GGT5 facilitates migration and invasion through the induction of epithelial-mesenchymal transformation in gastric cancer.

BACKGROUND: Gamma-glutamyltransferase 5 (GGT5), one of the two members in the GGT family (GGT1 and GGT5), plays a crucial role in oxidative regulation, inflammation promotion, and drug metabolism. Particularly in the tumorigenesis of various cancers, its significance has been recognized. Nevertheless, GGT5's role in gastric cancer (GC) remains ambiguous. This study delves into the function and prognostic significance of GGT5 in GC through a series of in vitro experiments. METHODS: Employing online bioinformatics analysis tools such as The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Kaplan-Meier plotter, and cBioPortal, we explored GGT5 characteristics and functions in GC. This encompassed aberrant expression, prognostic value, genomic alterations and mutations, immune cell infiltration, and associated signaling pathways. Immunohistochemistry was conducted to assess GGT5 expression in GC and adjacent normal tissues. Subsequently, univariate and multivariate logistic regression analyses were applied to investigate the associations between GGT5 and clinical characteristics. CCK8, wound healing, and migration assays were utilized to evaluate the impact of GGT5 on cell viability and migration. Additionally, Gene Set Enrichment Analysis (GSEA) and Western blot analysis were performed to scrutinize the activity of the epithelial-mesenchymal transformation (EMT) signaling pathway under GGT5 regulation. RESULTS: GGT5 exhibits upregulation in gastric cancer, with its overexpression significantly linked to histological differentiation in GC patients (P < 0.05). Multivariate analysis indicates that elevated GGT5 expression is an independent risk factor associated with poorer overall survival in gastric cancer patients (P < 0.05). In vitro experiments reveal that downregulation of GGT5 hampers the proliferation and migration of GC cell lines. Finally, GSEA using TCGA data highlights a significant correlation between GGT5 expression and genes associated with EMT, a finding further confirmed by Western blot analysis. CONCLUSIONS: GGT5 emerges as a promising prognostic biomarker and potential therapeutic target for GC.

Jiangu formula: A novel osteoclast-osteoblast coupling agent for effective osteoporosis treatment.

BACKGROUND: The discovering of an osteoclast (OC) coupling active agent, capable of suppressing OC-mediated bone resorption while concurrently stimulating osteoblast (OB)-mediated bone formation, presents a promising strategy to overcome limitations associated with existing antiresorptive agents. However, there is a lack of research on active OC coupling agents. PURPOSE: This study aims to investigate the potential of Jiangu Formula (JGF) in inhibiting OCs while maintaining the OCOB coupling function. METHODS: The anti-osteoporosis efficacy of JGF was evaluated in osteoporosis models induced by ovariectomy in C57BL/6 mouse and SD rats. The effect of JGF on OCs was evaluated by detecting its capacity to inhibit OC differentiation and bone resorption in an in vitro osteoclastogenesis model induced by RANKL. The OCOB coupling activity of JGF was evaluated by measuring the secretion levels of OC-derived coupling factors, OB differentiation activity of MC3T3-E1 interfered with conditioned medium, and the effect of JGF on OC inhibition and OB differentiation in a C3H10T1/2-RAW264.7 co-culture system. The mechanism of JGF was studied by network pharmacology and validated using western blot, immunofluorescence (IF), and ELISA. Following that, the active ingredients of JGF were explored through a chemotype-assembly approach, activity evaluation, and LC-MS/MS analysis. RESULTS: JGF inhibited bone resorption in murine osteoporosis without compromising the OCOB coupling effect on bone formation. In vitro assays showed that JGF preserved the coupling effect of OC on OB differentiation by maintaining the secretion of OC-derived coupling factors. Network analysis predicted STAT3 as a key regulation point for JGF to exert anti-osteoporosis effect. Further validation assays confirmed that JGF upregulated p-STAT3(Ser727) and its regulatory factors IL-2 in RANKL-induced RAW264.7 cells. Moreover, 23 components in JGF with anti-OC activity identified by chemotype-assembly approach and verification experiments. Notably, six compounds, including ophiopogonin D, ginsenoside Re, ginsenoside Rf, ginsenoside Rg3, ginsenoside Ro, and ononin were identified as OC-coupling compounds. CONCLUSION: This study first reported JGF as an agent that suppresses bone loss without affecting bone formation. The potential coupling mechanism of JGF involves the upregulation of STAT3 by its regulators IL-2. Additionally, the chemotype-assembly approach elucidated the activity compounds present in JGF, offering a novel strategy for developing an anti-resorption agent that preserves bone formation.

Bipoladien A, a Sesterterpenoid Containing an Undescribed 5/8/5/7 Carbon Skeleton from Bipolaris maydis.

Bipoladiens A-E (1-5), five new ophiobolin-derived sesterterpenoids, and a known compound 6 (bipolaricin R) were isolated from the cultures of the phytopathogenic fungus Bipolaris maydis. Their structures and absolute configurations were elucidated based on comprehensive spectroscopic analyses, HRESIMS, electronic circular dichroism (ECD) calculations, and single-crystal X-ray diffraction analyses. Notably, compound 1 has an undescribed tetracyclic 5/8/5/7 fused carbon skeleton, and compound 2 possesses a rare multicyclic caged ring system. The biosynthetic pathway of 1 was proposed starting from 6 via a series of oxidation and cyclization reactions. Compound 6 showed excellent antiproliferation and apoptosis induction effects against A549 cell line. Additionally, compounds 5 and 6 exhibited noticeable antimicrobial ability against Bacillus cereus, Staphylococcus aureus, and Staphylococcus epidermidis. These findings not only developed the chemical and bioactivities diversities of ophiobolin-sesterterpenoid but also provided an idea to boost the application of natural products in the control of food pathogens.

A 37-Year-Old Schizophrenic Woman With Abdominal Pain.

Introduction: Internal fistula across the posterior wall of stomach and the transverse colon caused by foreign bodies in the alimentary tract presents an extremely rare medical entity. Presentation of case: We report an aschizophrenia female patient with onset of internal fistula across the posterior wall of stomach and the transverse colon triggered by swallowed magnetic metal beads. The patient was admitted to the emergency room of Northern Jiangsu People's Hospital because of acute right lower abdominal pain. Emergency routine abdominal CT scan revealed acute appendicitis and a set of foreign body in digestive tract. Discussion: The foreign body in the stomach was removed by open surgery after tentative Endoscopic foreign body removal and laparoscopic appendectomy and exploration. In the process of exploring the gastric wall, it was found that one of magnet beads was embedded in the posterior wall of stomach and adhered to part of the transverse colon. After separation, it was found that an internal fistula was formed across the posterior wall of stomach and the transverse colon. As the patient ate only a small amount of food within 2 days, and the intestines were in good condition, we performed partial transverse colectomy, end-to-side anastomosis and gastric wall repair. Conclusion: This case shows that for long-term foreign bodies in the digestive tract, we should be beware of the onset of gastrointestinal perforation. Moreover, perforation caused by the force acting on a blunt foreign body often results in atypical imaging findings, and the diagnosis of perforation cannot be clearly determined by imaging findings such as the presence of free gas downstream of the diaphragm. This poses new challenges for clear diagnosis and treatment.

Triggering "signal-on" photoelectrochemical responses by heterojunction transition for selective detection of copper(II) based on Pd/MoS2@g-C3N4 nanocomposites.

Controlling the concentration of copper(II) in aquatic systems is of importance for human health. Numerous traditional technologies to detect Cu2+ may encounter with limitations, such as high signal background and complicated operation. Herein, a highly selective photoelectrochemical (PEC) sensor is proposed for the "signal-on" detection of Cu2+ employing g-C3N4 nanosheets with MoS2 and Pd quantum dots deposited (Pd/MoS2@g-C3N4). Pd/MoS2@g-C3N4 could present the enhanced photocurrents of specific responses to Cu2+ under light irradiation. MoS2 quantum dots on the sensor are agglomerated into MoS2 bulk during sensing Cu2+, forming an efficient Z-scheme heterojunction. The heterojunction transition induced photoelectrons transferring from the bulk MoS2 to g-C3N4, resulting in "signal-on" PEC responses. Such Z-scheme heterojunction has conquered the traditional heterojunction towards "signal-on" mechanism, that was further verified by band structure measurements and DMPO spin trapping ESR analysis. Photocurrent intensities increased gradually with the addition of incremental Cu2+ concentrations, achieving a detection limit of 0.21 µM and a broad linear interval range from 1 µM to 1 mM with high selectivity and stability. This work may open a new door towards the in situ construction of g-C3N4-based Z-scheme heterojunctions for the signal-on PEC sensing platform, providing wide applications in environmental monitoring and food safety.

Surgical outcomes of laparoscopic proximal gastrectomy for upper-third gastric cancer: esophagogastrostomy, gastric tube reconstruction, and double-tract reconstruction.

BACKGROUND: There is no consensus on the optimal reconstruction technique after proximal gastrectomy. The purpose of this study was to retrospectively compare the surgical outcomes among esophagogastrostomy (EG) anastomosis, gastric tube (GT) reconstruction and double-tract (DT) reconstruction in patients who underwent laparoscopic proximal gastrectomy (LPG) to clarify the superior reconstruction method. METHODS: This study enrolled 164 patients who underwent LPG at the Northern Jiangsu People's Hospital in Jiangsu between January 2017 to January 2022 (EG: 51 patients; GT: 77 patients; DT: 36 patients). We compared the clinical and pathological characteristics, surgical features, postoperative complications, nutritional status, and quality of life (QOL) among the above three groups. RESULTS: Mean operative time was longer with the DT group than the remaining two groups (p = 0.001). With regard to postoperative complications, considerable differences in the postoperative reflux symptoms (p = 0.042) and reflux esophagitis (p = 0.040) among the three groups were found. For the nutritional status, total protein, hemoglobin and albumin reduction rates in the GT group were significantly higher than the other two groups at 12 months postoperatively. In the PGSAS-45, three assessment items were better in the DT group significantly compared with the esophageal reflux subscale (p = 0.047, Cohen's d = 0.44), dissatisfaction at the meal (p = 0.009, Cohen's d = 0.58), and dissatisfaction for daily life subscale (p = 0.012, Cohen's d = 0.56). CONCLUSIONS: DT after LPG is a valuable reconstruction technique with satisfactory surgical outcomes, especially regarding reduced reflux symptoms, improving the postoperative nutritional status and QOL.

Enhancement of recombinant human interleukin-22 production by fusing with human serum albumin and supplementing N-acetylcysteine in Pichia Pastoris.

Interleukin-22 (IL-22) plays an important role in the treatment of organ failure, which can induce anti-apoptotic and proliferative signaling pathways; Nevertheless, the practical utilization of IL-22 is hindered by the restricted efficacy of its production. Pichia pastoris presents a viable platform for both industrial and pharmaceutical applications. In this study, we successfully generated a fusion protein consisting of truncated human serum albumin and human IL-22 (HSA-hIL-22) using P. pastoris, and examined the impact of antioxidants on HSA-hIL-22 production. We have achieved the production of HSA-hIL-22 in the culture medium at a yield of approximately 2.25 mg/ml. Moreover, 0-40 mM ascorbic acid supplementation did not significantly affect HSA-hIL-22 production or the growth rate of the recombinant strain. However, 80 mM ascorbic acid treatment had a detrimental effect on the expression of HSA-hIL-22. In addition, 5-10 mM N-acetyl-l-cysteine (NAC) resulted in an increase of HSA-hIL-22 production, accompanied by a reduction in the growth rate of the recombinant strain. Conversely, 20-80 mM NAC supplementation inhibited the growth of the recombinant strains and reduced intact HSA-hIL-22 production. However, neither NAC nor ascorbic acid exhibited any effect on superoxide dismutase (SOD) and malondialdehyde (MDA) levels, except that NAC increased GSH content. Furthermore, our findings indicate that recombinant HSA-hIL-22, which demonstrated the ability to stimulate the proliferation of HepG2 cells, possesses bioactivity. In addition, NAC did not affect HSA-hIL-22 bioactivity. In conclusion, our study demonstrates that NAC supplementation can enhance the secretion of functional HSA-hIL-22 proteins produced in P. pastoris without compromising their activity.

Development of flow battery technologies using the principles of sustainable chemistry.

Realizing decarbonization and sustainable energy supply by the integration of variable renewable energies has become an important direction for energy development. Flow batteries (FBs) are currently one of the most promising technologies for large-scale energy storage. This review aims to provide a comprehensive analysis of the state-of-the-art progress in FBs from the new perspectives of technological and environmental sustainability, thus guiding the future development of FB technologies. More importantly, we evaluate the current situation and future development of key materials with key aspects of green economy and decarbonization to promote sustainable development and improve the novel energy framework. Finally, we present an analysis of the current challenges and prospects on how to effectively construct low-carbon and sustainable FB materials in the future.

Single-cell transcriptomics reveals intestinal cell heterogeneity and identifies Ep300 as a potential therapeutic target in mice with acute liver failure.

Acute liver failure (ALF) is a severe life-threatening disease associated with the disorder of the gut-liver axis. However, the cellular characteristics of ALF in the gut and related therapeutic targets remain unexplored. Here, we utilized the D-GALN/LPS (D/L)-induced ALF model to characterize 33,216 single-cell transcriptomes and define a mouse ALF intestinal cellular atlas. We found that unique, previously uncharacterized intestinal immune cells, including T cells, B cells, macrophages, and neutrophils, are responsive to ALF, and we identified the transcriptional profiles of these subsets during ALF. We also delineated the heterogeneity of intestinal epithelial cells (IECs) and found that ALF-induced cell cycle arrest in intestinal stem cells and activated specific enterocyte and goblet cell clusters. Notably, the most significantly altered IECs, including enterocytes, intestinal stem cells and goblet cells, had similar activation patterns closely associated with inflammation from intestinal immune activation. Furthermore, our results unveiled a common Ep300-dependent transcriptional program that coordinates IEC activation during ALF, which was confirmed to be universal in different ALF models. Pharmacological inhibition of Ep300 with an inhibitor (SGC-CBP30) inhibited this cell-specific program, confirming that Ep300 is an effective target for alleviating ALF. Mechanistically, Ep300 inhibition restrained inflammation and oxidative stress in the dysregulated cluster of IECs through the P38-JNK pathway and corrected intestinal ecology by regulating intestinal microbial composition and metabolism, thereby protecting IECs and attenuating ALF. These findings confirm that Ep300 is a novel therapeutic target in ALF and pave the way for future pathophysiological studies on ALF.

New secondary metabolites with cytotoxicity from fungus Penicillium roqueforti.

Two novel compounds including a cyclohelminthol type polyketide (namely oxaleimide K, 1) and a maleimide derivative (namely peniroquefortine A, 2), and a new natural product (namely 2-(acetylamino)-N-[(1E)-2-phenylethenyl]-acetamide, 3), together with four known compounds (4-7), were isolated and identified from fungus Penicillium roqueforti, which was separated from the root soil of Hypericum beanii N. Robson collected from the Shennongjia Forestry District, Hubei Province. Their structures including absolute configurations were mainly established by the NMR spectroscopy analyses and single-crystal X-ray diffraction experiment. Compound 1 represents the second example of a cyclohelminthol type polyketide, which features a rare 6/6/5/5 tetracyclic system and a branched aliphatic chain containing a terminal olefin (oct-1-en-3-yl) moiety, and compound 2 possesses an unprecedented carbon skeleton that is uniquely defined by a maleimide moiety linked to the respective 4-methylene-2-(3-methylbut-2-en-1-yl)-phenol and para-substituted aromatic moieties via the carbon-carbon bonds. Remarkably, the absolute configuration of a cyclohelminthol type polyketide as exemplified by compound 1 is determined by the single-crystal diffraction analysis for the first time, highlighting an E-configuration for the linkage of a succinimide moiety and a tetrahydrofuran moiety for 1 rather than a Z-configuration as previously reported in the biosynthesis study, which gives a new insight into the structural elucidation of this category of polyketides. Additionally, compound 1 exhibited significant cytotoxic activity against multiple tumor cells, especially against the Farage and SU-DHL-2 cells (IC50 < 20 µM, 48 h). Further mechanism study revealed that compound 1 significantly induced cell cycle arrest in Farage and SU-DHL-2 cells by causing abnormal ROS level and triggering oxidative stress.

Spectasterols, Aromatic Ergosterols with 6/6/6/5/5, 6/6/6/6, and 6/6/6/5 Ring Systems from Aspergillus spectabilis.

Spectasterols A-E (1-5), aromatic ergosterols with unique ring systems, were isolated from Aspergillus spectabilis. Compounds 1 and 2 possess a 6/6/6/5/5 ring system with an additional cyclopentene, while 3 and 4 have an uncommon 6/6/6/6 ring system generated by the D-ring expansion via 1,2-alkyl shifts. Compound 3 exhibited cytotoxic activity (IC50 6.9 µM) and induced cell cycle arrest and apoptosis in HL60 cells. Compound 3 was anti-inflammatory; it decreased COX-2 levels at the transcription and protein levels and inhibited the nuclear translocation of NF-κB p65.

Identification of optimal primary tumor resection candidates for metastatic gastric cancer: Nomograms based on propensity score matching.

BACKGROUND: This study sought to develop and validate nomograms for screening patients with metastatic gastric cancer (mGC) who are candidates for primary tumor resection (PTR) and evaluating the prognosis of mGC patients after PTR. METHODS: From 2010 to 2016, we screened mGC patients with complete data from the Surveillance, Epidemiology, and End Results (SEER) database. Depending on whether or not PTR was performed, we categorized patients into surgery and non-surgery groups. A 1:1 propensity score matching (PSM) analysis was used to balance the characteristics of the two groups. The endpoints were overall survival (OS) and cancer-specific survival (CSS). Two predictive nomograms were developed using logistic regression to assess the likelihood of benefit. Two additional prognostic nomograms were developed to assess prognosis in mGC patients after PTR by Cox regression. Finally, nomograms were evaluated using a variety of methodologies. RESULTS: Our study included 3594 mGC patients who met the criteria. PTR was associated with improved OS and CSS time (median OS time after PSM: 15 vs. 7 months, P < 0.05; median CSS time after PSM: 17 vs. 7 months, P < 0.05). The OS-related predictive nomogram, including age, histologic type, grade, T stage, and chemotherapy, was developed. Moreover, the CSS-related predictive nomogram, including age, histologic type, grade, and chemotherapy, was developed. Sex, histologic type, grade, T stage, N stage, and chemotherapy were found to be correlated with OS. Furthermore, the CSS correlated with histologic type, grade, T stage, N stage, and chemotherapy. Both predictive and prognostic nomograms were found to be valuable and reliable after different types of validation. CONCLUSION: Predictive nomograms were developed and validated for identifying the optimal PTR mGC candidates. Prognostic nomograms were developed and validated for assessing the prognosis of mGC patients after PTR.

ß-Elemene enhances erlotinib sensitivity through induction of ferroptosis by upregulating lncRNA H19 in EGFR-mutant non-small cell lung cancer.

Nearly half of all Asian non-small cell lung cancer (NSCLC) patients harbour epidermal growth factor receptor (EGFR) mutations, and first-generation EGFR tyrosine kinase inhibitors (TKIs) are one of the first-line treatments that have improved the outcomes of these patients. Unfortunately, 20% of these patients can not benefit from the treatment. The basis of this primary resistance is poorly understood. Therefore, overcoming EGFR-TKI primary resistance and maintaining the efficacy of TKIs has become a key issue. ß-Elemene, a sesquiterpene compound extracted from Curcuma aromatica Salisb. (wenyujing), has shown potent antitumor effects. In this research, we found that ß-elemene combined with erlotinib enhanced the cytotoxicity of erlotinib to primary EGFR-TKI-resistant NSCLC cells with EGFR mutations and that ferroptosis was involved in the antitumor effect of the combination treatment. We found that lncRNA H19 was significantly downregulated in primary EGFR-TKI-resistant NSCLC cell lines and was upregulated by the combination treatment. Overexpression or knockdown of H19 conferred sensitivity or resistance to erlotinib, respectively, in both in vitro and in vivo studies. The high level of H19 enhanced the cytotoxicity of erlotinib by inducing ferroptosis. In conclusion, our data showed that ß-elemene combined with erlotinib could enhance sensitivity to EGFR-TKIs through induction of ferroptosis via H19 in primary EGFR-TKI-resistant lung cancer, providing a promising strategy to overcome EGFR-TKI resistance in NSCLC patients.

Pharmacokinetics and Tissue Distribution of Nasal Spray of a Novel Muscarinic Receptor Blocker, 101BHG-D01, in Dogs and Rats.

BACKGROUND: 101BHG-D01 is a novel selective anti-muscarinic (M) 3 receptor-blocking drug. 101BHG-D01 nasal spray is intended to be used to relieve sneezing and runny nose symptoms caused by allergic rhinitis. METHODS: In this study, we examined the plasma pharmacokinetics, tissue distribution, and major excretion mode of 101BHG-D01 in Beagle dogs and rats following nasal spray and intranasal administration, respectively, using HPLC-MS/MS. RESULTS/DISCUSSION: We found that the pharmacokinetics of 101BHG-D01 was linear in dogs. 101BHG-D01 entered the bloodstream rapidly following nasal spray. Its plasma half-life was approximately 6 h and resided at least 24 h in the body. Moreover, 101BHG-D01 retained a significant amount in the nasal cavity. Finally, we found that 101BHG-D01 was eliminated mainly in the form of stools in rats. CONCLUSION: In conclusion, we provided pertinent reference information regarding the design and optimization of drug delivery regimens for clinical trials.

Anoikis-Associated Lung Cancer Metastasis: Mechanisms and Therapies.

Tumor metastasis occurs in lung cancer, resulting in tumor progression and therapy failure. Anoikis is a mechanism of apoptosis that combats tumor metastasis; it inhibits the escape of tumor cells from the native extracellular matrix to other organs. Deciphering the regulators and mechanisms of anoikis in cancer metastasis is urgently needed to treat lung cancer. Several natural and synthetic products exhibit the pro-anoikis potential in lung cancer cells and in vivo models. These products include artonin E, imperatorin, oroxylin A, lupalbigenin, sulforaphane, renieramycin M, avicequinone B, and carbenoxolone. This review summarizes the current understanding of the molecular mechanisms of anoikis regulation and relevant regulators involved in lung cancer metastasis and discusses the therapeutic potential of targeting anoikis in the treatment of lung cancer metastasis.

Gastric Mucosa Pathology in Rats with Precancerous Lesions of Gastric Cancer with Spleen Deficiency and Blood Stasis.

Objective: This research aimed at better understanding the histopathological development of precancerous lesions of gastric cancer (PLGC) and organelle ultrastructure changes. Methods: Sprague-Dawley rats were randomly assigned to the model and control groups. Model rats drank N-methyl-N'-nitro-N-nitrosoguanidine solution, while control rats drank pure water ad libitum. At 1, 3, 5, 6, and 8 months after the start of feeding, eight rats were randomly chosen from each group, and gastric mucosa tissues were removed for histopathological analysis. H&E staining was applied to analyze the pathological histological structure of the rat gastric mucosa via a light microscope, and the ultrastructural changes were observed via a transmission electron microscope. Results: Gastric mucosal pathologies of model rats such as mucosal atrophy, intestinal metaplasia, inflammatory lesions, and even intraepithelial neoplasia deteriorated over time. The endoplasmic reticulum gap widened, the mitochondrial endothelial cristae were disrupted, the nuclear membrane thickened, and chromatin condensed with heterotypic alterations in the main and parietal cells. Additionally, endothelial cell enlargement and thickening of the microvascular intima were seen. Conclusion: Our research showed that the PLGC progression of rats is correlated with the pathological alteration axis of "normal gastric mucosa-gastric mucosa inflammatory changes-intestinal metaplasia with mild dysplasia-moderate to severe dysplasia." Ultrastructure analysis of model rats is compatible with the structural changes in the gastric mucosa with spleen deficiency and blood stasis. The pathological evolutionary axis and ultrastructural analysis are helpful for evaluating potential novel herbal therapies for PLGC.

Enzymatic integrated in-situ advanced anaerobic digestion of sewage sludge for the removal of antibiotics and antibiotic resistance genes.

Antibiotics and antibiotic resistance genes (ARGs) in sewage sludge can cause high ecotoxicological risks in the environment and public health concerns. The aims of this study were to establish enzymatic integrated in-situ advanced anaerobic digestion (AAD) by adding cellulase and papain as well as the two enzymes combined with zero valent iron (ZVI) directly into the anaerobic digesters to explore the removal of antibiotics and ARGs under the mesophilic condition (35 °C). The methane production potential during in-situ AAD was effectively improved. Papain and cellulase at 30 mg/gTSS were most effective in improving antibiotic removal. The removal of sulfamerazine (SMZ) and sulfadiazine (SMR) could reach 89.10 % and 71.75 %. Combined enzymes with ZVI also enhanced the removal of all target antibiotics, especially roxithromycin (ROX), SMZ and SMR most significantly. Except for sul1, tetA and tetB, the removal of ARGs by papain reached 6.33 %-82.15 %. The addition of cellulase effectively improved tetA removal. The combination of biological enzymes further enhanced the removal of qnrS and ermX. The tetG, tetB, sul3, ermX, ermT, qnrS, and aac(6')-IB-CR by combined enzymes with ZVI could even not be detected after digestion. Addition of papain, cellulase, and ZVI caused variations in the dominant bacteria. All target antibiotics presented significant positive correlations with the genera norank_f__Bacteroidetes_vadinHA17, norank_f__norank_o__SJA-15, norank_f__norank_o__Aminicenantales. Redundancy analysis showed archaea Methanosaeta and Candidatus_ Methanoacidiosum genera greatly contributed to antibiotics removal with the combination of enzymes and ZVI. Co-occurrence network analysis indicated the removal of ARGs was mainly based on the changes of existence of host bacteria.