xiaozheng Zhitong paste （XZP） alleviates bone cancer pain （BCP） through regulating the PTEN-induced putative kinase 1 （PINK1）/Parkin-mediated mitophagy-NOD-like receptor protein 3 （NLRP3） inflammasome pathway to suppress microglial pyroptosis. MethodsLipopolysaccharide （LPS） and LPS-adenosine triphosphate （ATP） were used to establish an inflammation and pyroptosis model in microglial cells. The cells were randomly divided into the following groups： control group， LPS group， LPS+low-dose XZP group， LPS+high-dose XZP group， LPS-ATP group， LPS-ATP+low-dose XZP group， LPS-ATP+high-dose XZP group， LPS-ATP+XZP group， and LPS-ATP+XZP+CsA group. Techniques including terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） staining， enzyme-linked immunosorbent assay （ELISA）， Western blot， and confocal fluorescence staining were employed to assess the effects of XZP on microglial apoptosis， inflammatory cytokine release， inflammasome activation， pyroptosis， and mitophagy. ResultsIn vitro experiments showed that compared with the blank group， the LPS group exhibited significantly increased levels of microglial apoptosis and pro-inflammatory factors interleukin （IL）-1β， IL-6， and tumor necrosis factor-α （TNF-α）（P<0.01）， along with significantly upregulated protein expression of inducible nitric oxide synthase （iNOS）， cyclooxygenase-2 （COX-2）， and phosphorylated nuclear factor-κB p65 （p-NF-κB p65） （P<0.01）. Compared with the LPS group， the high-dose LPS-XZP group significantly reduced the level of apoptosis （P<0.01） and the content of the aforementioned pro-inflammatory factors （P<0.01）. Both the low- and high-dose LPS-XZP groups dose-dependently downregulated the protein expression of iNOS， COX-2， and p-NF-κB p65 （P<0.05， P<0.01）. Compared with the blank group， the LPS-ATP group showed significantly upregulated expression of pyroptosis-related proteins， including Caspase-1/pro-Caspase-1， N-terminal fragment of gasdermin D （GSDMD-N）/full-length gasdermin D （GSDMD-F）， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， IL-1β precursor （pro-IL-1β）， and mature IL-1β （P<0.01）. The levels of pyroptotic factors IL-1β and IL-18 were significantly elevated （P<0.01）， and membrane pore formation and intracellular reactive oxygen species （ROS） levels were significantly increased （P<0.01）. Compared with the LPS-ATP group， both the low- and high-dose LPS-ATP+XZP groups dose-dependently downregulated the expression of the aforementioned pyroptosis-related proteins （P<0.05， P<0.01）. The low-dose LPS-ATP+XZP group reduced IL-1β levels （P<0.01）， while the high-dose group reduced both IL-1β and IL-18 levels （P<0.01） Both the low- and high-dose LPS-ATP+XZP groups dose-dependently reduced membrane pore formation and intracellular ROS production （P<0.01）. Compared with the blank group， the LPS-ATP group showed significantly reduced expression of mitophagy-related proteins PINK1 and Parkin， and a decreased ratio of microtubule-associated protein 1 light chain 3Ⅱ（LC3Ⅱ） to LC3Ⅰ（P<0.01）， while p62 expression was significantly increased （P<0.01）. Mitochondrial ROS levels were significantly enhanced （P<0.01）. Compared with the LPS-ATP group， both the low- and high-dose LPS-ATP+XZP groups dose-dependently reversed the expression of these proteins （P<0.05， P<0.01） and reduced mitochondrial ROS levels （P<0.01）. After treatment with the mitophagy inhibitor cyclosporin A （CsA）， the beneficial effects of XZP on mitochondrial function and its inhibitory effects on pyroptosis-related protein expression were significantly reversed （P<0.05， P<0.01）. ConclusionXZP reduces ROS levels by activating PINK1/Parkin-mediated mitophagy， thereby inhibiting NLRP3 inflammasome activation and microglial pyroptosis， which provides new molecular evidence for the mechanism by which XZP alleviates BCP.

ObjectiveTo investigate the effects and underlying mechanisms of Xiaozheng Zhitong Paste （XZP） on bone cancer pain （BCP）. MethodsThirty female BALB/c mice were randomly divided into five groups： a Sham group， a BCP group， a BCP+low-dose XZP group， a BCP+high-dose XZP group， and a BCP+high-dose XZP + protease-activated receptor 2 （PAR2） agonist GB-110 group. BCP mice model was constructed by injecting Lewis lung carcinoma cells into the femoral cavity of the right leg， which was followed by being treated with XZP for 21 d. After 21 d， the mice were sacrificed. Nissl staining was used to evaluate the survival of spinal cord neurons. Immunofluorescence staining was conducted to localize ionized calcium-binding adapter molecule 1 （Iba1） and neuronal nuclear antigen （NeuN） in spinal cord tissue， thereby assessing microglial activation and neuronal survival. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， transforming growth factor-β （TGF-β）， interleukin-4 （IL-4）， and interleukin-10 （IL-10） in spinal cord tissue. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect mRNA expression levels associated with M1/M2 polarization of microglia. Western blot analysis was performed to examine the expression of proteins related to microglial polarization as well as those involved in the PAR2/nuclear factor kappa B （NF-κB）/NOD-like receptor protein 3 （NLRP3） signaling pathway in the spinal cord. ResultsCompared with the Sham group， the spinal cord neurons were damaged， the number of Nissl-positive spinal cord neurons in the spinal cord tissue was significantly reduced （P<0.01）， and the rate of NeuN-positive cells was significantly decreased （P<0.01）. The spinal cord microglia were activated， the inflammatory level of the spinal cord tissue was enhanced， and Iba1 staining was significantly enhanced （P<0.01）. The levels of IL-1β， TNF-α， IL-6， TGF-β， IL-4 and IL-10 were significantly increased （P<0.01）. The mRNA expressions of IL-1β， TNF-α and inducible nitric oxide synthase （iNOS） were significantly increased （P<0.01）， and the expression of PAR2， NLRP3， ASC and NF-κB p65 proteins in the spinal cord tissue of the BCP mice was significantly enhanced （P<0.01）. Compared with the BCP group， high-dose XZP treatment significantly increased the number of Nissl-positive spinal cord neurons in the BCP mice （P<0.01）， significantly enhanced the rate of NeuN-positive cells in the spinal cord tissue， and significantly weakened Iba1 staining （P<0.01）. In addition， the levels of IL-1β， TNF-α， and IL-6 were significantly decreased， while the levels of TGF-β， IL-4， and IL-10 were significantly increased （P<0.05， P<0.01）. The mRNA expression levels of IL-1β， TNF-α， and iNOS were decreased， whereas those of cluster of differentiation 206 （CD206）， arginase-1 （Arg-1）， and YM1/2 were significantly increased （P<0.05， P<0.01）. Low-dose and high-dose XZP treatment significantly decreased the expression of PAR2， NLRP3， ASC， and NF-κB p65 proteins in the spinal cord tissue （P<0.05， P<0.01）. These effects could all be significantly eliminated by the PAR2 agonist GB-110. ConclusionXZP can mitigate BCP in mice， which may be achieved through blocking the activated PAR2/NF-κB/NLRP3 pathway.

Cancer pain is one of the most common complications in patients with malignant tumors， severely affecting their quality of life. Its pathogenesis involves complex interactions among the tumor microenvironment， peripheral sensitization， and central sensitization. The tumor microenvironment initiates peripheral pain sensitization by secreting algogenic mediators， activating ion channels and related receptor signaling pathways， driving abnormal osteoclast activation， and mediating neuro-immune crosstalk. Persistent nociceptive input further triggers increased excitability of central neurons， activation of glial cells， and neuroinflammatory cascade reactions， ultimately leading to central pain sensitization. Although traditional opioid drugs can alleviate pain to some extent， they still have many limitations， such as incomplete analgesia， drug tolerance， and adverse reactions. In recent years， traditional Chinese medicine （TCM） compounds have made continuous progress in the treatment of cancer pain. Studies have shown that they can not only effectively relieve cancer pain and reduce the dosage of opioids but also significantly improve patients' quality of life. TCM treatment of cancer pain follows the principle of syndrome differentiation and treatment. Based on this， targeted therapeutic principles have been proposed， including promoting blood circulation， removing stasis， regulating Qi， and unblocking collaterals； tonifying the kidney， replenishing essence， warming Yang， and dispersing cold， activating blood， resolving phlegm， detoxifying， and dispersing nodules， as well as strengthening the body， replenishing deficiency， and harmonizing Qi and blood. Modern research indicates that TCM compounds can exert synergistic effects through multiple pathways， inhibiting inflammatory responses， regulating nerve conduction， intervening in bone metabolism and related gene expression， thereby producing anti-inflammatory and bone-protective effects to achieve the goal of alleviating cancer pain. This article systematically elaborates on the pathogenesis of cancer pain， the clinical application of TCM in treating cancer pain， and its related mechanisms of action， aiming to provide a theoretical basis and new strategies for the integration of TCM into comprehensive cancer pain management.

Pain， as one of the most common symptoms in cancer patients， seriously affects the survival quality of patients. The three-step pain relief program currently used in clinical practice cannot completely relieve pain in cancer patients and is accompanied by many problems. From the perspective of Jueyin syndrome in traditional Chinese medicine （TCM）， this paper believed that the core pathogenesis of cancer pain was declined healthy Qi and cold and heat in complexity， and used Wumeiwan as the main formula with modification according to syndrome for clearing the upper， warming the lower part of the body， and harmonizing the cold and heat. It can regulate the pathological environment of deficiency， cold， stasis， toxicity， and heat， and restore the physiological function of Yang transforming Qi while Yin constituting form， so as to prevent， relieve， and even eliminate cancer pain， having achieved good clinical efficacy. It can not only help cancer patients relieve pain， but also control tumor and eliminate tumor， achieving a dual benefit of pain relief and tumor suppression. It gives full play to the characteristics and advantages of syndrome differentiation and treatment in TCM， and expands the scope of ZHANG Zhongjing's treatment for Jueyin syndrome， which provides ideas for the clinical diagnosis and treatment of cancer pain from the perspective of deficiency-excess in complexity and cold and heat in complexity.

Objective:Molecular mechanisms underlying compound heterozygous mutations in a patient with inherited antithrombin (AT) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in November 2018 with a one-day history of sudden syncope and limb twitching. Peripheral venous blood was collected from the proband and members of his lineages, totaling nine persons across three generations, and a family lineage survey was conducted. AT activity (AT:A) was measured using a chromogenic substrate assay, while AT antigen (AT:Ag) was detected through an immunoturbidimetric assay. Mutation sites were identified by means of Sanger sequencing of the SERPINC1 gene, and silico tools were applied to predict the mutational conservation and hydrophobicity changes. Recombinant plasmid expression vectors were constructed and transfected into HEK293T cells for in vitro overexpression studies. The recombinant AT protein was characterized using Western Blotting, ELISA, and cellular immunofluorescence assays.Results:The proband was a 21-year-old man with type Ⅰ AT deficiency. His AT:A was 33%, along with a corresponding reduction in AT:Ag. The genetic analysis revealed there was a heterozygous insertion mutation at c.318_319insT (p.Asn107*) and a heterozygous missense mutation at c.922G>T (p.Gly308Cys) in exons 2 and 5, respectively. These mutation sites were entirely conserved among the homologous species. Additionally, hydrophobicity studies showed that the p.Gly308Cys mutation will decrease the hydrophilicity of amino acid residues 307-313. The in vitro expression studies indicated a reduction of approximately 46.98%±2.94% and 41.35%±1.48% in the amount of recombinant protein AT-G308C in transfected cell lysates and culture supernatants, respectively. Treatment with the proteasome inhibitor (MG132) restored the cytoplasmic levels of AT-G308C protein to a level similar to that of wild-type protein. However, neither cell lysate nor culture supernatant demonstrated the presence of the recombinant protein AT-N107*. Conclusions:The heterozygous insertion mutation of p.Asn107* and the heterozygous missense mutation of p.Gly308Cys have been associated with reduced AT levels in proband. The p.Asn107* heterozygous insertion mutation may initiate the degradation of mRNA via nonsense mutation-mediated mechanisms, which would remove the defective transcripts, as well as the p.The Gly308Cys heterozygous missense mutation may cause the AT protein to undergo proteasome-dependent degradation by modifying the hydrophobicity of nearby residues in the cytoplasm.

Objective:To investigate the relationship between inherited protein S deficiency (PSD) and cerebral venous sinus thrombosis (CVST) by phenotype and gene mutation analysis of 2 inherited PSD pedigrees with nonsense mutations.Methods:Retrospective analysis of clinical and imaging data of 2 patients diagnosed with CVST who were treated in the Department of Neurology, the First Affiliated Hospital of Wenzhou Medical University in July and October 2023 was carried out. The peripheral blood samples were collected from proband 1 and her family members (3 subjects, 2 generations in total), and proband 2 and his family members (8 subjects of 3 generations in total). Their protein S (PS) activity (PS:A), the content of total PS antigen (TPS:Ag) and free PS antigen (FPS:Ag) were measured for definite diagnosis. Polymerase chain reaction was done in all 15 exons of the active PROS1 gene and its 5′ and 3′ untranslated regions and the amplification products were analyzed by direct sequencing. The results were compared with human PROS1 reference sequences, using Chromas software to find the mutation sites. Results:The proband 1 is a female, and the proband 2 is a male. Both of them had young onset and the clinical presentation of CVST. The PS:A level was reduced to 29% in the proband 1 and reduced to about 35% in her mother; PS:A was reduced to 21%-27% in the proband 2 and his 6 family members; a decline in the same proportion of TPS:Ag and FPS:Ag was found in the 2 probands and their family members, therefore they were primarily diagnosed as typeⅠPSD. Gene analysis showed that the proband 1 and her mother had a nonsense mutation of c.1680T>A in exon 14 (p.Tyr560 *) of the PROS1 gene; the proband 2 and his 6 family members had a nonsense mutation of c.1687C>T in exon 14 (p.Gln563 *) of the PROS1 gene. Conclusion:The reduced protein S levels in PSD patients and their family members may be associated with the p.Tyr560 * and p.Gln563 * nonsense mutations of the PROS1 gene, and the clinical manifestations of CVST in PSD patients may be related to these 2 nonsense mutations.

In order to explore the role of BspE protein in Brucella infection,yeast two-hybrid tech-nique was used to screen host cell proteins that interact with BspE protein.The constructed BspE recombinant plasmid pGBKT7-BspE was used as bait plasmid to hybridize with the RAW264.7-cD-NA library of mouse mononuclear macrophages by yeast two-hybridization technique.The positive clones were extracted by plasmid,sequenced and co-immunoprecipitation to determine the host cell proteins that could interact with BspE.The subcellular localization of BspE proteins was analyzed by confocal laser microscopy.The physical and chemical properties,protein structure and function of BspE interacting proteins were analyzed by bioinformatics.The siRNA for one of the BspE inter-acting proteins was synthesized,the expression of its gene was silenced in HEK293T cells,and the silenced cells was infected with Brucella M5-90 and the number of intracellular bacteria was coun-ted.The results showed that the decoy plasmid pGBKT7-BspE was successfully constructed,and the plasmid could express BspE protein in yeast.Eight positive clones were obtained from the host cell genome library by yeast two-hybridization.The positive clones were identified as RBM27 and PCBP1 by sequencing,backcross and co-immunoprecipitation.Bioinformatics was used to predict the cell location,protein structure and amino acid composition of RBM27 and PCBP1.After siRNA interference,the expression level of PCBP1 was significantly decreased and the amount of M5-90 in the cell was increased.Brucellosis secreted protein BspE interacts with host proteins RBM27 and PCBPl,and PCBP1 negatively regulates the proliferation of Brucellosis.

Objective:To investigate the influence of the interaction between gastric cancer (GC) cell-derived exosomes and hepatic Kupffer cells on GC with liver metastasis and analyze the potential mechanism.Methods:Cells with high hepatic metastatic potential (MKN 45-HL) were constructed from a parental GC cell line (MKN 45) using a nude mouse model and methods of viral transfection and flow sorting. Exosomes were collected using ultra-centrifugation and characterized by electron microscopy, nanoparticle tracking system and Western blot. A nude mouse model of liver metastasis induced by GC cell-derived exosomes was constructed, and the development of liver metastases was monitored by live imaging. The regulatory effects of GC cell-derived exosomes on macrophage polarization were assessed by cell culture, qRT-PCR, and immunofluorescence staining. Using the omics analysis of exosomal miRNA and qRT-PCR, the molecular targets by which exosomes specifically promoting macrophage M2 polarization were screened and validated.Results:GC cell-derived exosomes were mainly concentrated in the liver, most of which were ingested by intrahepatic macrophages, and could promote macrophages to M2 polarization in both in vitro culture and nude mice. Both groups of mice trained with MKN 45 and MKN 45-HL exosomes showed obvious liver metastases after mouse forestomach carcinoma (MFC) cells injection through the spleen, and MKN 45-HL exosomes showed a much stronger ability to promote hepatic macrophage M2 polarization and liver metastasis of MFC cells. Moreover, the miRNA omics analysis revealed a lot of differentially expressed miRNAs between MKN 45-derived and MKN 45-HL-derived exosomes. The expression of miR-519a-3p increased significantly in the exosomes derived from MKN 45-HL cell line and the clinical serum of GC patients with liver metastasis. It was found that miR-519a-3p could be internalized by macrophages through exosomes delivery. Furthermore, the miR-519a-3p in exosomes from patient′s serum had a predictive value for GC with liver metastasis and was closely associated with the prognosis of GC patients with liver metastasis. Conclusions:GC cell-derived exosomes trigger M2-like polarization of hepatic Kupffer cells via miR-519a-3p, thus promoting the progression of liver metastasis in GC and playing a critical role in shaping the pre-metastatic liver niche in gastric cancer. This study provides a new perspective on the mechanism of GC with liver metastasis and reveal potential targets for future therapeutic strategies.

Oral and maxillofacial-head and neck soft tissue defects affect the appearance of patients, as well as pronunciation, swallowing and other functions. Introduction of the propeller flap in 1991 has improved reconstruction procedures for oral and maxillofacial-head and neck soft tissue defects. A propeller flap has several advantages over traditional local flaps. It improves mobility, colour and texture matching for maxillofacial defect, surgical procedure, and individual satisfaction. Therefore, it can be used as a complement to the traditional flap by providing surgeons with more options. This paper reviews the classification, surgical procedures, and recent clinical applicatiosn and indications of the propeller flap.

Objective:To analyze 12 antithrombins (AT) gene mutations that cause AT deficiency and discuss the relationship between the SERPINC1 gene. mutations and venous thrombotic events.Methods:This study belongs to case series of observational studies. Collected the clinical data of 12 AT deficiency cases in the First Affiliated Hospital of Wenzhou Medical University from April 2014 to April 2021 and collected the blood samples before treatment. The AT activity (AT: A) and AT antigen (AT: Ag) was detected by chromogenic substrate and immunoturbidimetry, respectively. The 7 exons and flanking sequences of the SERPINC1 gene were sequenced directly by PCR, the suspected mutations were validated by reverse sequencing. Analyzed the correlation between the SERPINC1 gene. mutations and venous thrombotic events and figured out the proportion.Results:The AT: A of the 12 patients all decreased significantly, ranging from 30% to 66%, and the AT: Ag of the 7 patients decreased accordingly, showing type Ⅰ AT deficiency, and the AT: Ag of the other 5 patients were normal, presented type Ⅱ AT deficiency. 12 mutations were found including 6 heterozygous mutations which were discovered for the first time: c.456_458delCTT(p.phe121del), c.318_319insT(p.Asn75stop), c.922G>T(p.Gly276Cys), c.938T>C (p.Met281Thr), c.1346T>A(p.Leu417Gln)and c.851T>C(p.Met252Thr). All 12 patients had venous thrombosis, and 3 cases including 2 compound heterozygotes and 1 single heterozygote all suffered from deep venous thrombosis (DVT) when they were younger without obvious triggers. The other 9 patients all combined with the other thrombotic factors including old age, hypertensive, smoking, pregnancy, and prolonged immobilization.Conclusion:Patients with AT deficiency caused by SERPINC1 gene defects are prone to venous thrombosis, especially combined with other thrombotic factors.