This study was designed to systematically assess the active ingredients of Tongguanteng injection (TGT) and its molecular mechanisms in CRC, with further experimental validation of the implicated pathways. The active ingredients of TGT were obtained from the Herb database. The SwissTargetPrediction database was used to predict the potential targets of these active ingredients. Targets of CRC were obtained from GeneCards, OMIM, DisGeNET, and TTD databases. Cytoscape 3.9.1 was used to build ingredient-target-disease and PPI networks, followed by GO and KEGG enrichment analyses. The interaction between active ingredients and receptor proteins was analyzed through molecular docking using Autodock Tools 1.5.7. Bioinformatics validation of core targets was carried out with UALCAN, KM-plotter, HPA, TIMER, and cBioPortal. Furthermore, in vitro studies were performed to verify the therapeutic efficacy of TGT and its active constituent Tenacissoside G (TG) against CRC and to confirm the critical mechanisms. Network analysis revealed 54 TGT ingredients and 177 CRC-related targets, among which AKT1, EGFR, BCL2, and PTGS2 emerged as the key core targets. KEGG pathway analysis revealed significant involvement of the PD-L1/PD-1 checkpoint pathway. Molecular docking analysis showed high-affinity binding of the active compounds TG to PTGS2. Bioinformatics analysis verified differential expression patterns of PTGS2, EGFR and BCL2 relating to prognostic significance between colorectal cancer and normal tissues. In vitro validation demonstrated TGT and its active ingredients TG induce CRC cell apoptosis by suppressing PTGS2-mediated PD-L1 signaling. TGT and TG play an anti-CRC role by targeting PTGS2 and inhibiting PD-L1/PD-1 to promote apoptosis. This study indicates the therapeutic potential of TGT in CRC.

Background Tongguanteng Injection (TGT), derived from the stems of Marsdenia tenacissima, has been demonstrated to exert anti-tumor effects. Aurora Kinase A (AURKA) is a key regulator of cell cycle progression and has become a promising target for breast cancer treatment. However, the role of AURKA in ferroptosis is unclear, particularly regarding its contribution to the therapeutic effect of TGT in breast cancer. Methods The chemical components of TGT were analyzed by Ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). The anti-breast cancer effect of TGT were detected in vitro and in vivo. RNA-seq analysis were used to identify the targets of TGT treatment. Subsequently, flow cytometry, RT-PCR, western blot, Co-IP, immunofluorescence and immunohistochemistry were employed to elucidate the regulatory mechanisms of TGT on ferroptosis and cell cycle. Concurrently, ferroptosis and cell cycle associated pathways were investigated in the plasmids and siRNAs transfected cells. Furthermore, tumor bearing mouse model was established by 4T1 cells stably transduced with lentivirus for further validating the findings. Results A total of 136 compounds were identified from TGT. The study demonstrates that TGT exerts an anti-breast cancer effect by inducing G2/M phase arrest and ferroptosis, evidenced by downregulation of CDK1 and Cyclin B, as well as increased Fe2+ accumulation, glutathione (GSH) depletion, and elevated lipid peroxidation. RNA-seq analysis revealed that AURKA was significantly downregulated and identified as a key target of TGT treatment. Mechanistically, TGT disrupts the AURKA-Kelch-like ECH-associated protein 1 (KEAP1) interaction, leading to nuclear factor E2-related factor 2 (NRF2) nuclear translocation and subsequent heme oxygenase 1 (HO-1)-mediated ferroptosis in vitro. Knockdown of AURKA or treatment with its inhibitor alisertib both induce G2/M phase arrest and ferroptosis, while AURKA overexpression reverses the effects of TGT and accelerates tumor progression. Combining TGT with docetaxel enhances ferroptosis sensitivity and amplifies the regulatory effect of AURKA on cell cycle and ferroptosis. Consistent with in vitro results, TGT significantly inhibited tumor growth, arrested cell cycle and promoted ferroptosis in 4T1-AURKALV cell bearing mice. Conclusion These findings propose that TGT targets AURKA/KEAP1/NRF2 axis to exert anti-breast cancer effect, and AURKA inhibition represents a potential strategy for breast cancer treatment through inducing cell cycle arrest and driving ferroptosis.

Oral lichen planus (OLP) is a prevalent chronic inflammatory condition that affects the oral mucosa. Histologically, it is characterized by the liquefaction and degeneration of basal epithelial cells, indicating a disruption of the basal layer architecture, which may represent an early event in the disease’s pathogenesis. However, the molecular mechanisms underlying this epithelial damage remain poorly understood. In our study, histological staining and transmission electron microscopy revealed aberrant cell death in the basal epithelial layer of OLP tissues. Immunodetection demonstrated the presence of phosphorylated mixed lineage kinase domain-like protein (pMLKL), a key marker of necroptosis, specifically localized to basal keratinocytes. Notably, interferon type I (IFN-I), particularly IFNα, was significantly upregulated in OLP mucosa compared to healthy controls. The expression of Z-DNA binding protein 1 (ZBP1), an IFN-stimulated gene and an upstream regulator of necroptosis, was elevated in pMLKL-positive epithelial cells. In vitro stimulation with IFNα2a induced the expression of ZBP1 and pMLKL in HaCaT keratinocytes, while ZBP1 knockdown abrogated MLKL phosphorylation. Collectively, these results suggest that, in the context of increased IFN-I signaling, ZBP1 is aberrantly upregulated in the OLP epithelium, promoting necroptosis in basal keratinocytes. This necroptotic activity may contribute to the damage and disruption of the basal layer, providing novel insights into the pathogenesis of OLP and highlighting potential molecular targets for therapeutic intervention.

INTRODUCTION AND AIMS:Erosive oral lichen planus (E-OLP) is a chronic inflammatory condition that poses a therapeutic challenge due to its recurrent nature and potential for malignant transformation. This study aims to evaluate the efficacy and safety of methylene blue-mediated photodynamic therapy (MB-PDT) combined with topical corticosteroid in treating E-OLP. METHODS:This single-centre, single-blinded, randomized, controlled trial was performed from March 2024 to August 2025. A total of 74 patients with E-OLP were randomly assigned to the MB-PDT group (n = 37) receiving MB-PDT plus 0.1% triamcinolone acetonide (TA) and the TA group (n = 37) treating with topical TA alone. The primary outcome was the clinical efficacy rate between the two groups. RESULTS:After 4 weeks of treatment, the clinical efficacy rate was significantly higher in the MB-PDT group than in the TA group (P = .006). Change in the Numeric Rating Scale scores were significantly greater in the MB-PDT group compared to the TA group consistently from week 4 to week 12 (P < .01). The recurrence rate was also significantly lower in MB-PDT group compared to TA group at the 12-week follow-up (P = .038). No adverse events were observed during and after treatment. CONCLUSION:MB-PDT combined with topical corticosteroid appears to be a safe and effective therapeutic approach for E-OLP, with findings suggesting superior pain relief, lesion healing, and reduced recurrence compared with topical corticosteroid. CLINICAL RELEVANCE:This combination therapy is expected to become a promising management option for patients with E-OLP, with potential to improve long-term disease control and quality of life.

Introduction and aims Oral lichen planus (OLP) is a chronic inflammatory disease of unknown etiology. Recent studies have implicated microbial dysbiosis in its pathogenesis. Our previous research revealed an increased abundance of Fusobacterium nucleatum (F.nucleatum) in OLP tissues, suggesting its potential involvement. Methods We utilized single-cell RNA sequencing, spatial transcriptomics, and immunohistochemistry to analyse Claudin-4 expression and its spatial distribution in OLP lesions. In vitro, HaCaT keratinocytes exposed to outer membrane vesicles (OMVs) derived from F.nucleatum (F.n-OMVs) were evaluated for epithelial barrier function via transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FD4) permeability assays. Claudin-4 expression and JNK/c-JUN pathway activation were evaluated by qPCR, Western blotting, and RNA sequencing. The JNK inhibitor SP600125 was used to explore pathway involvement. Results Claudin-4 expression was significantly decreased and exhibited disrupted spatial organization in OLP lesions. F.n-OMVs compromised HaCaT epithelial barrier function, downregulated Claudin-4 expression, and altered its localization. Western blotting demonstrated activation of the JNK/c-JUN signaling pathway. Inhibition of JNK with SP600125 restored Claudin-4 expression and barrier function. Conclusion Our study demonstrates that F.n-OMVs induce epithelial barrier dysfunction in OLP by activating the JNK/c-JUN pathway to downregulate Claudin-4, highlighting a novel microbial mechanism contributing to OLP pathogenesis. Clinical Relevance These findings identify F.n-OMVs as microbial drivers of epithelial barrier disruption in OLP, suggesting that targeting OMVs or the JNK/c-JUN/Claudin-4 axis may offer new diagnostic and therapeutic strategies for OLP management.

Osteosarcoma is a malignant bone tumor that occurs in adolescents and for which surgical resection and chemotherapy are the treatments of first choice. However, tumor cell metastasis and the toxic effects of chemotherapeutic agents bring great challenges to the treatment of osteosarcoma. Fisetin, a dietary flavonoid derived from vegetables and fruits, plays a therapeutic role in many cancers, but its role and underlying mechanism on osteosarcoma remain unrevealed. This study found that fisetin significantly inhibited the proliferation of osteosarcoma cells and organoids. RNA sequencing revealed that forkhead box O3 (FOXO3) may be a key target of fisetin in its anti-osteosarcoma activity, which was also verified by immunofluorescence. siFOXO3 reversed the effects of fisetin on proliferation, apoptosis, and migration of osteosarcoma cells. Fisetin exerts its effects by targeting the ROS/FOXO3 pathway to promote the binding of FOXO3 to the microtubule-associated protein light chain 3 (LC3) promoter, thereby upregulating LC3 levels and inducing autophagy. Subsequently, activated autophagy further triggers ferroptosis, while silencing LC3 reversed fisetin-induced ferroptosis. In vivo, fisetin inhibited tumor growth while promoting FOXO3 and LC3, and inhibiting SLC7A11 protein expression. Therefore, fisetin-induced autophagy in osteosarcoma cells may be associated with the reactive oxygen species (ROS)/FOXO3 axis, thereby promoting ferroptosis. These findings highlight fisetin's potential as a unique therapy for osteosarcoma.

ETHNOPHARMACOLOGICAL RELEVANCE:Marsdenia tenacissima extract (MTE) from the stem of the Traditional Chinese herbal medicine of Marsdenia tenacissima (Roxb.) Wight et Arn. has been used as an anticancer remedy for decades. AIM OF THE STUDY:To investigate the beneficial effects of MTE in osteosarcoma in vitro and in vivo and its potential mechanisms. MATERIAL AND METHODS:Nude mice with ectopic xenograft tumors were treated with MTE of 10 mg/kg and 20 mg/kg for 2 weeks. Human osteosarcoma (OS) cell lines 143B and MG63 were treated with 40, 60, and 80 mg/mL MTE for 24 h. RESULTS:MTE significantly inhibited OS cell proliferation in vitro and in vivo. Specifically, intracellular ferrous ion accumulation is closely related to the upregulation of HO-1 expression levels regulated by MTE. NAC and DFO could restore the effects of MTE on OS cell viability. In addition, the Fe2+ accumulation caused cellular oxidative stress and promoted HO-1 translocation to mitochondria. Correspondingly, the mitochondria were damaged, lysosomes were recruited around, and the expression of autophagy-related proteins PINK1 and Parkin was upregulated. Moreover, the autophagy inhibitor 3-MA reversed the effect of MTE on HO-1 and GPX4 protein expression levels in OS cells. In vivo experiments demonstrated that MTE inhibited the growth of xenograft osteosarcoma while increasing the expression of LC3B, HO-1 and FTH1, and simultaneously suppressing the level of GPX4 in tumor tissues. CONCLUSION:MTE exerts anti-osteosarcoma effects by modulating HO-1 and activating mitophagy, thereby accelerating the ferroptosis.

Huachansu injection (HCSI) shows effective medicinal functions against osteosarcoma. This study aimed to reveal the underlying mechanisms of HCSI against osteosarcoma by integrating metabolomics, network pharmacology and bioinformatics. Metabolomics was used to identify different metabolites and pathways. Network pharmacology was utilized to predict the potential targets of HCSI against osteosarcoma. Differentially expressed lncRNAs and miRNAs were screened and the corresponding lncRNAs-miRNAs-mRNAs network were constructed through the GEO database and miRcode database. Machine learning and immune infiltration analysis were performed on the key target obtained from the intersection of network pharmacology and bioinformatics. The binding affinity between active compounds of HCSI and potential targets was evaluated by molecular docking. The underlying mechanisms were further validated by RT-qPCR and immunoblotting. Lipid metabolism pathways were obtained by non-target metabolomics enrichment. A total of 44 HCSI targets associated with osteosarcoma were collected by network pharmacology. Intersection of the mRNAs obtained from ceRNA network with the above 44 targets yielded eight common targets. The main target HMGCR were obtained by machine learning and RT-qPCR. The BCYRN1-miR-27a-3p-HMGCR axis was subsequently screened as the primary ceRNA regulatory network in HSCI against osteosarcoma. Molecular docking also showed an excellent affinity between the active compounds of HCSI and HMGCR. In vitro experiments demonstrated that HCSI down-regulated HMGCR, thereby reduced intracellular cholesterol levels, and ultimately promoting osteosarcoma cell apoptosis. HCSI could inhibit osteosarcoma progression by regulating lipid metabolism through BCYRN1-miR-27a-3p-HMGCR axis, indicating that HCSI may provide insights for developing herbal medicine injection-based therapies for osteosarcoma.

OBJECTIVE:Huachansu injection (HCSI), a promising anti-cancer Chinese medicine injection, has been reported to have the potential for reducing the toxicity of chemotherapy and improving the quality of life for colorectal cancer (CRC) patients. The objective of this study is to explore the synergistic and detoxifying effects of HCSI when used in combination with irinotecan (CPT-11). METHODS:To investigate the effect of HCSI on anti-CRC efficacy and intestinal toxicity of CPT-11, we measured changes in the biological behavior of LoVo cells in vitro, and anti-tumor effects in LoVo cell xenograft nude mice models in vivo. Meanwhile, the effect of HCSI on intestinal toxicity and the uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1) expression was investigated in the CPT-11-induced colitis mouse model. Subsequently, we measured the effect of HCSI and its 13 constituent bufadienolides on the expression of UGT1A1 and organic anion transporting polypeptides 1B3 (OATP1B3) in HepG2 cells. RESULTS:The combination index (CI) results showed that the combination of HCSI and CPT-11 exhibited a synergistic effect (CI < 1), which significantly suppressing the LoVo cell migration, enhancing G2/M and S phase arrest, and inhibiting tumor growth in vivo. Additionally, the damage to intestinal tissues was attenuated by HCSI in CPT-11-induced colitis model, while the increased expression of UGT1A1 in HepG2 cells and in mouse was observed. CONCLUSION:The co-therapy with HCSI alleviated the intestinal toxicity induced by CPT-11 and exerted an enhanced anti-CRC effect. The detoxifying mechanism may be related to the increased expression of UGT1A1 and OATP1B3 by HCSI and its bufadienolides components. The findings of this study may serve as a theoretical insights and strategies to improve CRC patient outcomes. Please cite this article as: Jiang B, Meng ZY, Hu YJ, Chen JJ, Zong L, Xu LY, Zhang XQ, Zhang JX, Han YL. Huachansu injection enhances anti-colorectal cancer efficacy of irinotecan and alleviates its induced intestinal toxicity through upregulating UGT1A1-OATP1B3 expression in vitro and in vivo. J Integr Med. 2025; 23(5):576-590.

Ovarian cancer (OC) is the most common malignant gynecologic tumor, with the highest mortality rate among female reproductive system cancers. Resistance to chemotherapy drugs, which often develops after long-term use, is a major cause of treatment failure. In recent years, traditional Chinese medicine has been widely used in the treatment of tumor for their advantages in improving the efficacy of chemotherapy and alleviating the toxic side effects. Tenacissoside G (Tsd-G), as one of the main active ingredients of Marsdenia tenacissima, exhibits anti-tumor effects. However, its impact on ovarian cancer is not well understood. To assess the role and mechanism of Tsd-G in reversing paclitaxel (PTX) resistance, the reversal fold of Tsd-G in combination with PTX on OC PTX-resistant (A2780/T) cells was determined using CCK-8 assay. The apoptosis level and migration ability of A2780/T cells after 24 h treatment with Tsd-G and PTX were assessed by Hoechst 33,342, flow cytometry, and wound healing assay. Western Blot and Src overexpression plasmid were used to explore the relationship between Src and PTX resistance. The relationship between Src expression and human OC was analyzed by gene expression database. The effect of Tsd-G on P-gp activity was detected by flow cytometry. Western blot and RT-PCR experiments were performed to detect the differences in mRNA and protein expression of Src/PTN/P-gp signaling axis to validate the mechanism of Tsd-G in reversing the resistance to PTX in ovarian cancer. The results showed that Tsd-G reverses PTX resistance in ovarian cancer cells by regulating cell proliferation, cell cycle, inducing apoptosis, and inhibiting migration. The mechanism might associate with the inhibition of Src expression and phosphorylation activation, which in turn inhibits the expression and activity of downstream PTN and P-gp. This study provides a new idea for the treatment of PTX-resistant OC patients and provides theoretical support for revealing the anti-ovarian cancer active ingredients in Marsdenia tenacissima. Tsd-G reverses PTX resistance by inhibiting the Src/PTN/P-gp signaling axis and inducing PTX accumulation in ovarian cancer paclitaxel-resistant cells.

Background Oral lichen planus (OLP) is commonly accepted as an interface mucositis. The immunogen from keratinocytes is considered as the agonist priming the topical immunity. Our previous study has identified increased dendritic cell (DC) infiltration and TLR9 expression in OLP lesions. Heat shock protein 90 (HSP90) has been widely considered as an autoantigen and TLR9 agonist that could activate DCs. However, the significance and impact of HSP90 as an immunogen in OLP remain unclear. Objective To investigate the effects of the HSP90 complex from OLP lesion tissues on DCs activation and the polarization of naïve T cells. Methods The expression pattern of HSP90 in OLP lesions and healthy control mucosa was evaluated by single-cell RNA sequence, immunohistochemistry, quantitative reverse transcription polymerase chain reaction, and immunoblotting. The HSP90 complex was further extracted by immunoprecipitation from the OLP lesions and healthy control mucosa as the agonist of DCs. The peripheral DCs were collected and stimulated with the extractives. When challenged, DCs’ expression of interferon-alpha and MHC molecules was detected by flow cytometry. Hence, after cocultured with pre-stimulated DCs, polarization of naïve T cells was investigated by cytokines profile analysis. Results HSP90 expression was significantly greater in the lamina propria of OLP lesion and closely related with lymphocyte infiltration. The HSP90 complex in OLP lesion tissues activated DCs via TLR9 and increased their interferon-α secretion and MHC II expression. Prestimulated DCs increased the proportion of Th17 cells. Conclusions The HSP90 complex isolated from OLP lesion tissue activated a TLR9/interferon-α pathway of DC and further promoted the polarization of naïve T cells towards Th17 immunity.

Objectives:To search for a new classification scheme for oral lichen planus (OLP) and oral lichenoid lesions (OLL) based on innate lymphoid cells (ILCs) and to evaluate the clinical significance of this classification for diagnosis and treatment. Subjects and methods:This study was based on a clinical cohort and applied flow cytometry to prospectively analyze the ILC subgroups and proportions in OLP and OLL lesions using SPSS software (version 26.0) to attempt cluster analysis to classify diseases at the cellular level based on the phenotype and quantity of ILCs cells, analyze the correlation between the new classification of diseases and clinical risk factors based on the patient's clinical background information and classification results, and evaluate the differences in therapeutic effects among patients in different groups in corresponding clinical cohorts. Results:In the OLP and OLL groups, the ILC compartment consisted mainly of ILC1 (75.02% ± 27.55% and 72.99% ± 25.23%, respectively), ILC2 (1.49% ± 4.12% and 1.72% ± 3.18%, respectively), and ILC3 (16.52% ± 19.47% and 18.77% ± 18.12%, respectively). Using k-means clustering and two-step clustering, patients could be clustered into three groups that did not respond equally to the same treatment. Using k-means clustering, there was a statistically significant difference in REU scores between the ILC1 advantage group and the OLL subgroup before and after treatment (P = 0.02), which was not observed in two-step clustering. This indicates that k-means clustering may have greater value in the clinical application of OLL. In the ILC1 absolute advantage group, using HCQ + TGP for one month could effectively treat the patients regardless of the use of k-means clustering or two-step clustering (P ≤0.001), whereas the other groups did not. Conclusions:This study provides a preliminary OLP and OLL classification method based on ILC subgroups that can guide the cytological classification of diseases to a certain extent. Further clinical application values should be verified in subsequent cohort studies.

BackgroundOral lichen planus (OLP) is a chronic, immune-mediated disorder affecting 2%-4% of the population. Corticosteroids are commonly used for treatment, but responses vary, likely due to differences in lymphocyte profiles. This study evaluated the impact of CD20+ B cells and CD38+ plasma cells on corticosteroid efficacy in OLP patients.Patients and MethodsThis retrospective study included 101 patients with symptomatic, diffuse, and severe OLP. Immunohistochemical staining identified CD3+ T cells, CD20+ B cells, and CD38+ plasma cells in tissue lesions. All patients received prednisone, and outcomes were assessed using Reticulation/Erosion/Ulceration (REU) and Visual Analog Scale (VAS) scores. Statistical analyses compared treatment responses based on lymphocyte profiles.ResultsAll patients showed CD3-positive T cell infiltration. Patients in the CD20- group showed significantly greater reductions in REU and VAS scores than the CD20+ group, indicating that B cell infiltration may reduce corticosteroid efficacy. Conversely, CD38+ patients exhibited greater score reductions than CD38- patients, suggesting that plasma cells may enhance responsiveness to treatment. The CD38+/CD20- group exhibited the most favorable response; the CD38-/CD20+ group had the least.ConclusionsInfiltration by B cells and plasma cells influences corticosteroid response in OLP. B cells are associated with reduced efficacy, while plasma cells correlate with improved treatment outcomes.

Oral lichen planus (OLP) is a prevalent chronic inflammatory condition that affects the oral mucosa. Histologically, it is characterized by the liquefaction and degeneration of basal epithelial cells, indicating a disruption of the basal layer architecture, which may represent an early event in the disease’s pathogenesis. However, the molecular mechanisms underlying this epithelial damage remain poorly understood. In our study, histological staining and transmission electron microscopy revealed aberrant cell death in the basal epithelial layer of OLP tissues. Immunodetection demonstrated the presence of phosphorylated mixed lineage kinase domain-like protein (pMLKL), a key marker of necroptosis, specifically localized to basal keratinocytes. Notably, interferon type I (IFN-I), particularly IFNα, was significantly upregulated in OLP mucosa compared to healthy controls. The expression of Z-DNA binding protein 1 (ZBP1), an IFN-stimulated gene and an upstream regulator of necroptosis, was elevated in pMLKL-positive epithelial cells. In vitro stimulation with IFNα2a induced the expression of ZBP1 and pMLKL in HaCaT keratinocytes, while ZBP1 knockdown abrogated MLKL phosphorylation. Collectively, these results suggest that, in the context of increased IFN-I signaling, ZBP1 is aberrantly upregulated in the OLP epithelium, promoting necroptosis in basal keratinocytes. This necroptotic activity may contribute to the damage and disruption of the basal layer, providing novel insights into the pathogenesis of OLP and highlighting potential molecular targets for therapeutic intervention.

Objective The main objectives of this study were to identify the active components of Tongguanteng injection (TGT) and investigate the preclinical efficacy and mechanism of TGT on osteosarcoma using a combination of network pharmacology and experimental validation. Methods To identify the active constituents and targets of TGT against osteosarcoma using network pharmacology, we constructed a network consisting of an 'active ingredient-disease-target-pathway' and a protein–protein interaction (PPI) network. The target organ network was utilized to investigate the distribution of core targets in tissues. Afterwards, the core targets underwent Gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The binding energy between receptors and ligands was compared using molecular docking. In addition, SwissADME was employed to forecast the pharmacokinetic characteristics of the substances. Finally, real-time polymerase chain reaction (RT-PCR), cell proliferation assay, morphological analysis, apoptosis assay, mitochondrial membrane potential (MMP) detection, and Western blotting were utilized to confirm the potential mechanisms of TGT treatment in osteosarcoma cell lines 143B and SAOS2. Results A total of 54 chemical constituents of TGT and 71 targets associated with osteosarcoma were acquired. Through the molecular docking technology, Tenacigenin B, Marsdekoiside, Taraxasterol, Tenacissoside G, Tenacissoside L, and Tenacissoside J were identified as the primary active components of TGT among the various compounds. Analysis of target organs suggests that TGT may play an anti-osteosarcoma role through immune regulation. The GO and KEGG enrichment analysis revealed that TGT could trigger osteosarcoma cell apoptosis by inhibiting the HIF-1 signalling pathway and modulating PD-1 expression and the PD-1 checkpoint pathway in cancer. SwissADME database predicted that Tenacigenin B and Taraxasterol had the best drug-likeness. In vitro studies also demonstrated that TGT suppressed the activity and induced alterations in the morphology of osteosarcoma cells. It decreased MMP levels, triggered apoptosis by increasing Bax expression and Caspase-3 activity, and decreased Bcl-2 expression, thereby exerting an anti-osteosarcoma effect. In the meantime, RT-PCR tests demonstrated that TGT could control immune response against tumors and hinder the proliferation and spread of cancerous cells by impacting the levels of critical factors, including JUN, HSP90AA1, HDAC1, and CDK1. Conclusion The study accurately anticipated the active components, targets, and pathways of TGT in the management of osteosarcoma. The molecular mechanism of TGT-induced apoptosis in osteosarcoma cells was demonstrated by in vitro experiments. These results provide theoretical and technical support for TGT as a clinical adjuvant drug for osteosarcoma.

BACKGROUND:In China, Tongguanteng injection (TGT) is widely used in the treatment or adjuvant treatment of various types of cancer. However, the effect and mechanism of TGT in osteosarcoma is not clear. METHODS:The 143B and MG-63 cells were treated with different concentrations of TGT. Cell proliferation, migration, invasion and apoptosis were detected using CCK8 assay, transwell assay and flow cytometry. Differentially expressed genes (DEGs) were screened using RNA sequencing (RNA-seq). The identified mRNA and protein expression associated with the IRE1/CHOP pathway was validated by RT-PCR and western blot assay. To explore the underlying mechanisms, 4-phenylbutyric acid (4-PBA) was selected as a specific endoplasmic reticulum (ER) stress inhibitor. Small interfering RNA (siRNA) or pEX-3-ERN1 plasmid was transfected into 143B cells to silence or overexpress IRE1, respectively. The potential downstream proteins, including CHOP, and apoptosis associated proteins, caspase-3 and PARP1 were determined. Furthermore, the effect of TGT was demonstrated in 143B cell tumor-bearing mice in vivo. H&E staining, TUNEL staining and immunohistochemistry were conducted in tumor tissues obtained from the xenograft mouse model. RESULTS:TGT was shown to dramatically suppress the proliferation, migration and invasion, and induce apoptosis of osteosarcoma 143B and MG-63 cells in vitro. The identified DEGs included HSPA5 (encoding BiP) and ERN1 (encoding the IRE1 protein), as well as apoptosis-associated gene DDIT3 (encoding the CHOP protein). The term "IRE1-mediated unfolded protein response" was screened to be the most enriched biological process GO term. The expression of ER stress-associated proteins including ATF6, BiP, p-IRE1, XBP1s and CHOP, as well as apoptosis-associated cleaved caspase-3 and cleaved PARP1 proteins, was significantly upregulated by TGT treatment in osteosarcoma 143B cells, suggesting that TGT might promote the apoptosis of osteosarcoma 143B cells through the IRE1/CHOP pathway. Furthermore, knocking down IRE1 with si-IRE1 or inhibiting of ER stress with 4-PBA suppressed the expression of ATF6, BiP, XBP1s and CHOP induced by TGT, as well as the expression of cleaved caspase-3 and cleaved PARP1. On the contrary, overexpressing IRE1 promoted CHOP expression and induced osteosarcoma cell apoptosis. Consistent with in vitro results, TGT dramatically inhibited the tumor growth and promoted the expression of p-IRE1 and CHOP in tumor-bearing mice. CONCLUSION:The findings suggest that TGT exerts an anti-osteosarcoma effect in vitro and in vivo. The underlying mechanism might be associated with the activation of IRE1/CHOP pathway in ER stress. Our findings suggest that targeting IRE1/CHOP pathway might be a potential novel approach for osteosarcoma treatment.

ObjectivesTo investigate the effects of the heat shock protein 90 (HSP90) complex from oral lichen planus (OLP) lesion tissues on dendritic cell (DC) activation and the polarization of na & iuml;ve T cells.MethodsExpression of HSP90 in OLP lesions and healthy control (HC) mucosa was evaluated by single-cell RNA sequence, IHC, qRT-PCR, and immunoblotting. HSP90 complex was extracted by immunoprecipitation from oral mucosa as the agonist of DCs. Expression of IFN-alpha and MHC was detected by flow cytometry. After cocultured with pre-stimulated DCs, polarization of na & iuml;ve T cells was investigated by cytokine analysis.ResultsHSP90 was significantly higher in the lamina propria of OLP lesion and closely related to lymphocyte infiltration. HSP90 complex of OLP lesion activated DCs via TLR9 and increased their IFN-alpha secretion and MHC II expression. Pre-stimulated DCs increased the proportion of Th17 cells.ConclusionsHSP90 complex isolated from OLP lesion activated TLR9/IFN-alpha of DCs and further promoted the polarization of na & iuml;ve T cells toward Th17 immunity.

Introduction: The study aimed to compare the outcomes of nirmatrelvir and ritonavir drug combination (Paxlovid) therapy in patients who received treatment within or after five days of COVID-19 confirmed in the elderly. Methodology: This was a single-center, retrospective cohort study of older COVID-19 patients (≥ 60 years) admitted from April 7 to May 30, 2022. Patients were categorized into the EP group (starting Paxlovid within five days) and the LP group (starting Paxlovid after five days) following symptoms onset. Length of stay and positive SARS-CoV-2 duration were compared between the two groups. Severe case conversion from mild and moderate COVID-19 patients were also analyzed. Results: In total, 273 patients were included: 137 in the EP group and 136 in the LP group. Compared to the LP group, the EP group had a significantly shorter length of stay (12.4 vs. 14.7 days, p = 0.001) and positive SARS-CoV-2 duration (11.7 vs. 15.8 days, p < 0.001). The EP group had lower severe case conversion (4.4% vs. 15.4%, p = 0.002). Additionally, abnormal IL-6 and lower lymphocyte count indicated increased length of stay. Older age was associated with a decreased risk in SARS-CoV-2 negative test (HR = 0.98) and an increased risk in severe case conversion (OR = 1.11). Conclusions: Starting Paxlovid within five days of COVID-19 symptoms onset reduced the length of stay and SARS-CoV-2 duration compared to initiating treatment after five days. While severe case conversion among mild COVID-19 patients might be comparable whether starting Paxlovid within or after five days.

P-glycoprotein (P-gp)-mediated herb-drug interactions (HDIs) may impact drug efficacy and safety. Tenacissoside G (Tsd-G), a major active component of Marsdenia tenacissima, exhibits anticancer activity. To analyze the effect of Tsd-G on the pharmacokinetics of paclitaxel (PTX), researchers selected 30 Sprague-Dawley (SD) rats, randomized into a solvent control group, a verapamil positive control group, and 20, 40, and 60 mg/kg Tsd-G groups. After seven consecutive days of intraperitoneal injection of verapamil or Tsd-G, a single dose of 6 mg/kg PTX was injected intravenously. Plasma samples were collected at different time points, and proteins were precipitated using a methanol-acetonitrile solution. An ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed, with docetaxel as an internal standard, and quantified using positive ion multiple reaction monitoring (MRM) mode. This analytical method's specificity, accuracy, precision, recovery, matrix effect, and sample stability meet the requirements for biological sample determination. After Tsd-G administration in rats, the mean residence time of PTX was significantly prolonged. And Tsd-G can stably bind to P-gp by forming hydrogen bonds and inhibiting the expression of P-gp in rat liver. Although the metabolites of PTX were not detected in this study, the above results still indicate the existence of HDIs between Tsd-G and PTX, and P-gp may be the main target to mediate HDIs.