BACKGROUND: Temozolomide (TMZ) resistance is a significant challenge in treating glioblastoma (GBM). Collagen remodeling has been shown to be a critical factor for therapy resistance in other cancers. This study aimed to investigate the mechanism of TMZ chemoresistance by GBM cells reprogramming collagens. METHODS: Key extracellular matrix components, including collagens, were examined in paired primary and recurrent GBM samples as well as in TMZ-treated spontaneous and grafted GBM murine models. Human GBM cell lines (U251, TS667) and mouse primary GBM cells were used for in vitro studies. RNA-sequencing analysis, chromatin immunoprecipitation, immunoprecipitation-mass spectrometry, and co-immunoprecipitation assays were conducted to explore the mechanisms involved in collagen accumulation. A series of in vitro and in vivo experiments were designed to assess the role of the collagen regulators prolyl 4-hydroxylase subunit alpha 1 (P4HA1) and yes-associated protein (YAP) in sensitizing GBM cells to TMZ. RESULTS: This study revealed that TMZ exposure significantly elevated collagen type I (COL I) expression in both GBM patients and murine models. Collagen accumulation sustained GBM cell survival under TMZ-induced stress, contributing to enhanced TMZ resistance. Mechanistically, P4HA1 directly binded to and hydroxylated YAP, preventing ubiquitination-mediated YAP degradation. Stabilized YAP robustly drove collagen type I alpha 1 ( COL1A1) transcription, leading to increased collagen deposition. Disruption of the P4HA1-YAP axis effectively reduced COL I deposition, sensitized GBM cells to TMZ, and significantly improved mouse survival. CONCLUSION P4HA1 maintained YAP-mediated COL1A1 transcription, leading to collagen accumulation and promoting chemoresistance in GBM.
Temozolomide ; Humans ; Glioblastoma/drug therapy* ; Animals ; Mice ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics* ; YAP-Signaling Proteins ; Hydroxylation ; Dacarbazine/pharmacology* ; Adaptor Proteins, Signal Transducing/metabolism* ; Transcription Factors/metabolism* ; Collagen/biosynthesis* ; Collagen Type I/metabolism* ; Prolyl Hydroxylases/metabolism* ; Antineoplastic Agents, Alkylating/therapeutic use*

High-performance liquid chromatography(HPLC) was used to determine the content of three major components in Citri Reticulatae Semen(CRS), including limonin, nomilin, and obacunone. The chromaticity of the CRS sample during salt processing and stir-frying was measured using a color difference meter. Next, the relationship between the color and content of the salt-processed CRS sample was investigated through correlation analysis. By integrating the oil bath technique for processing simulation with HPLC, the changes in the relative content of nomilin and its transformation products were analyzed, with its structural transformation pattern during processing identified. Additionally, RAW264.7 cells were induced with lipopolysaccharides(LPSs) to establish an inflammatory model, and the anti-inflammatory activity of nomilin and its transformation product, namely obacunone was evaluated. The results indicated that as processing progressed, E~*ab and L~* values showed a downward trend; a~* values exhibited a slow increase over a certain period, followed by no significant changes, and b~* values remained stable with no significant changes over a certain period and then started to decrease. The limonin content remained barely unchanged; the nomilin content decreased, and the obacunone increased significantly. The changing trends in content and color parameters during salt-processing and stir-frying were basically consistent. The content of nomilin and obacunone was significantly correlated with the colorimetric values(L~*, a~*, b~*, and E~*ab), while limonin content showed no significant correlation with these values. By analyzing HPLC patterns of nomylin at different heating temperatures and time, it was found that under conditions of 200-250 ℃ for heating of 5-60 min, the content of nomilin significantly decreased, while the obacunone content increased pronouncedly. The in vitro anti-inflammatory activity results indicated that compared to the model group, the group with a high concentration of nomilin and the groups with varying concentrations of obacunone showed significantly reduced release of nitric oxide(NO)(P<0.01). When both were at the same concentration, obacunone showed better performance in inhibiting NO release. In this study, the obvious correlation between the color and content of major components during the processing of CRS samples was identified, and the dynamic patterns of quality change in CRS samples during processing were revealed. Additionally, the study revealed and confirmed the transformation of nomilin into obacunone during processing, with the in vitro anti-inflammatory activity of obacunone significantly greater than that of nomilin. These findings provided a scientific basis for CRS processing optimization, tablet quality control, and its clinical application.
Mice ; Animals ; Drugs, Chinese Herbal/pharmacology* ; RAW 264.7 Cells ; Limonins/chemistry* ; Chromatography, High Pressure Liquid ; Citrus/chemistry* ; Color ; Benzoxepins/chemistry* ; Anti-Inflammatory Agents/chemistry*

This study investigates the effect of glycyrrhetinic acid(GA) combined with doxorubicin(DOX) on apoptosis in HepG2 cells and its possible mechanisms. HepG2 cells were cultured in vitro, and cell viability was assessed using the cell counting kit-8(CCK-8) method. Flow cytometry was used to measure apoptosis levels in HepG2 cells. The cells were divided into the following groups: control group(0 μmol·L~(-1)), DOX group(2 μmol·L~(-1)), GA group(150 μmol·L~(-1)), and DOX + GA combination group(2 μmol·L~(-1) DOX + 150 μmol·L~(-1) GA), with treatments given for 24 hours. The colocalization level between the endoplasmic reticulum(ER) and mitochondria was assessed by colocalization fluorescence imaging. Fluorescence probes were used to measure the Ca~(2+) content in the ER and mitochondria. The qRT-PCR and Western blot were used to determine the mRNA and protein expression of sirtuin-3(SIRT3). Co-immunoprecipitation(CO-IP) was applied to investigate the interactions between voltage-dependent anion channel 1(VDAC1) and SIRT3, as well as between VDAC1, glucose-regulated protein 75(GRP75), and inositol 1,4,5-trisphosphate receptor(IP3R). The results showed that the combination of DOX and GA promoted apoptosis in HepG2 liver cancer cells. The colocalization level between the ER and mitochondria was significantly reduced, the Ca~(2+) content in the ER was significantly increased, and the Ca~(2+) content in the mitochondria was significantly decreased. The relative expression of VDAC1, GRP75, and IP3R was significantly reduced, and interactions between VDAC1, GRP75, and IP3R were observed. SIRT3 mRNA and protein expression levels were significantly increased, and an interaction between SIRT3 and VDAC1 was detected. The acetylation level of VDAC1 was significantly decreased. In conclusion, GA combined with DOX induces apoptosis in HepG2 cells by mediating the deacetylation of VDAC1 through SIRT3, weakening the interactions among VDAC1, GRP75, and IP3R. This regulates the formation of endoplasmic reticulum-mitochondrial membrane domains(ERMMDs), affects Ca~(2+) transport between the ER and mitochondria, and ultimately triggers cell apoptosis.
Humans ; Apoptosis/drug effects* ; Hep G2 Cells ; Glycyrrhetinic Acid/pharmacology* ; Doxorubicin/pharmacology* ; Liver Neoplasms/genetics* ; Carcinoma, Hepatocellular/physiopathology* ; Mitochondria/metabolism* ; Endoplasmic Reticulum/metabolism* ; Cell Survival/drug effects* ; Membrane Proteins/genetics*

OBJECTIVE: To investigate the effectiveness of posterior minimally invasive approach in the treatment of posterior wall acetabular fractures. METHODS: The clinical data of 17 patients with posterior wall acetabular fractures treated with posterior minimally invasive approach between March 2019 and June 2023 were retrospectively analyzed. There were 14 males and 3 females with an average age of 41 years ranging from 28 to 57 years. The causes of injury were traffic accident in 12 cases and falling from height in 5 cases. There were 3 cases complicated with posterior hip dislocation and 2 cases complicated with sciatic nerve injury. According to AO/Orthopaedic Trauma Association (AO/OTA) classification, there were 11 cases of type A1.1 and 6 cases of type A1.2. The time from injury to operation was 5-8 days, with an average of 6.2 days. The incision length, intraoperative blood loss, and operation time were recorded. The quality of posterior wall fracture reduction were evaluated by Matta criteria, and hip function were evaluated by modified Merle d'Aubign-Postel score criteria at 6 months after operation and last follow-up. RESULTS: The operation was successfully completed in 17 cases. The length of incision ranged from 7 to 9 cm, with an average of 8.3 cm, and all incisions healed by first intention. The intraoperative blood loss ranged from 200 to 350 mL, with an average of 281 mL. The operation time ranged from 45 to 70 minutes, with an average of 57 minutes. Two patients had sciatic nerve injury before operation, and the sciatic nerve function recovered completely at 3 months after operation; the other 15 patients had no symptoms of sciatic nerve injury after operation. All the 17 patients were followed up 14-27 months, with an average of 19.5 months. At 1 week after operation, according to the Matta criteria, anatomical reduction was achieved in 12 cases and satisfactory reduction in 5 cases, with a satisfaction rate of 100%. According to the modified Merle d'Aubign-Postel scoring system, the hip function score was 13-18 (mean, 16.1) at 6 months after operation. Among them, 5 cases were excellent, 9 were good, and 3 were fair, with an excellent and good rate of 82.4%. At last follow-up, the hip function score was 7-18 (mean, 13.7), of which 3 cases were excellent, 9 were good, 3 were fair, and 2 were poor, with an excellent and good rate of 70.6%. During the follow-up, there was no infection, failure of internal fixation, and femoral head necrosis, and heterotopic ossification occurred in 2 cases. CONCLUSION The posterior minimally invasive approach has the advantages of less trauma, shorter operation time, less blood loss, without cutting off the external rotator muscle. Exposure through the gluteus medius-piriformis space and piriformis-supercilium space can provide sufficient safe exposure for the posterior wall acetabulum fracture, which is a reliable alternative approach for the posterior acetabular fracture.
Humans ; Acetabulum/surgery* ; Male ; Female ; Adult ; Middle Aged ; Minimally Invasive Surgical Procedures/methods* ; Retrospective Studies ; Fracture Fixation, Internal/instrumentation* ; Fractures, Bone/diagnostic imaging* ; Treatment Outcome ; Operative Time

OBJECTIVE: To explore the effectiveness of additional anti-rotation steel plate assisted intramedullary nail technology in treatment of aseptic femoral non-union patients. METHODS: A retrospective analysis was conducted on 21 patients with aseptic femoral non-union who admitted between September 2020 and October 2024 and treated with additional anti-rotation steel plate assisted intramedullary nail technology. There were 17 males and 4 females, aged 25-67 years (mean, 44 years). There were 19 cases of femoral anterograde intramedullary nail fixation, 1 case of femoral retrograde intramedullary nail fixation, and 1 case of steel plate fixation with fatigue fracture. There were 9 cases of hypertrophic non-union and 12 cases of atrophic non-union. All patients had varying degrees of fracture end atrophy/sclerosis. Among them, 20 patients who were fixed with intramedullary nails underwent removal of soft tissue and hardened bone at the fracture end, and cortical treatment resulted in the appearance of "chili sign" at the fracture end. Iliac bone grafting and anti-rotation steel plate fixation were performed. One patient with steel plate fixation was removed the steel palte and fixed with a retrograde intramedullary nail, while the hardened bone at the fracture end was removed, iliac bone grafting and anti-rotation steel plate fixation were performed. Postoperative follow-up observation included the incision healing, maximum knee flexion range of motion, bone healing, length of lower limbs, and subjective satisfaction. The lower extremity functional scale (LEFS) score was used to evaluate the lower limb function. RESULTS: All incisions healed by first intention. All patients were followed up 7-26 months (mean, 15.5 months). At last follow-up, the femoral fracture healed with the obvious callus formation at the fracture end; the maximum knee flexion range of motion was 95°-127° (mean, 112.67°). The LEFS score increased from 29.9±6.7 before operation to 75.9±3.0 at last follow-up, and the difference was significant (t=-29.622, P<0.001). Except for 1 patient who underwent intramedullary nail dynamic treatment before operation and had a lower limb shortening of about 0.9 cm, the other patients had bilateral lower limbs of equal length. All patients had no postoperative infections, mal-union of fractures, deep vein thrombosis, joint stiffness, or other complications. CONCLUSION The use of additional anti-rotation steel plate assisted intramedullary nail technology in the treatment of aseptic femoral non-union not only overcomes the drawbacks of insufficient stability at the fracture end of intramedullary nails, but also overcomes the shortcomings of biased fixation with steel plates. It has the advantages of minimal trauma, effective maintenance of fracture stability, and ideal postoperative functional recovery, making it an effective treatment for aseptic femoral non-union.
Humans ; Male ; Fracture Fixation, Intramedullary/instrumentation* ; Female ; Bone Plates ; Middle Aged ; Adult ; Femoral Fractures/surgery* ; Retrospective Studies ; Bone Nails ; Aged ; Fractures, Ununited/surgery* ; Treatment Outcome ; Bone Transplantation/methods* ; Steel ; Fracture Healing

OBJECTIVE: To explore the changes of stress and displacement of intervertebral discs and vertebral bodies in adolescent idiopathic cervical kyphosis models caused by Wuqinxi () Huju() and extension movement after torque loading by finite element analysis. METHODS: One healthy male volunteer aged 24-year-old (heighted 178 cm and weighted 65 kg) was selected, software such as Mimics 21.0, Geomagic wrap 2017, SolidWorks 2017, and Ansys Workbench 17.0 were used to simulate adolescent idiopathic cervical spine model, an axial compressive load of 266 N was applied to the center of the end plate on C₂ for head physical gravity simulation, the lower part of C₇ vertebral body was set as the point of freedom constraint, a torque of 1.5 N·m was applied with C₂ as the reference point to simulate the stress on intervertebral discs and vertebral bodies after 45° movement of Wuqinxi () Huju (). RESULTS: The normal C₂-C₇ cervical spine model and adolescent idiopathic cervical kyphosis model were successfully constructed. The maximum stress value of intervertebral disc when the Huju()was raised and extended at 45° and loaded with torque occurred in C_3,4 intervertebral disc (3.588 1) MPa. The maximum stress values of each intervertebral disc were C_3,4(3.588 1 MPa)>C_2,3 (3.467 5 MPa) >C_4,5(2.597 7 MPa) >C_5,6 (2.378 8 MPa) >C_6,7 (1.404 9 MPa), respectively. The maximum stress of C6 vertebral body was 5.842 9 MPa, while the stresses of C₂, C₃, C₄, and C₅ vertebral bodies was 4.184 8, 4.437 8, 4.148 7, and 2.852 4 MPa respectively. The overall stress of vertebral body was mainly concentrated in the front of vertebral body. CONCLUSION After long-term practice of Huju()movement, the stress concentration in intervertebral discs and the front of vertebral body changes the stress load state of intervertebral discs and vertebral body. As time goes by, intervertebral discs may change, forming a shape that is higher in the front and lower in the back. The vertebral body may also undergo remodeling, resulting in a relative increase in the height of the anterior edge of vertebral body and promoting the recovery of cervical kyphosis to a physiological lordosis state.
Humans ; Finite Element Analysis ; Male ; Cervical Vertebrae/physiopathology* ; Kyphosis/therapy* ; Young Adult ; Adolescent ; Adult

OBJECTIVE: To explore the effect of bear bile powder (BBP) on acute lung injury (ALI) and the underlying mechanism. METHODS: The chemical constituents of BBP were analyzed by ultra-high-pressure liquid chromatography-mass spectrometry (UPLC-MS). After 7 days of adaptive feeding, 50 mice were randomly divided into 5 groups by a random number table (n=10): normal control (NC), lipopolysaccharide (LPS), dexamethasone (Dex), low-, and high-dose BBP groups. The dosing cycle was 9 days. On the 12th and 14th days, 20 µL of Staphylococcus aureus solution (bacterial concentration of 1 × 10^-7 CFU/mL) was given by nasal drip after 1 h of intragastric administration, and the mice in the NC group was given the same dose of phosphated buffered saline (PBS) solution. On the 16th day, after 1 h intragastric administration, 100 µL of LPS solution (1 mg/mL) was given by tracheal intubation, and the same dose of PBS solution was given to the NC group. Lung tissue was obtained to measure the myeloperoxidase (MPO) activity, the lung wet/dry weight ratio and expressions of CD14 and other related proteins. The lower lobe of the right lung was obtained for pathological examination. The concentrations of inflammatory cytokines including interleukin (IL)-6, tumour necrosis factor α (TNF-α ) and IL-1β in the bronchoalveolar lavage fluid (BALF) were detected by enzyme linked immunosorbent assay, and the number of neutrophils was counted. The colonic contents of the mice were analyzed by 16 sRNA technique and the contents of short-chain fatty acids (SCFAs) were measured by gas chromatograph-mass spectrometer (GC-MS). RESULTS: UPLC-MS revealed that the chemical components of BBP samples were mainly tauroursodeoxycholic acid and taurochenodeoxycholic acid sodium salt. BBP reduced the activity of MPO, concentrations of inflammatory cytokines, and inhibited the expression of CD14 protein, thus suppressing the activation of NF-κB pathway (P<0.05). The lung histopathological results indicated that BBP significantly reduced the degree of neutrophil infiltration, cell shedding, necrosis, and alveolar cavity depression. Moreover, BBP effectively regulated the composition of the intestinal microflora and increased the production of SCFAs, which contributed to its treatment effect (P<0.05). CONCLUSIONS BBP alleviates lung injury in ALI mouse through inhibiting activation of NF-κB pathway and decreasing expression of CD14 protein. BBP may promote recovery of ALI by improving the structure of intestinal flora and enhancing metabolic function of intestinal flora.
Animals ; Acute Lung Injury/pathology* ; Lipopolysaccharides ; Ursidae ; Gastrointestinal Microbiome/drug effects* ; Bile/chemistry* ; Lipopolysaccharide Receptors/metabolism* ; Powders ; Male ; Lung/drug effects* ; Mice ; Peroxidase/metabolism* ; Signal Transduction/drug effects* ; Cytokines/metabolism*

OBJECTIVE: To explore the role of the natural compound hesperidin in glycolysis, the key ratelimiting enzyme, in colorectal cancer (CRC) cell lines. METHODS: In vitro, HCT116 and SW620 were treated with different doses of hesperidin (0-500 µmol/L), cell counting kit-8 and colone formation assays were utilized to detected inhibition effect of hesperidin on CRC cell lines. Transwell and wound healing assays were performed to detect the ability of hesperidin (0, 25, 50 and 75 µmol/L) to migrate CRC cells. To confirm the apoptotic-inducing effect of hesperidin, apoptosis and cycle assays were employed. Western blot, glucose uptake, and lactate production determination measurements were applied to determine inhibitory effects of hesperidin (0, 25 and 50 µmol/L) on glycolysis. In vivo, according to the random number table method, nude mice with successful tumor loading were randomly divided into vehicle, low-dose hesperidin (20 mg/kg) and high-dose hesperidin (60 mg/kg) groups, with 6 mice in each group. The body weights and tumor volumes of mice were recorded during 4-week treatment. The expression of key glycolysis rate-limiting enzymes was determined using Western blot, and glucose uptake and lactate production were assessed. Finally, protein interactions were probed with DirectDIA Quantitative Proteomics, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. RESULTS: Hesperidin could inhibit CRC cell line growth (P<0.05 or P<0.01). Moreover, hesperidin presented an inhibitory effect on the migrating abilities of CRC cells. Hesperidin also promoted apoptosis and cell cycle alterations (P<0.05). The immunoblotting results manifested that hesperidin decreased the levels of hexokinase 2, glucose transporter protein 1 (GLUT1), GLUT3, L-lactate dehydrogenase A, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), PFKFB3, and pyruvate kinase isozymes M2 (P<0.01). It remarkably suppressed tumor xenograft growth in nude mice. GO and KEGG analyses showed that hesperidin treatment altered metabolic function. CONCLUSION Hesperidin inhibits glycolysis and is a potential therapeutic choice for CRC treatment.
Hesperidin/therapeutic use* ; Colorectal Neoplasms/metabolism* ; Glycolysis/drug effects* ; Animals ; Humans ; Apoptosis/drug effects* ; Mice, Nude ; Cell Movement/drug effects* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Glucose/metabolism* ; Cell Cycle/drug effects* ; Mice, Inbred BALB C ; Mice ; HCT116 Cells ; Lactic Acid

OBJECTIVES: To explore the underlying pharmacological mechanisms and its potential effects of Chinese medicine herbal formula Sini Powder (SNP) on hepatocellular carcinoma (HCC). METHODS: The active components of SNP and their in vivo distribution were identified using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Construction of component-target-disease networks, protein-protein interaction network, Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and molecular docking were employed to analyze the active components and anti-HCC mechanisms of SNP. Cell viability assay and wound healing assay were utilized to confirm the effect of SNP-containing serum (2.5%, 5.0%, 10%, 20%, and 40%), isoprenaline or propranolol (both 10, 100, and 1,000 µ mol/L) on proliferation and migration of HepG 2 or Huh7 cells. Meanwhile, the effect of isoprenaline or propranolol on the β 2 adrenergic receptor (ADRB2) mRNA expression on HepG2 cells were measured by real-time quantitative reverse transcription (RT-qPCR). Mice with subcutaneous tumors were either subjected to chronic restraint stress (CRS) followed by SNP administration (364 mg/mL) or directly treated with SNP (364 mg/mL). These two parallel experiments were performed to validate the effects of SNP on stress responses. Stress-related proteins and hormones were quantified using RT-qPCR, enzyme-linked immunosorbent assay, and immunohistochemistry. Metagenomic sequencing was performed to confirm the influence of SNP on the gut microbiota in the tumor-bearing CRS mice. RESULTS: The distribution of the 12 active components of SNP was confirmed in various tissues and feces. Network pharmacology analysis confirmed the anti-HCC effects of the 5 active components. The potential anti-HCC mechanisms of SNP may involve the epidermal growth factor receptor (EGFR), proto-oncogene tyrosine-protein kinase Src (SRC) and signal transducer and activator of transcription 3 (STAT3) pathways. SNP-containing serum inhibited the proliferation of HepG2 and Huh7 cells at concentrations of 2.5% and 5.0%, respectively, after 24 h of treatment. Furthermore, SNP suppressed tumor progression in tumor-bearing mice exposed to CRS. SNP treatment also downregulated the expressions of stress-related proteins and pro-inflammatory cytokines, primarily by modulating the gut microbiota. Specifically, the abundance of Alistipes and Prevotella, which belong to the phylum Bacteroidetes, increased in the SNP-treated group, whereas Lachnospira, in the phylum Firmicutes, decreased. CONCLUSION SNP can combat HCC by alleviating stress responses through the regulation of gut microbiota.
Animals ; Gastrointestinal Microbiome/drug effects* ; Liver Neoplasms/microbiology* ; Carcinoma, Hepatocellular/microbiology* ; Humans ; Drugs, Chinese Herbal/therapeutic use* ; Powders ; Cell Proliferation/drug effects* ; Mice ; Molecular Docking Simulation ; Cell Line, Tumor ; Hep G2 Cells ; Receptors, Adrenergic, beta-2/genetics* ; Stress, Physiological/drug effects* ; Cell Movement/drug effects* ; Male ; Protein Interaction Maps/drug effects* ; Cell Survival/drug effects* ; Proto-Oncogene Mas

Homo- or heterodimeric compounds that affect dimeric protein function through interaction between monomeric moieties and protein subunits can serve as valuable sources of potent and selective drug candidates. Here, we screened an in-house dimeric natural product collection, and panepocyclinol A (PecA) emerged as a selective and potent STAT3 inhibitor with profound anti-tumor efficacy. Through cross-linking C712/C718 residues in separate STAT3 monomers with two distinct Michael receptors, PecA inhibits STAT3 DNA binding affinity and transcription activity. Molecular dynamics simulation reveals the key conformation changes of STAT3 dimers upon the di-covalent binding with PecA that abolishes its DNA interactions. Furthermore, PecA exhibits high efficacy against anaplastic large T cell lymphoma in vitro and in vivo, especially those with constitutively activated STAT3 or STAT3^Y640F. In summary, our study describes a distinct and effective di-covalent modification for the dimeric compound PecA to disrupt STAT3 function.