Structural properties of glucan from Russula griseocarnosa and its immunomodulatory activities mediated via T cell differentiation.

The polysaccharide, RGP2, was isolated from Russula griseocarnosa and its immunostimulatory effects were confirmed in cyclophosphamide (CTX)-induced immunosuppressed mice. Following purification via chromatography, structural analysis revealed that RGP2 had a molecular weight of 11.82 kDa and consisted of glucose (Glc), galactose (Gal), mannose, glucuronic acid and glucosamine. Bond structure analysis and nuclear magnetic resonance characterization confirmed that the main chain of RGP2 was formed by →6)-ß-D-Glcp-(1→, →3)-ß-D-Glcp-(1→ and →6)-α-D-Galp-(1→, which was substituted at O-3 of →6)-ß-D-Glcp-(1→ by ß-D-Glcp-(1→. RGP2 was found to ameliorate pathological damage in the spleen and enhance immune cell activity in immunosuppressed mice. Based on combined multiomics analysis, RGP2 altered the abundance of immune-related microbiota (such as Lactobacillus, Faecalibacterium, and Bacteroides) in the gut and metabolites (uridine, leucine, and tryptophan) in the serum. Compared with immunosuppressed mice, RGP2 also restored the function of antigen-presenting cells, promoted the polarization of macrophages into the M1 phenotype, positively affected the differentiation of helper T cells, and inhibited regulatory T cell differentiation through the protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway, ultimately exerting an immune boosting function. Overall, our findings highlight therapeutic strategies to alleviate CTX-induced immunosuppression in a clinical setting.

CD20 expression regulates CD37 levels in B-cell lymphoma - implications for immunotherapies.

Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies.

Preclinical evaluation of cyclophosphamide and fludarabine combined with CD19 CAR-T in the treatment of B-cell hematologic malignancies in vivo.

Background: Chimeric antigen receptor T (CAR-T) cell therapy has achieved marked therapeutic success in ameliorating hematological malignancies. However, there is an extant void in the clinical guidelines concerning the most effective chemotherapy regimen prior to chimeric antigen receptor T (CAR-T) cell therapy, as well as the optimal timing for CAR-T cell infusion post-chemotherapy. Materials and Methods: We employed cell-derived tumor xenograft (CDX) murine models to delineate the optimal pre-conditioning chemotherapy regimen and timing for CAR-T cell treatment. Furthermore, transcriptome sequencing was implemented to identify the therapeutic targets and elucidate the underlying mechanisms governing the treatment regimen. Results: Our preclinical in vivo evaluation determined that a combination of cyclophosphamide and fludarabine, followed by the infusion of CD19 CAR-T cells five days subsequent to the chemotherapy, exerts the most efficacious therapeutic effect in B-cell hematological malignancies. Concurrently, RNA-seq data indicated that the therapeutic efficacy predominantly perturbs tumor cell metabolism, primarily through the inhibition of key mitochondrial targets, such as C-Jun Kinase enzyme (C-JUN). Conclusion: In summary, the present study offers critical clinical guidance and serves as an authoritative reference for the deployment of CD19 CAR-T cell therapy in the treatment of B-cell hematological malignancies.

The STING inhibitor (ISD-017) reduces glomerulonephritis in 129.B6.Fcgr2b-deficient mice.

The absence of stimulator of interferon genes (STING) in 129.B6.Fcgr2b-deficient mice rescue lupus phenotypes. The administration of a STING inhibitor (ISD017) into the young 129.B6.Fcgr2b-deficient mice prevents lupus nephritis development. This study mainly aimed to evaluate the effects of STING inhibition (ISD107) on established SLE in mice to prove that ISD017 could be a good therapeutic drug to reverse the already set-up autoimmunity and kidney impairment. Twenty-four-week-old Fcgr2b-deficient mice were treated with cyclophosphamide (25 mg/kg, intraperitoneal, once per week), ISD017 (10 mg/kg, intraperitoneal, three times per week), or control vehicle for 8 weeks, and were analyzed for phenotypes. Both ISD017 and cyclophosphamide treatment increased long-term survival and reduced the severity of glomerulonephritis in Fcgr2b-deficient mice. While cyclophosphamide reduced activated B cells (B220+GL-7+), ISD017 decreased activated T cells (CD4+CD69+) and neutrophils (Ly6c+Ly6g+) in Fcgr2b-deficient mice. In addition, ISD017 reduced IL-1ß and interferon-inducible genes. In summary, ISD017 treatment in symptomatic 129.B6.Fcgr2b-deficient mice reduced the severity of glomerulonephritis and increased long-term survival. ISD017 worked comparably to cyclophosphamide for treating lupus nephritis in 129.B6.Fcgr2b-deficient mice. ISD017 reduced activated T cells and neutrophils, while cyclophosphamide targeted activated B cells. These results suggested that STING inhibitors can potentially be a new therapeutic drug for treating lupus.

Effect of Flavonols of Aronia melanocarpa Fruits on Morphofunctional State of Immunocompetent Organs of Rats under Cyclophosphamide-Induced Immunosuppression.

Aronia melanocarpa berries contain many compounds with potential benefits for human health. The food flavonoids quercetin and rutin, found in significant amounts in the fruits of A. melanocarpa, are known to have favourable effects on animal and human organisms. However, data on the effect of flavonols isolated from black chokeberry on immune functions during immunosuppression are not available in the literature. Thus, the aim of this study was to evaluate the effect of flavonol fraction isolated from A. melanocarpa fruits, in comparison with pure quercetin and rutin substances, on the dysfunctional state of rat thymus and spleen in immunodeficiency. The study was performed on Wistar rats. The animals were orally administered solutions of the investigated substances for 7 days: water, a mixture of quercetin and rutin and flavonol fraction of A. melanocarpa. For induction of immunosuppression, the animals were injected once intraperitoneally with cyclophosphamide. Substance administration was then continued for another 7 days. The results showed that under the influence of flavonols, there was a decrease in cyclophosphamide-mediated reaction of lipid peroxidation enhancement and stimulation of proliferation of lymphocytes of thymus and spleen in rats. At that, the effect of the flavonol fraction of aronia was more pronounced.

Cyclophosphamide induces ovarian granulosa cell ferroptosis via a mechanism associated with HO-1 and ROS-mediated mitochondrial dysfunction.

Abnormal granulosa cell (GC) death contributes to cyclophosphamide (CTX) induced primary ovarian insufficiency (POI). To investigate the contribution of GCs to POI, gene profiles of GCs exposed to CTX were assessed using RNA-Seq and bioinformatics analysis. The results showed the differentially expressed genes (DEGs) were enriched in the ferroptosis-related pathway, which is correlated with upregulated heme oxygenase 1 (HO-1) and downregulated glutathione peroxidase-4 (GPX4). Using CTX-induced cell culture (COV434 and KGN cells), the levels of iron, reactive oxygen species (ROS), lipid peroxide, mitochondrial superoxide, mitochondrial morphology and mitochondrial membrane potential (MMP) were detected by DCFDA, MitoSOX, C11-BODIPY, MitoTracker, Nonylacridine Orange (NAO), JC-1 and transmission electron microscopy respectively. The results showed iron overload and disrupted ROS, including cytoROS, mtROS and lipROS homeostasis, were associated with upregulation of HO-1 and could induce ferroptosis via mitochondrial dysfunction in CTX-induced GCs. Moreover, HO-1 inhibition could suppress ferroptosis induced GPX4 depletion. This implies a role for ROS in CTX-induced ferroptosis and highlights the effect of HO-1 modulators in improving CTX-induced ovarian damage, which may provide a theoretical basis for preventing or restoring GC and ovarian function in patients with POI.

Structural characterization and immune-enhancing effects of a novel polysaccharide extracted from Sargassum fusiforme.

To alleviate the adverse effects of chemotherapy and bolster immune function, a novel polysaccharide derived from Sargassum fusiforme named as SFP-αII. The structural composition of SFP-αII predominantly consisted of guluronic and mannuronic acids in a molar ratio of 33.8:66.2, with an average molecular weight of 16.5 kDa. Its structure was primarily characterized by →4)-α-GulA-(1 â†’ and →4)-ß-ManA-(1 â†’ linkages confirmed by FT-IR, methylation, and NMR analyses. The absence of a triple-helix structure was in SFP-αII was confirmed using circular dichroism and Congo red dye assays. The dimensions varied with lengths ranging from 20 nm up to 3 µm revealed by atomic force microscopy (AFM). SFP-αII has been found to enhance immunomodulatory activity in cyclophosphamide (CTX)-induced immunosuppressed mice. This was evidenced by improvements in immune organ indices, cytokine levels, and the release of nitric oxide (NO). Specifically, SFP-αII mitigated immunosuppression by upregulating the secretion of IL-1ß (167.3 %) and TNF-α (227.1 %) at a dose of 400 mg/kg, compared with the CTX group in macrophages. Ultimately, SFP-αII may serve as a mechanism for immune enhancement through modulation of TLR4-mediated NF-κB and MAPK signaling pathways. This integration of traditional Chinese and Western medicine, leveraging SFP-αII as a potential functional food could be pivotal in alleviating immunosuppressive side effects in CTX treatment.

An alkali-extracted neutral heteropolysaccharide from Phellinus nigricans used as an immunopotentiator in immunosuppressed mice by activating macrophages.

A neutral heteropolysaccharide (PNANb) was isolated with alkali (0.1 M NaOH) from mycelia of Phellinus nigricans, and the structure, immunostimulating activity and some of the underlying molecular mechanisms of action of PNANb were explored in the current study. PNANb (14.95 kDa) predominantly consisted of Gal, Glc, and Man with minor Fuc. GC-MS and NMR analyses indicated that the backbone of PNANb was mainly composed of 6-α-Galp, 2,6-α-Galp with minor 3,6-ß-Glcp, which was substituted with complex side chains at C-2 of 2,6-α-Galp and C-3 of 3,6-ß-Glcp. Notably, PNANb (50 or 100 mg/kg) possessed immunoprotective effects in cyclophosphamide (Cy)-induced immunosuppressed C57BL/6 mice, which was supported by evidence including the enhancement of spleen and thymus indices, levels of serum immunoglobulins (IgG, IgM) and cytokines (IFN-γ, IL-2, IL-4, IL-10), and macrophage activity. However, the immunostimulation effects of PNANb were decreased when macrophages were depleted, underscoring the essential role of macrophages in the beneficial effects of PNANb in Cy-induced immunosuppressed mice. Further investigations in vitro indicated that PNANb activated macrophages through MAPK/NF-κB signaling pathways mediated by Toll-like receptor 4. Therefore, PNANb can serve as a prospective immunopotentiator in immunosuppression.

Combination low-dose cyclophosphamide with check-point blockade and ionizing radiation promote an abscopal effect in mouse models of melanoma.

PURPOSE: The complex strategy of hypo-fractionated radiotherapy (HFRT) in combination with an immune checkpoint inhibitor (ICI) can stimulate a potential systemic antitumor response; however, the abscopal effect is always precluded by the tumor microenvironment, which may limit sufficient T-cell infiltration of distant nonirradiated tumors for certain kinds of inhibitory factors, such as regulatory T-cells (Tregs). Additionally, low-dose cyclophosphamide (LD-CYC) can specifically kill regulatory Tregs and strongly synergize antigen-specific immune responses, which could promote an abscopal effect. MATERIALS AND METHODS: We explored whether a triple regimen consisting of HFRT, ICI, and LD-CYC could achieve a better systemic antitumor response in bilateral mouse tumor models. RESULT: Our data demonstrate that LD-CYC combined with HFRT and antiprogrammed cell death ligand 1 (PDL-1) therapy could enhance the abscopal effect than only HFRT/antiPDL-1 or HFRT alone. Surprisingly, repeat CYC doses cannot further restrain tumor proliferation but can prolong murine overall survival, as revealed by the major pathologic responses. These results are associated with increased CD8 + effector T-cell infiltration, although LD-CYC did not upregulate PDL-1 expression in the tumor. CONCLUSIONS: Compared with traditional strategies, for the first time, we demonstrated that a triple treatment strategy remarkably increased the number of radiation-induced tumor-infiltrating CD8 + T-cells, effectively decreasing infiltrating Tregs, and promoting an abscopal effect. Thus, we describe a novel and effective therapeutic approach by combining multiple strategies to target several tumor-mediated immune inhibitory mechanisms.

Exploration of the underlying mechanism of Astragaloside III in attenuating immunosuppression via network pharmacology and vitro/vivo pharmacological validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus mongholicus Bunge (AM, recorded in http://www.worldfloraonline.org, 2023-08-03) is a kind of medicine food homology plant with a long medicinal history in China. Astragaloside III (AS-III) has immunomodulatory effects and is one of the most active components in AM. However, its underlying mechanism of action is still not fully explained. AIM OF THE STUDY: The research was designed to discuss the protective effects of AS-III on immunosuppression and to elucidate its prospective mechanism. MATERIALS AND METHODS: Molecular docking methods and network pharmacology analysis were used to comprehensively investigate potential targets and relative pathways for AS-III and immunosuppression. In order to study and verify the pharmacological activity and mechanism of AS-III in alleviating immunosuppression, immunosuppression mouse model induced by cyclophosphamide (CTX) in vivo and macrophage RAW264.7 cell model induced by hypoxia/lipopolysaccharide (LPS) in vitro were used. RESULTS: A total of 105 common targets were obtained from the AS-III-related and immunosuppression-related target networks. The results of network pharmacology and molecular docking demonstrate that AS-III may treat immunosuppression through by regulating glucose metabolism-related pathways such as regulation of lipolysis in adipocytes, carbohydrate digestion and absorption, cGMP-PKG signaling pathway, central carbon metabolism in cancer together with HIF-1 pathway. The results of molecular docking showed that AS-III has good binding relationship with LDHA, AKT1 and HIF1A. In CTX-induced immunosuppressive mouse model, AS-III had a significant protective effect on the reduction of body weight, immune organ index and hematological indices. It can also protect immune organs from damage. In addition, AS-III could significantly improve the expression of key proteins involved in energy metabolism and serum inflammatory factors. To further validate the animal results, an initial inflammatory/immune response model of macrophage RAW264.7 cells was constructed through hypoxia and LPS. AS-III improved the immune function of macrophages, reduced the release of NO, TNF-α, IL-1ß, PDHK-1, LDH, lactate, HK, PK and GLUT-1, and restored the decrease of ATP caused by hypoxia. Besides, AS-III was also demonstrated that it could inhibit the increase of HIF-1α, PDHK-1 and LDH by adding inhibitors and agonists. CONCLUSIONS: In this study, the main targets of AS-III for immunosuppressive therapy were initially analyzed. AS-III was systematically confirmed to attenuates immunosuppressive state through the HIF-1α/PDHK-1 pathway. These findings offer an experimental foundation for the use of AS-III as a potential candidate for the treatment of immunosuppression.

Modulating effects of heat-killed and live Limosilactobacillus reuteri PSC102 on the immune response and gut microbiota of cyclophosphamide-treated rats.

In the present study, we investigated the potential immunomodulatory effects of heat-killed (hLR) and live Limosilactobacillus reuteri PSC102 (LR; formerly Lactobacillus reuteri PSC102) in RAW264.7 macrophage cells and Sprague-Dawley rats. RAW264.7 murine macrophage cells were stimulated with hLR and LR for 24 h. Cyclophosphamide (CTX)-induced immunosuppressed Sprague-Dawley rats were orally administered with three doses of hLR (L-Low, M-Medium, and H-High) and LR for 3 weeks. The phagocytic capacity, production of nitric oxide (NO), and expression of cytokines in RAW264.7 cells were measured, and the different parameters of immunity in rats were determined. hLR and LR treatments promoted phagocytic activity and induced the production of NO and the expression of iNOS, TNF-α, IL-1ß, IL-6, and Cox-2 in macrophage cells. In the in vivo experiment, hLR and LR treatments significantly increased the immune organ indices, alleviated the spleen injury, and ameliorated the number of white blood cells, granulocytes, lymphocytes, and mid-range absolute counts in immunosuppressive rats. hLR and LR increased neutrophil migration and phagocytosis, splenocyte proliferation, and T lymphocyte subsets (CD4+, CD8+, CD45RA+, and CD28+). The levels of immune factors (IL-2, IL-4, IL-6, IL-10, IL-12A, TNF-α, and IFN-γ) in the hLR and LR groups were upregulated compared with those in the CTX-treatment group. hLR and LR treatments could also modulate the gut microbiota composition, thereby increasing the relative abundance of Bacteroidetes and Firmicutes but decreasing the level of Proteobacteria. hLR and LR protected against CTX-induced adverse reactions by modulating the immune response and gut microbiota composition. Therefore, they could be used as potential immunomodulatory agents.

Augmentation of NK-cell activity and immunity by combined natural polyphenols and saccharides in vitro and in vivo.

Curcuma longa and Sargassum coreanum are commonly used in traditional pharmaceutical medicine to improve immune function in chronic diseases. The present study was designed to systematically elucidate the in vitro and in vivo immuno-enhancing effects of a combination of C. longa and S. coreanum extracts (CS) that contain polyphenols and saccharides as functional molecules in a cyclophosphamide (Cy)-induced model of immunosuppression. In primary splenocytes, we observed the ameliorative effects of CS on a Cy-induced immunosuppression model with low cytotoxicity and an optimal mixture procedure. CS treatment enhanced T- and B-cell proliferation, increased splenic natural killer-cell activity, and restored cytokine release. Wistar rats were orally administered low (30 mg/kg), intermediate (100 mg/kg), or high (300 mg/kg) doses of CS for four weeks, followed by oral administration of Cy (5 mg/kg) for four weeks. Compared with the vehicle group, low-, intermediate-, and high-dose CS treatment accelerated dose-dependent recovery of the serum level of tumor necrosis factor-α, interferon-γ, interleukin-2, and interleukin-12. These results suggest that CS treatment accelerates the amelioration of immune deficiency in Cy-treated primary splenocytes and rats, which supports considering it for immunity maintenance. Our findings provide experimental evidence for further research and clinical application in immunosuppressed patients.

Noni enhances the anticancer activity of cyclophosphamide and suppresses myelotoxicity and hepatotoxicity in tumor-bearing mice.

BACKGROUND AND AIM: Morinda citrifolia fruit juice (noni) is an herbal remedy documented to have antioxidant properties. It has been suggested that prevention of carcinogen-DNA adduct formation and the antioxidant activity of NJ may contribute to the cancer preventive effect. In the present study, the antitumor activity of noni was investigated in the presence of cyclophosphamide (CYL) in vitro and in vivo. METHODS: In vitro breast cancer cells (MDA-MB-468) were used to measure the percentage of inhibition and the IC50. The in vivo antitumor activity of noni was studied by monitoring the mean survival time (MST), percentage increase in life span (%ILS), viable and non-viable cell count, tumor volume, body weight, and hematological and serum biochemical parameters in mice. Treatment with noni and CYL exhibited dose- and time-dependent cytotoxicity toward breast cancer cells. RESULTS: Individual treatment of noni and CYL exhibited dose- and time-dependent cytotoxicity on breast cancer cell lines, while in combination therapy of noni and CYL, noni enhances cytotoxic effect of CYL at 48 h than that at 24 h. Similar result was found in in vivo studies, the results of which revealed that alone treatment of CYL and noni suppressed tumor growth. However, combination treatment with CYL and noni presented better tumor inhibition than that of alone treatment of CYL and noni. On the contrary, CYL alone drastically attenuated hematological parameters, i.e., RBC, WBC, and Hb compared to normal and control groups, and this change was reversed and normalized by noni when given as combination therapy with CYL. Moreover, the levels of serum biochemical markers, i.e., AST, ALP, and ALT, were significantly increased in the control and CYL-treated groups than those in the normal group. In the combination treatment of noni and CYL, the above biochemical marker levels significantly decreased compared to CYL alone-treated group. CONCLUSIONS: The present study suggested that CYL treatment can cause serious myelotoxicity and hepatic injury in cancer patients. In conclusion, the combined use of noni with CYL potentially enhances the antitumor activity of CYL and suppresses myelotoxicity and hepatotoxicity induced by CYL in tumor-bearing mice.

The ameliorative effect on chemotherapy-induced injury and tumor immunosuppressive microenvironment of the polysaccharide from the rhizome of Menispermum dauricum DC.

Combined medication has attracted increasing attention as an important treatment option for tumors due to the serious adverse effects of chemotherapy. In this study, as a new therapy strategy, a combination treatment of MDP (a polysaccharide from the rhizome of Menispermum dauricum DC.) with cyclophosphamide (CTX) was investigated. The results showed that combination treatment with MDP and CTX exerted a significantly synergistic anti-tumor effect in Lewis tumor-bearing mice, improved CTX-induced emaciation and hair loss, as well as increased the number of leukocytes, erythrocytes, hemoglobin, and platelets in the peripheral blood. In addition, compared with CTX alone, the thymus index and spleen index of the MDP + CTX group were increased, the number of CD3 + T cells, CD8 + T cells, white blood cells and B cells in spleen also increased significantly. MDP could also ameliorate the increase in liver and kidney index caused by CTX. In the Lewis lung cancer model, MDP showed a certain degree of anti-tumor effects, which may be related to its promotion of tumor-associated macrophages (TAMs) to M1 phenotype polarisation, enhancement of the number of T cells in tumor tissues and promotion of Th cells in tumor tissues to Th1 phenotype polarisation, thus alleviating the immunosuppressive microenvironment in tumor tissues. This study laid the foundation for the development of MDP as a polysaccharide drug for the treatment or adjuvant therapy of tumors and has important significance for the further clinical application of polysaccharides.

Minnelide suppresses GVHD and enhances survival while maintaining GVT responses.

Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent antiinflammatory and antitumor activity in several preclinical models and has proven both safe and efficacious in clinical trials for advanced gastrointestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared with posttransplant cyclophosphamide (PTCy). Strikingly, we found Minnelide improved survival, weight loss, and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score, and colonic length. Notably, in addition to decreased donor T cell infiltration early after aHSCT, several regulatory cell populations, including Tregs, ILC2s, and myeloid-derived stem cells in the colon were increased, which together may account for Minnelide's GVHD suppression after aHSCT. Importantly, Minnelide's GVHD prevention was accompanied by preservation of graft-versus-tumor activity. As Minnelide possesses anti-acute myeloid leukemia (anti-AML) activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for patients with AML undergoing aHSCT.

Immunoenhancement effect of cinobufagin on macrophages and the cyclophosphamide-induced immunosuppression mouse model.

Cinobufagin (CBG) is a natural active substance. Although its various pharmacological activities have been explored, the immunomodulatory activity of CBG remains unexplored. Therefore, this study aimed to investigate the anti-inflammatory and immunomodulatory activities of CBG ex vivo and in vivo. The immunomodulatory activity of CBG was investigated in RAW 264.7 cells. CBG showed no significant toxicity to cells. Additionally, 0.5-8 µg/mL CBG significantly increased the phagocytosis ability of macrophages and the secretion levels of IL-1ß and TNF-α. Thus, it exerted immunomodulatory effects. We established the immunosuppressive model induced by cyclophosphamide (CTX) in mice and studied the immunomodulatory activity of CBG in vivo. The experimental results showed that the intervention of CBG alleviated the CTX-induced weight loss, restored the lymphocyte nuclear cell number, and promoted the secretion and mRNA expression of cytokines IFN-γ, IL-4, IL-6, and IL-12. Moreover, CBG increased the immune organ index, protected the growth of the spleen and thymus, and improved the pathological changes in immunosuppressed mice. Western blot results showed that different concentrations of CBG upregulated the phosphorylation level of PI3K/Akt/mTOR in the spleen of CTX-induced immunosuppressed mice. This suggests that the immunomodulatory effect of CBG may be related to the regulation of PI3K/Akt/mTOR signaling pathway. This study provides a theoretical basis for developing CBG immune enhancers and opens up new ideas for the comprehensive utilization and development of CBG in factories.

Paraprobiotic derived from Bacillus velezensis GV1 improves immune response and gut microbiota composition in cyclophosphamide-treated immunosuppressed mice.

Paraprobiotics that benefit human health have the capacity to modulate innate and adaptive immune systems. In this study, we prepared the paraprobiotic from Bacillus velezensis GV1 using the heat-killing method and investigated its effects on immunity and gut microbiota in vitro and in vivo. The morphology of inactivated strain GV1 was observed using scanning electron microscopy. Treatment with GV1 promoted nitric oxide production and augmented cytokine (IL-6, IL-1ß, and TNF-α) expression and secretion in RAW 264.7 macrophages. Moreover, the strain GV1 could alleviate cyclophosphamide monohydrate (CTX)-induced immunosuppression by reversing spleen damage and restoring the immune organ index, as well as by increasing the expression of immune-related cytokines (TNF-α, IL-1ß, IFN-γ, and IL-2) in the spleen and thymus, respectively. Furthermore, GV1 treatment dramatically healed the CTX-damaged colon and regulated gut microbiota by increasing the relative abundance of beneficial bacterial families (Lactobacillaceae, Akkermansiaceae, and Coriobacteriaceae) and decreasing that of harmful bacterial families (Desulfovibrionaceae, Erysipelotrichaceae, and Staphylococcaceae). Thus, the heat-killed GV1 can be considered a potential immunoregulatory agent for use as a functional food or immune-enhancing medicine.

Trifluoperazine mitigates cyclophosphamide-induced hepatic oxidative stress, inflammation, and apoptosis in mice by modulating the AKT/mTOR-driven autophagy and Nrf2/HO-1 signaling cascades.

AIM: This study aims to investigate the hepatoprotective effect of the antipsychotic drug trifluoperazine (TFP) against cyclophosphamide (CPA)-induced hepatic injury by exploring its effect on autophagy and the Nrf2/HO-1 signaling pathway. MAIN METHODS: The hepatotoxicity of CPA was assessed by biochemical analysis of the serum hepatotoxicity markers (ALT, AST, and direct bilirubin), histopathological examination, and ultrastructure analysis by transmission electron microscopy (TEM). The ELISA technique was used to assess the hepatic content of oxidative stress (MDA and SOD) and inflammatory markers (IL-1ß and TNF-α). Immunohistochemical assessment was used to investigate the hepatic expression of NF-κB, Nrf2, caspase-3, as well as autophagy flux markers (p62 and LC3B). The mRNA expression of HO-1 was assessed using RT-qPCR. Western blot assay was used to determine the expression of p-AKT and p-mTOR. KEY FINDINGS: TFP improved CPA-induced hepatotoxicity by reducing the elevated hepatotoxicity markers, and alleviating the histopathological changes with improving ultrastructure alterations. It also reduced oxidative stress by reducing MDA content and upregulating SOD activity. In addition, it exhibited anti-inflammatory and anti-apoptotic effects by decreasing NF-κB expression, IL-1ß, TNF-α levels, and caspase-3 expression. Furthermore, TFP-induced hepatoprotection was mediated by favoring Nrf2 expression and increasing the mRNA level of HO-1. As well, it improved autophagy by increasing LC3B expression concurrently with reducing p62 expression. Moreover, TFP modulated the AKT/mTOR pathway by reducing the expression of p-AKT and p-mTOR. SIGNIFICANCE: TFP significantly protected against CPA-induced hepatotoxicity by upregulating Nrf2/HO-1 signaling along with enhancement of protective autophagy via inhibition of the AKT/mTOR signaling pathway.

Rujifang inhibits triple-negative breast cancer growth via the PI3K/AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rujifang (RJF) constitutes a traditional Chinese medicinal compound extensively employed in the management of triple-negative breast cancer (TNBC). However, information regarding its potential active ingredients, antitumor effects, safety, and mechanism of action remains unreported. AIM OF THE STUDY: To investigate the efficacy and safety of RJF in the context of TNBC. MATERIALS AND METHODS: We employed the ultra high-performance liquid chromatography-electrospray four-pole time-of-flight mass spectrometry technique (UPLC/Q-TOF-MS/MS) to scrutinize the chemical constituents of RJF. Subcutaneously transplanted tumor models were utilized to assess the impact of RJF on TNBC in vivo. Thirty female BLAB/c mice were randomly divided into five groups: the model group, cyclophosphamide group, and RJF high-dose, medium-dose, and low-dose groups. A total of 1 × 106 4T1 cells were subcutaneously injected into the right shoulder of mice, and they were administered treatments for a span of 28 days. We conducted evaluations on blood parameters, encompassing white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), neutrophils, lymphocytes, and monocytes, as well as hepatorenal indicators including alkaline phosphatase (ALP), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), albumin, and creatinine (CRE) to gauge the safety of RJF. Ki67 and TUNEL were detected via immunohistochemistry and immunofluorescence, respectively. We prepared RJF drug-containing serum for TNBC cell lines and assessed the in vitro inhibitory effect of RJF on tumor cell growth through the CCK8 assay and cell cycle analysis. RT-PCR was employed to detect the mRNA expression of cyclin-dependent kinase and cyclin-dependent kinase inhibitors in tumor tissues, and Western blot was carried out to ascertain the expression of cyclin and pathway-related proteins. RESULTS: 100 compounds were identified in RJF, which consisted of 3 flavonoids, 24 glycosides, 18 alkaloids, 3 amino acids, 8 phenylpropanoids, 6 terpenes, 20 organic acids, and 18 other compounds. In animal experiments, both CTX and RJF exhibited substantial antitumor effects. RJF led to an increase in the number of neutrophils in peripheral blood, with no significant impact on other hematological indices. In contrast, CTX reduced red blood cell count, hemoglobin levels, and white blood cell count, while increasing platelet count. RJF exhibited no discernible influence on hepatorenal function, whereas Cyclophosphamide (CTX) decreased ALP, GOT, and GPT levels. Both CTX and RJF reduced the expression of Ki67 and heightened the occurrence of apoptosis in tumor tissue. RJF drug-containing serum hindered the viability of 4T1 and MD-MBA-231 cells in a time and concentration-dependent manner. In cell cycle experiments, RJF diminished the proportion of G2 phase cells and arrested the cell cycle at the S phase. RT-PCR analysis indicated that RJF down-regulated the mRNA expression of CDK2 and CDK4, while up-regulating that of P21 and P27 in tumor tissue. The trends in CDKs and CDKIs protein expression mirrored those of mRNA expression. Moreover, the PI3K/AKT pathway displayed downregulation in the tumor tissue of mice treated with RJF. CONCLUSION: RJF demonstrates effectiveness and safety in the context of TNBC. It exerts anti-tumor effects by arresting the cell cycle at the S phase through the PI3K-AKT pathway.

Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines.

Cyclophosphamide (CP) is an alkylating chemotherapeutic agent commonly used in cancer treatments. In this study, we aimed to investigate the effects of 4-Hydroperoxy cyclophosphamide (4-HC), which is active form of CP, on glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phospho-protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (p-PERK), phospho-inositol-requiring enzyme 1 alpha (p-IRE1α), eukaryotic translation initiation factor 2 alpha (eIF2α), and caspase-3 messenger ribonucleic acids (mRNAs) and proteins that play roles in the ER stress pathway and apoptosis in U87 and T98 human glioblastoma cell lines. U87 and T98 human glioblastoma cell lines were divided into control and 4-HC-treated groups. Cell viability assay was used to detect the half maximal inhibitory concentration (IC50) for 24 hours of 4-HC. Immunocytochemistry and quantitative polymerase chain reaction (qPCR) methods were used to evaluate the levels of proteins and their mRNAs. The IC50 values of U87 and T98 cells were calculated as 15.67±0.58 µM and 19.92±1 µM, respectively. The levels of GRP78, ATF6, p-PERK, p-IRE1α, eIF2α, and caspase-3 protein expressions in the 4-HC-treated group compared to that in the control group. These increased protein expressions also were correlated with the mRNA levels. The ER stress signal pathway could be active in 4-HC-induced cell death. Further studies of ER-related stress mechanisms in anticancer treatment would be important for effective therapeutic strategies.