RESUMEN
Acute kidney injury (AKI) is a common clinical condition associated with increased incidence and mortality rates. Hederasaponin C (HSC) is one of the main active components of Pulsatilla chinensis (Bunge) Regel. HSC possesses various pharmacological activities, including anti-inflammatory activity. However, the protective effect of HSC against lipopolysaccharide (LPS)-induced AKI in mice remains unclear. Therefore, we investigated the protective effect of HSC against LPS-induced renal inflammation and the underlying molecular mechanisms. Herein, using MTT and LDH assays to assess both cell viability and LDH activity; using dual staining techniques to identify different cell death patterns; conducting immunoblotting, QRT-PCR, and immunofluorescence analyses to evaluate levels of protein and mRNA expression; employing immunoblotting, molecular docking, SPR experiments, and CETSA to investigate the interaction between HSC and TLR4; and studying the anti-inflammatory effects of HSC in the LPS-induced AKI. The results indicate that HSC inhibits the expression of TLR4 and the activation of NF-κB and PIP2 signaling pathways, while simultaneously suppressing the activation of the NLRP3 inflammasome. In animal models, HSC ameliorated LPS-induced AKI and diminished inflammatory response and the level of renal injury markers. These findings suggest that HSC has potential as a therapeutic agent to mitigate sepsis-related AKI.
Asunto(s)
Lesión Renal Aguda , FN-kappa B , Saponinas , Animales , Ratones , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/tratamiento farmacológico , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Lipopolisacáridos/farmacología , Simulación del Acoplamiento Molecular , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal , Receptor Toll-Like 4/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Saponinas/farmacología , Saponinas/uso terapéutico , Fosfoinositido Fosfolipasa CRESUMEN
NF-κB and MAPK are classic inflammation signaling pathways which regulate inflammation signal transmission and induce the expression of many inflammatory factors. Based on the potent anti-inflammatory activity of benzofuran and its derivatives, several new heterocyclic/benzofuran hybrids were first designed and synthesized by molecular hybridization. Their structure was confirmed by 1H NMR, 13C NMR, HRMS or X-single crystal diffraction. The anti-inflammatory activity of these new compounds was screened by compounds; compound 5d exhibited an excellent inhibitory effect on the generation of NO (IC50 = 52.23 ± 0.97 µM), and low cytotoxicity (IC50 > 80 µM) against the RAW-264.7 cell lines. To further elucidate the possible anti-inflammatory mechanisms of compound 5d, the hallmark protein expressions of the NF-κB and MAPK pathways were studied in LPS-stimulated RAW264.7 cells. The results indicate that compound 5d not only significantly inhibits the phosphorylation levels of IKKα/IKKß, IKßα, P65, ERK, JNK and P38 in the classic MAPK/NF-κB signaling pathway in a dose-dependent manner, but also down-regulates the secretion of pro-inflammatory factors such as NO, COX-2, TNF-α and IL-6. Further, the in vivo anti-inflammatory activity of compound 5d indicated that it could regulate the involvement of neutrophils, leukocytes and lymphocytes in inflammation processes, and reduce the expression of IL-1ß, TNF-α and IL-6 in serum and tissues. These results strongly suggest that the piperazine/benzofuran hybrid 5d has a good potential for developing an anti-inflammatory lead compound, and the anti-inflammatory mechanism might be related to the NF-κB and MAPK signaling pathways.
Asunto(s)
Antiinflamatorios , Benzofuranos , Sistema de Señalización de MAP Quinasas , FN-kappa B , Animales , Ratones , Antiinflamatorios/química , Antiinflamatorios/farmacología , Benzofuranos/química , Benzofuranos/farmacología , Inflamación/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Células RAW 264.7 , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacologíaRESUMEN
Lung cancer is one of the leading causes of cancer death. Non-small-cell lung cancer (NSCLC) accounts for the majority of lung cancer diagnoses. Dihydrotanshinone (DHT) is a compound extract from Salvia miltiorrhiza, which has favorable anti-inflammatory and anti-cancer activities. However, the role of DHT in NSCLC has not been fully studied. The anti-cancer drugs used for treating lung cancer often lead to apoptosis; however, the drug resistance of apoptosis restricts the effect of these drugs. Oncosis is a passive form of cell death that is different from apoptosis. It is characterized by cell swelling, and Porimin is a specific marker for oncosis. In this study, the role of DHT in mediating oncosis in A549 cells was investigated. In vitro, the MTS assay was used to detect cell activity after DHT treatment. Microscopy and electron microscopy were used to observe cell morphology changes. Western blotting was used to detect protein expression. Flow cytometry was used to detect intracellular reactive oxygen species (ROS) level, calcium ion (Ca2+) level, and cell mortality. The intracellular Lactic dehydrogenase (LDH) level was detected by an LDH detection kit after DHT treatment. The ATP level was detected using an ATP detection kit. In vivo, Lewis lung cancer (LLC) xenograft mice were used to evaluate the anti-tumor effect of DHT. Hematoxylin and eosin (HE) staining was used to detect the pathology of lung cancer tumors. The detection of Porimin in the tumor tissues of the mice after DHT administration was assessed by immunohistochemistry (IHC). The results of this study showed that DHT treatment changed the cell morphology; destroyed the mitochondrial structure; increased the expression of Porimin; increased the levels of LDH, ROS, and Ca2+; decreased the mitochondrial membrane potential and ATP level; and played an anti-tumor role in vitro by mediating oncosis in A549 cells. The in vivo studies showed that DHT could effectively inhibit tumor growth. The results of protein detection and IHC detection in the tumor tissues showed that the expression of Porimin was increased and that oncosis occurred in the tumor tissues of mice. DHT triggered Porimin-dependent oncosis by ROS-mediated mitochondrial dysfunction in NSCLC. The in vivo studies showed that DHT could inhibit tumor growth in LLC xenograft mice by triggering oncosis. This study indicates the potential for DHT to treat NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Humanos , Ratones , Adenosina Trifosfato/metabolismo , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Neoplasias Pulmonares/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Pyroptosis, a type of programmed cell death (PCD), is characterized by cell swelling with bubbles, and the release of inflammatory cell cytokines. Cucurbitacin B (CuB), extracted from muskmelon pedicel, is a natural bioactive product that could effectively exert anti-tumor activities in lung cancer. However, the exact molecular mechanisms and the direct targets of CuB in non-small cell lung cancer (NSCLC) remain to be discovered. Here, we firstly found that CuB exerted an anti-tumor effect via pyroptosis in NSCLC cells and NSCLC mice models. Next, based on the molecular docking and cellular thermal shift assay (CETSA), we identified that CuB directly bound to Toll-like receptor 4 (TLR4) to activate the NLRP3 inflammasome, which further caused the separation of N- and C-terminals of Gasdermin D (GSDMD) to execute pyroptosis. Moreover, CuB enhanced the mitochondrial reactive oxygen species (ROS), mitochondrial membrane protein Tom20 accumulation, and cytosolic calcium (Ca2+) release, leading to pyroptosis in NSCLC cells. Silencing of TLR4 inhibited CuB-induced pyroptosis and decreased the level of ROS and Ca2+ in A549 cells. In vivo study showed that CuB treatment suppressed lung tumor growth in mice via pyroptosis without dose-dependent manner, and CuB at 0.75 mg/kg had a better anti-tumor effect compared to the Gefitinib group. Taken together, our findings revealed the mechanisms and targets of CuB triggering pyroptosis in NSCLC, thus supporting the notion of developing CuB as a promising therapeutic agent for NSCLC.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Inflamasomas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptosis/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Triterpenos/farmacología , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Transducción de Señal , Receptor Toll-Like 4/genética , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute lung injury (ALI) is a serious clinical disease. Rotundic acid (RA), a natural ingredient isolated from Ilex rotunda Thunb, exhibits multiple pharmacological activities. However, RA's therapeutic effect and mechanism on ALI remain to be elucidated. The present study aimed to further clarify its regulating effects on inflammation in vitro and in vivo. Our results indicated that RA significantly inhibited the overproduction of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). RA decreased ROS production and calcium influx. In addition, RA inhibited the activation of PI3K, MAPK, and NF-κB pathways and enhanced the activity of nuclear factor E2-related factor 2 (Nrf2) signaling. The cellular thermal shift assay and docking results indicated that RA bind to TLR4 to block TLR4 dimerization. Furthermore, RA pretreatment effectively inhibited ear edema induced by xylene and LPS-induced endotoxin death and had a protective effect on LPS-induced ALI. Our findings collectively indicated that RA has anti-inflammatory effects, which may serve as a potential therapeutic option for pulmonary inflammation.
Asunto(s)
Lesión Pulmonar Aguda , Antiinflamatorios , Receptor Toll-Like 4 , Triterpenos/farmacología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Citocinas/metabolismo , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Células RAW 264.7 , Transducción de Señal , Receptor Toll-Like 4/metabolismoRESUMEN
Dihydrotanshinone (DHT), one of the major ingredients of Salvia miltiorrhiza Bunge (Danshen), displays many bioactivities. However, the activity and underlying mechanism of DHT in anti-inflammation have not yet been elucidated. In this study, we investigated the anti-inflammatory activity and molecular mechanism of action of DHT both in vitro and in vivo. Our data showed that DHT significantly decreased the release of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1ß in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, THP-1 cells, and bone marrow-derived macrophages (BMDMs), and altered the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). In addition, flow cytometry results indicated that DHT reduced the calcium influx, and generation of reactive oxygen species (ROS), and nitric oxide (NO) generation in LPS-stimulated RAW264.7 cells. Moreover, DHT suppressed the transcription of nuclear factor-κB (NF-κB), the expressions of NF-κB proteins, and nuclear translocation of NF-κB/p65, thereby suggesting that the NF-κB pathway played a role in the anti-inflammatory action of DHT. In addition, DHT attenuated LPS-challenged activator protein-1 (AP-1) activity, resulting from interference of the mitogen-activated protein kinase (MAPK) pathway. The molecular docking simulation of DHT to toll-like receptor 4 (TLR4) suggested that DHT binds to the active sites of TLR4 to block TLR4 dimerization, which was further corroborated by cellular thermal shift assay and co-immunoprecipitation (Co-IP) experiments. Furthermore, the recruitment of myeloid differentiation primary response gene 88 (MyD88) and the expression of transforming growth factor-b (TGF-b)-activated kinase 1 (p-TAK1) were disturbed by the inhibition of TLR4 dimerization. Thus, investigating the molecular mechanism of DHT indicated that TLR4-MyD88-NF-κB/MAPK signaling cascades were involved in the anti-inflammatory activity of DHT in vitro. In in vivo mouse models, DHT significantly ameliorated LPS-challenged acute kidney injury, inhibited dimethylbenzene-induced mouse ear oedema, and rescued LPS-induced sepsis in mice. Taken together, our results indicated that DHT exhibited significant anti-inflammatory activity both in vitro and in vivo, suggesting that DHT may be a potential therapeutic agent for inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Fenantrenos/farmacología , Receptor Toll-Like 4/metabolismo , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/tratamiento farmacológico , Animales , Antiinflamatorios/uso terapéutico , Citocinas/genética , Citocinas/metabolismo , Dimerización , Edema/inducido químicamente , Edema/tratamiento farmacológico , Furanos , Células HEK293 , Humanos , Lipopolisacáridos , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fenantrenos/uso terapéutico , Quinonas , Células RAW 264.7 , Sepsis/inducido químicamente , Sepsis/tratamiento farmacológico , Células THP-1 , XilenosRESUMEN
Background: Indoleamine-2,3-dioxygenase 1 (IDO1) is responsible for tumor immune escape by regulating T cell-associated immune responses and promoting the activation of immunosuppressive. Given the vital role of IDO1 in immune response, further investigation on the regulation of IDO1 in tumors is needed. Methods: Herein, we used ELISA kit to detect the interferon-gamma (IFN-γ), Tryptophan (Trp), and kynurenic acid (Kyn) levels; western blot, Flow cytometry, and immunofluorescence assays detected the expression of the proteins; Molecular docking assay, SPR assay and Cellular Thermal Shift Assay (CETSA) were used to detect the interaction between IDO1 and Abrine; nano live label-free system was used to detect the phagocytosis activity; tumor xenografts animal experiments were used to explore the anti-tumor effect of Abrine; flow cytometry detected the immune cells changes. Results: The important immune and inflammatory response cytokine interferon-gamma (IFN-γ) up-regulated the IDO1 expression in cancer cells through the methylation of 6-methyladenosine (m6A) m6A modification of RNA, metabolism of Trp into Kyn, and JAK1/STAT1 signaling pathway, which could be inhibited by IDO1 inhibitor Abrine. CD47 is IFN-γ-stimulated genes (ISGs) and prevents the phagocytosis of macrophages, leading to the cancer immune escape, and this effect could be inhibited by Abrine both in vivo and in vitro. The PD-1/PD-L1 axis is an important immune checkpoint in regulating immune response, overexpression of PD-1 or PD-L1 promotes immune suppression, while in this study Abrine could inhibit the expression of PD-L1 in cancer cells or tumor tissue. The combination treatment of Abrine and anti-PD-1 antibody has a synergistic effect on suppressing the tumor growth through up-regulating CD4+ or CD8+ T cells, down-regulating the Foxp3+ Treg cells, and inhibiting the expression of IDO1, CD47, and PD-L1. Conclusion: Overall, this study reveals that Abrine as an IDO1 inhibitor has an inhibition effect on immune escape and has a synergistic effect with the anti-PD-1 antibody on the treatment of HCC.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Humanos , Antígeno B7-H1/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Antígeno CD47/metabolismo , Linfocitos T CD8-positivos , Inmunoterapia , Alcaloides Indólicos/metabolismo , Interferón gamma/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Simulación del Acoplamiento Molecular , Triptófano/metabolismoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bunge is a widely used traditional Chinese medicine with anticholinesterase, antitumor, and anti-inflammatory. Total Tanshinones (TTN), the most significant active ingredient of Salvia miltiorrhiza Bunge, exerts anti-inflammatory activity. However, the protective mechanism of total Tanshinones on acute lung injury (ALI) still needs to be explored. AIM OF THIS STUDY: In this study, the underlying mechanisms of TTN to treat with ALI were investigated in vitro and in vivo. MATERIALS AND METHODS: Cell experiments established an in vitro model of LPS-induced J774A.1 and MH-S macrophages to verify the mechanism. The levels of inflammatory cytokines (TNF-α, IL-6 and IL-1ß) were estimated by ELISA. The changes of ROS, Ca2+ and NO were detected by flow cytometry. The expression levels of proteins related to the NLRP3 inflammasome were determined by Western blotting. The effect of TTN on NLRP3 inflammasome activation was examined by immunofluorescence analysis of caspase-1 p20. Male BALB/c mice were selected to establish the ALI model. The experiment was randomly divided into six groups: control, LPS, LPS + si-NC, LPA + si-Nek7, LPS + TTN, and DEX. Pathological alterations were explored by H&E staining. The expression levels of proteins related to the NLRP3 inflammasome were analyzed by Western blotting. RESULTS: TTN decreased pro-inflammatory cytokines levels like TNF-α, IL-6, IL-1ß, NO, and ROS in alveolar macrophages. TTN bound to NIMA-related kinase 7 (NEK7), a new therapeutic protein to modulate NLRP3 inflammasome and PLCγ2-PIP2 signaling pathway. In ALI mice, LPS enhanced IL-1ß levels in the serum, lung tissues, and bronchoalveolar lavage fluid (BALF),which were reversed by TTN. TTN decreased cleaved-caspase-1 and NLRP3 expressions in lung tissues. When Nek7 was knocked down in mice by siRNA, the syndrome of ALI in mice was significantly suppressed, of which the effect was similar to that of TTN. CONCLUSIONS: This research demonstrates that TTN alleviated ALI by binding to NEK7 in vitro and in vivo to modulate NLRP3 inflammasome activation and PLCγ2-PIP2 signaling pathways.
Asunto(s)
Lesión Pulmonar Aguda , Inflamasomas , Masculino , Ratones , Animales , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Interleucina-6 , Lipopolisacáridos/farmacología , Fosfolipasa C gamma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/prevención & control , Transducción de Señal , Citocinas/farmacología , Antiinflamatorios/efectos adversos , Caspasas/metabolismo , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: The successful launch of icaritin, a therapeutic drug for liver cancer derived from Epimedium brevicornu, has provided new impetus for the development of prenylated flavonoids in the field of oncology. Flemingia macrophylla is reported to contain characteristic prenylated flavonoids which can regulate the p53 protein. We aimed to isolate these constituents and conduct activity evaluation, structure-activity relationship, and mechanism studies to provide candidate compounds for antitumor drug development. METHODS: In this study, chromatographic techniques combined with spectroscopic methods were used to separate, purify, and identify the constituents of Flemingia macrophylla methanol extract. The cytotoxic activity of the constituents was evaluated using an MTT assay with A549 and H1975 cells as the model. The binding mechanism between the compounds and the p53 protein was investigated with molecular docking and validated with cellular thermal shift assay (CETSA). Western blotting (WB) was employed to detect the expression of p53 protein and apoptosis-related proteins in cells. RESULTS: Chiral HPLC separation of racemates 1 and 7 provided two pairs of undescribed enantiomers (1a/1b and 7a/7b), along with eight known compounds (2 - 9) isolated from Flemingia macrophylla roots. Their structures were elucidated by spectroscopic analysis, and the absolute configurations of the enantiomers were determined from experimental and calculated electronic circular dichroism data. Compounds 1 - 7, and the non-prenyl analogues 10 - 13, were evaluated for cytotoxic activity against the human lung cancer A549 and H1975 cell line. Compounds 5 - 7 displayed better cytotoxicity than the positive control icaritin in A549 and H1975, with IC50 values ranging from 4.50 to 19.83 µmol·L-1 and < 5 µmol·L-1, respectively. The structure-activity relationships of the chromone or flavonoid analogues against A549 cells were discussed. Molecular docking results demonstrated that compound 7a has strong interaction with p53 and WB indicated that 7a induced apoptosis by increasing the p53 protein, decreasing the anti-apoptotic protein Bcl-2, and activating the caspase family in A549 cells. These results suggest that prenylated flavonoids are potential p53 protein activators. CONCLUSION: This study demonstrates that Flemingia macrophylla is rich in prenylated flavonoid constituents, among which compounds 5 and 7 exhibited significant cytotoxic activity against A549 cells and served as reference candidates for the design and development of prenylated compounds as antitumor therapeutic drugs.
RESUMEN
BACKGROUND: Lung cancer is the primary cause of cancer-related mortality worldwide owing to its strong metastatic ability. EGFR-TKI (Gefitinib) has demonstrated efficacy in metastatic lung cancer therapy, but most patients ultimately develop resistance to Gefitinib, leading to a poor prognosis. Pedunculoside (PE), a triterpene saponin extracted from Ilex rotunda Thunb., has shown anti-inflammatory, lipid-lowering and anti-tumor effects. Nevertheless, the therapeutic effect and potential mechanisms of PE on NSCLC treatment are unclear. PURPOSE: To investigate the inhibitory effect and prospective mechanisms of PE on NSCLC metastases and Gefitinib-resistant NSCLC. METHODS: In vitro, A549/GR cells were established by Gefitinib persistent induction of A549 cells with a low dose and shock with a high dose. The cell migratory ability was measured using wound healing and Transwell assays. Additionally, EMT-related Markers or ROS production were assessed by RT-qPCR, immunofluorescence, Western blotting, and flow cytometry assays in A549/GR and TGF-ß1-induced A549 cells. In vivo, B16-F10 cells were intravenously injected into mice, and the effect of PE on tumor metastases were determined using hematoxylin-eosin staining, Caliper IVIS Lumina, DCFH2-DA staining, and western blotting assays. RESULTS: PE reversed TGF-ß1-induced EMT by downregulating EMT-related protein expression through MAPK and Nrf2 pathways, decreasing ROS production, and inhibiting cell migration and invasion ability. Moreover, PE treatment enabled A549/GR cells to retrieve the sensitivity to Gefitinib and mitigate the biological characteristics of EMT. PE also significantly inhibited lung metastasis in mice by reversing EMT proteins expression, decreasing ROS production, and inhibiting MAPK and Nrf2 pathways. CONCLUSIONS: Collectively, this research presents a novel finding that PE can reverse NSCLC metastasis and improve Gefitinib sensitivity in Gefitinib-resistant NSCLC through the MAPK and Nrf2 pathways, subsequently suppressing lung metastasis in B16-F10 lung metastatic mice model. Our findings indicate that PE is a potential agent for inhibiting metastasis and improving Gefitinib resistance in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Triterpenos , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/patología , Gefitinib/farmacología , Neoplasias Pulmonares/patología , Factor de Crecimiento Transformador beta1/farmacología , Factor 2 Relacionado con NF-E2 , Transición Epitelial-Mesenquimal , Especies Reactivas de Oxígeno , Línea Celular Tumoral , Triterpenos/farmacología , Resistencia a AntineoplásicosRESUMEN
Background: Necroptosis, a novel form of programmed cell death wherein the necrotic morphology is characterized by swelling of the cells, rupture of the plasma membrane, and dysfunction of the organelle, has been always observed in cardiovascular diseases. Sugarcane leaf polysaccharide (SLP) are primary components present in sugarcane leaves that exert cardiovascular protective effects. However, the positive effect of SLP and underlying mechanisms in myocardial ischemia-reperfusion (MI/R) remain unexplored. Aim: In this study, the protective effects of SLP on MI/R injury were investigated under in vitro and in vivo conditions. Methods: The protective effects of SLP on MI/R injury were assessed using tertiary butyl hydrogen peroxide (TBHP)-stimulated-H9c2 cells in the in vitro assay and using Sprague Dawley rats in the in vivo assay. Results: In vitro, SLP significantly reversed TBHP-induced H9c2 cell death by inhibiting necroptosis and oxidative stress. SLP exerted antioxidant activity through the Nrf2/HO-1 pathway. SLP suppressed necroptosis by decreasing phosphorylation of RIP1, RIP3, and MLKL in TBHP-stimulated H9c2 cells. In vivo, SLP attenuated MI/R injury by decreasing the myocardial infarct area; increasing myeloperoxidase and superoxide dismutase levels; and reducing malondialdehyde, interleukin-6, and tumor necrosis factor-α levels.
RESUMEN
BACKGROUND: Necroptosis is a type of programmed necrosis mediated by receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3), which is morphologically characterized by enlarged organelles, ruptured plasma membrane, and subsequent loss of intracellular contents. Cryptotanshinone (CPT), a diterpene quinone compound extracted from the root of Salvia miltiorrhiza Bunge, has been reported to have significant anticancer activities. However, the detailed mechanism of CPT has not been clearly illustrated. OBJECTIVE: The present study aimed to explore the cell death type and mechanisms of CPT-induced in non-small cell lung cancer (NSCLC) cells. METHODS: The cytotoxicity of CPT on A549 cells was assessed by MTS assay. Ca2+ release and reactive oxygen species (ROS) generation were detected by flow cytometry. The changes in mitochondrial membrane potential (MMP) were observed through JC-1 staining. The expressions of p- RIP1, p-RIP3, p-MLKL, and MAPKs pathway proteins were analyzed by western blotting analysis. The efficacy of CPT in vivo was evaluated by the Lewis lung carcinoma (LLC) xenograft mice model. Blood samples were collected for hematology analysis. ELISA investigated the effects of CPT on tumor necrosis factor α (TNF-α). Hematoxylin and eosin staining (HE) determined the tumor tissues. Proteins' expression of tumor tissues was quantified by western blotting. RESULTS: CPT inhibited the cell viability of A549 cells in a time- and concentration-dependent manner, which was reversed by Necrostatin-1 (Nec-1). In addition, CPT treatment increased the expression of p-RIP1, p-RIP3, p-MLKL, the release of Ca2+, ROS generation, and the MAPKs pathway activated in A549 cells. Moreover, animal experiment results showed that intraperitoneal injection of CPT (15 mg/kg and 30 mg/kg) significantly inhibited tumor growth in C57BL/6 mice without affecting the bodyweight and injuring the organs. CONCLUSION: Our findings suggested that CPT-induced necroptosis via RIP1/RIP3/MLKL signaling pathway both in vitro and in vivo, indicating that CPT may be a promising agent in the treatment of NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Necroptosis , Fenantrenos , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
Pneumonia, such as acute lung injury (ALI), has been a type of lethal disease that is generally caused by uncontrolled inflammatory response and excessive generation of reactive oxygen species (ROS). Herein, we report Fe-curcumin-based nanoparticles (Fe-Cur NPs) with nanozyme functionalities in guiding the intracellular ROS scavenging and meanwhile exhibiting anti-inflammation efficacy for curing ALI. The nanoparticles are noncytotoxic when directing these biological activities. Mechanism studies for the anti-inflammation aspects of Fe-Cur NPs were systematically carried out, in which the infected cells and tissues were alleviated through downregulating levels of several important inflammatory cytokines (such as TNF-α, IL-1ß, and IL-6), decreasing the intracellular Ca2+ release, inhibiting NLRP3 inflammasomes, and suppressing NF-κB signaling pathways. In addition, we performed both the intratracheal and intravenous injection of Fe-Cur NPs in mice experiencing ALI and, importantly, found that the accumulation of such nanozymes was enhanced in lung tissue (better than free curcumin drugs), demonstrating its promising therapeutic efficiency in two different administration methods. We showed that the inflammation reduction of Fe-Cur NPs was effective in animal experiments and that ROS scavenging was also effectively achieved in lung tissue. Finally, we revealed that Fe-Cur NPs can decrease the level of macrophage cells (CD11bloF4/80hi) and CD3+CD45+ T cells in mice, which could help suppress the inflammation cytokine storm caused by ALI. Overall, this work has developed the strategy of using Fe-Cur NPs as nanozymes to scavenge intracellular ROS and as an anti-inflammation nanodrugs to synergistically cure ALI, which may serve as a promising therapeutic agent in the clinical treatment of this deadly disease. Fe-Cur NP nanozymes were designed to attenuate ALI by clearing intracellular ROS and alleviating inflammation synergistically. Relevant cytokines, inflammasomes, and signaling pathways were studied.
RESUMEN
Gene transcription is governed by epigenetic regulation that is essential for the pro-inflammatory mediators surge following pathological triggers. Acute lung injury (ALI) is driven by pro-inflammatory cytokines produced by the innate immune system, which involves the nod-like receptor 3 (NLRP3) inflammasome and nuclear factor-κB (NF-κB) pathways. These two pathways are interconnected and share a common inducer the phosphatidylinositol 4,5-bisphosphate (PIP2), an epigenetic regulator of (Ribosomal ribonucleic acid (rRNA) gene transcription, to regulate inflammation by the direct inhibition of NF-κB phosphorylation and NLRP3 inflammasome activation. Herein, we report that hederasaponin C (HSC) exerted a therapeutic effect against ALI through the regulation of the PIP2/NF-κB/NLRP3 signaling pathway. In lipopolysaccharide (LPS)/lipopolysaccharide + adenosine triphosphate (LPS+ATP)-stimulated macrophages, our results showed that HSC remarkably inhibited the secretion of interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α (TNF-α). Moreover, HSC inhibited NF-κB/p65 nuclear translocation and the binding of PIP2 to transforming growth factor-ß activated kinase 1 (TAK1). The intracellular calcium (Ca2+) level was decreased by HSC via the PIP2 signaling pathway, which subsequently inhibited the activation of NLRP3 inflammasome. HSC markedly alleviated LPS-induced ALI, restored lung function of mice, and rescued ALI-induced mice death. In addition, HSC significantly reduced the level of white blood cells (WBC), neutrophils, and lymphocytes, as well as pro-inflammatory mediators like IL-6, IL-1ß, and TNF-α. Hematoxylin and eosin (H&E) staining results suggested HSC has a significant therapeutic effect on lung injury of mice. Interestingly, the PIP2/NF-κB/NLRP3 signaling pathway was further confirmed by the treatment of HSC with ALI, which is consistent with the treatment of HSC with LPS/LPS+ATP-stimulated macrophages. Overall, our findings revealed that HSC demonstrated significant anti-inflammatory activity through modulating the PIP2/NF-κB/NLRP3 axis in vitro and in vivo, suggesting that HSC is a potential therapeutic agent for the clinical treatment of ALI.
Asunto(s)
Lesión Pulmonar Aguda , Lipopolisacáridos , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/genética , Adenosina Trifosfato , Animales , Epigénesis Genética , Inflamasomas/metabolismo , Mediadores de Inflamación/uso terapéutico , Interleucina-6/metabolismo , Lipopolisacáridos/toxicidad , Ratones , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Lung cancer is the leading cause of cancer mortality worldwide, and most of the patients after treatment with EGF-TKIs develop drug resistance, which is closely correlated with EMT. Cucurbitacin B (CuB) is a natural product of the Chinese herb Cucurbitaceae plant, which has a favorable role in anti-inflammation and anti-cancer activities. However, the effect of CuB on EMT is still far from fully explored. In this study, the inhibition effect of CuB on EMT was investigated. METHODS: In this study, TGF-ß1 was used to induce EMT in A549 cells. MTS assay was used to detect the cell viability of CuB co-treated with TGF-ß1. Wound healing assay and transwell assay were used to determine the migration and invasion capacity of cells. Flow cytometry and fluorescence microscope were used to detect the ROS level in cells. Western blotting assay and immunofluorescence assay were used to detect the proteins expression. Gefitinib was used to establish EGF-TKI resistant NSCLC cells. B16-F10 intravenous injection mice model was used to evaluate the effect of CuB on lung cancer metastasis in vivo. Caliper IVIS Lumina and HE staining were used to detect the lung cancer metastasis of mice. RESULTS: In this study, the results indicated that CuB inhibited TGF-ß1-induced EMT in A549 cells through reversing the cell morphology changes of EMT, increasing the protein expression of E-cadherin, decreasing the proteins expression of N-cadherin and Vimentin, suppressing the migration and invasion ability. CuB also decreased the ROS production and p-PI3K, p-Akt and p-mTOR expression in TGF-ß1-induced EMT in A549 cells. Furthermore, Gefitinib resistant A549 cells (A549-GR) were well established, which has the EMT characteristics, and CuB could inhibit the EMT in A549-GR cells through ROS and PI3K/Akt/mTOR pathways. In vivo study showed that CuB inhibited the lung cancer metastasis effectively through intratracheal administration. CONCLUSION: CuB inhibits EMT in TGF-ß1-induced A549 cells and Gefitinib resistant A549 cells through decreasing ROS production and PI3K/Akt/mTOR signaling pathway. In vivo study validated that CuB inhibits lung cancer metastasis in mice. The study may be supporting CuB as a promising therapeutic agent for NSCLC and Gefitinib resistant NSCLC.
RESUMEN
Acute kidney injury (AKI) is a common critical illness that involves multiple systems and multiple organs with a rapid decline in kidney function over short period. It has a high mortality rate and presents a great treatment challenge for physicians. Oleuropein, the main active constituent of Ilex pubescens Hook. et Arn. var. kwangsiensis Hand.-Mazz. displays significant anti-inflammatory activity, although oleuropein's therapeutic effect and mechanism of action in AKI remain to be elucidated. The present study aimed to further clarify the mechanism by which oleuropein exerts effects on inflammation in vitro and in vivo. In vitro, the inflammatory effect and mechanism were investigated through ELISA, Western blotting, the thermal shift assay, co-immunoprecipitation, and immunofluorescence staining. Lipopolysaccharide (LPS) induced acute kidney injury was employed in an animal model to investigate oleuropein's therapeutic effect on AKI and mechanism in vivo. The underlying mechanisms were investigated by Western blot analysis of kidney tissue. In LPS-stimulated macrophages, our data demonstrated that oleuropein significantly reduced the expression of inflammatory mediators like NO, IL-6, TNF-α, iNOS, and COX-2. Moreover, oleuropein inhibited NF-κB/p65 translocation, and had a negative regulatory effect on key proteins in the NF-κB and MAPK pathways. In addition, the thermal shift and co-immunoprecipitation assays revealed that oleuropein played an essential role in binding to the active sites of TLR4, as well as inhibiting TLR4 dimerization and suppressing the binding of TLR4 to MyD88. Oleuropein markedly alleviated LPS induced acute kidney injury, decreased serum creatinine and blood urea nitrogen (BUN) levels and proinflammatory cytokines. More importantly, the TLR4-MyD88-NF-κB/MAPK pathways were confirmed to play an important role in the oleuropein treatment of AKI. In this study, oleuropein exhibited excellent anti-inflammatory effects by regulating TLR4-MyD88-NF-κB/MAPK axis in vitro and in vivo, suggesting oleuropein as a candidate molecule for treating AKI.
RESUMEN
BACKGROUND: Tanshinone I (TI) is a primary component of Salvia miltiorrhiza Bunge (Danshen), which confers a favorable role in a variety of pharmacological activities including cardiovascular protection. However, the exact mechanism of the cardiovascular protection activity of TI remains to be illustrated. In this study, the cardiovascular protective effect and its mechanism of TI were investigated. METHODS: In this study, tert-butyl hydroperoxide (t-BHP)-stimulated H9c2 cells model was employed to investigate the protective effect in vitro. The cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) kit. The reactive-oxygen-species (ROS) level and mitochondrial membrane potential (MMP) were investigated by the flow cytometry and JC-1 assay, respectively. While in vivo experiment, the cardiovascular protective effect of TI was determined by using myocardial ischemia-reperfusion (MI/R) model including hematoxylin-eosin (H&E) staining assay and determination of superoxide dismutase (SOD) and malondialdehyde (MDA). Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release were detected by Enzyme-linked immunosorbent assay (ELISA). Receptor interacting protein kinase 1 (RIP1), receptor interacting protein kinase 3 (RIP3), receptor interacting protein kinase 3 (MLKL), protein kinase B (Akt), Nuclear factor erythroid 2 related factor 2 (Nrf2), Heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO-1) were determined by western blotting. RESULTS: Our data demonstrated that TI pretreatment attenuated t-BHP and MI/R injury-induced necroptosis by inhibiting the expression of p-RIP1, p-RIP3, and p-MLKL. TI activated the Akt/Nrf2 pathway to promote the expression of antioxidant-related proteins such as phosphorylation of Akt, nuclear factor erythroid 2 related factor 2 (Nrf2), quinone oxidoreductase-1 (NQO-1) and heme oxygenase-1 (HO-1) expression in t-BHP-stimulated H9c2 cells. TI relieved oxidative stress by mitigating ROS generation and reversing MMP loss. In vivo experiment, TI made electrocardiograph (ECG) recovery better and lessened the degree of myocardial tissue damage. The counts of white blood cell (WBC), neutrophil (Neu), lymphocyte (Lym), and the release of TNF-α and IL-6 were reversed by TI treatment. SOD level was increased, while MDA level was decreased by TI treatment. CONCLUSION: Collectively, our findings indicated that TI exerted cardiovascular protective activities in vitro and in vivo through suppressing RIP1/RIP3/MLKL and activating Akt/Nrf2 signaling pathways, which could be developed into a cardiovascular protective agent.
RESUMEN
BACKGROUND: Nuezhenide (NZD), an iridoid glycoside isolated from Ilex pubescens Hook. & Arn. var. kwangsiensis Hand.-Mazz., used as a traditional Chinese medicine for clearing away heat and toxic materials, displays a variety of biological activities such as anti-tumor, antioxidant, and other life-protecting activities. However, a few studies involving anti-inflammatory activity and the mechanism of NZD have also been reported. In the present study, the anti-inflammatory and antioxidative effects of NZD are illustrated. OBJECTIVE: This study aims to test the hypothesis that NZD suppresses LPS-induced inflammation by targeting the NF-κB pathway in RAW264.7 cells. METHODS: LPS-stimulated RAW264.7 cells were employed to detect the effect of NZD on the release of cytokines by ELISA. Protein expression levels of related molecular markers were quantitated by western blot analysis. The levels of ROS, NO, and Ca2+ were detected by flow cytometry. The changes in mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were observed and verified by fluorescence microscopy. Using immunofluorescence assay, the translocation of NF-κB/p65 from the cytoplasm into the nucleus was determined by confocal microscopy. RESULTS: NZD exhibited anti-inflammatory activity and reduced the release of inflammatory cytokines such as nitrite, TNF-α, and IL-6. NZD suppressed the expression of the phosphorylated proteins like IKKα/ß, IκBα, and p65. Besides, the flow cytometry results indicated that NZD inhibited the levels of ROS, NO, and Ca2+ in LPS-stimulated RAW264.7 cells. JC-1 assay data showed that NZD reversed LPS-induced MMP loss. Furthermore, NZD suppressed LPS-induced NF-B/p65 translocation from the cytoplasm into the nucleus. CONCLUSION: NZD exhibits anti-inflammatory effects through the NF-κB pathway on RAW264.7 cells.
Asunto(s)
Antiinflamatorios/química , Glucósidos/química , Piranos/química , Factor de Transcripción ReIA/metabolismo , Animales , Antiinflamatorios/farmacología , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Glucósidos/farmacología , Humanos , Interleucina-6/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Inhibidor NF-kappaB alfa/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Piranos/farmacología , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Acute lung injury (ALI) is an acute inflammatory process in the lung parenchyma. Anemoside B4 (B4) was isolated from Pulsatilla, a plant-based drug against inflammation and commonly applied in traditional Chinese medicine. However, the anti-inflammatory effect and the mechanisms of B4 are not clear. In this study, we explored the potential mechanisms and anti-inflammatory activity of B4 both in vitro and in vivo. The results indicated that B4 suppressed the expression of iNOS, COX-2, NLRP3, caspase-1, and IL-1ß. The ELISA assay results showed that B4 significantly restrained the release of inflammatory cytokines like TNF-α, IL-6, and IL-1ß in macrophage cells. In addition, B4 rescued mitochondrial membrane potential (MMP) loss in (lipopolysaccharide) LPS plus ATP stimulated macrophage cells. Co-IP and molecular docking results illustrated that B4 disrupted the dimerization of TLR4. For in vivo results, B4 exhibited a protective effect on LPS and bleomycin- (BLM-) induced ALI in mice through suppressing the lesions of lung tissues, the release of inflammatory cytokines, and the levels of white blood cells, neutrophils, and lymphoid cells in the blood. Collectively, B4 has a protective effect on ALI via blocking TLR4 dimerization and NLRP3 inflammasome activation, suggesting that B4 is a potential agent for the treatment of ALI.
Asunto(s)
Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Sustancias Protectoras/farmacología , Multimerización de Proteína/efectos de los fármacos , Saponinas/farmacología , Receptor Toll-Like 4/metabolismo , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Adenosina Trifosfato , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Sustancias Protectoras/química , Saponinas/química , Relación Estructura-Actividad , Receptor Toll-Like 4/químicaRESUMEN
BACKGROUND: Pneumonia refers to the inflammation of the terminal airway, alveoli and pulmonary interstitium, which can be caused by pathogenic microorganisms, physical and chemical factors, immune damage, and drugs. Anemoside B4, the major ingredient of Pulsatilla chinensis (Bunge) Regel, exhibited anti-inflammatory activity. However, the therapeutic effect of anemoside B4 on pneumonia has not been unraveled. This study aims to investigate that anemoside B4 attenuates the inflammatory responses in Klebsiella pneumonia (KP)- and influenza virus FM1 (FM1)-induced pneumonia mice model. METHODS: The network pharmacology and molecular docking assays were employed to predict the targets of anemoside B4's treatment of pneumonia. Two models (bacterial KP-infected mice and virus FM1-infected mice) were employed in our study. BALB/c mice were divided into six groups: control, model group (KP-induced pneumonia or FM1-induced pneumonia), anemoside B4 (B4)-treated group (2.5, 5, 10 mg/kg), and positive drug group (ribavirin or ceftriaxone sodium injection). Blood samples were collected for hematology analysis. The effects of B4 on inflammation-associated mediators were investigated by Enzyme-linked immunosorbent assay (ELISA) and hematoxylin and eosin staining (HE) staining. Proteins expression was quantified by western blotting. RESULTS: The network results indicated that many pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) participated in anemoside B4's anti-inflammatory activity. The counts of neutrophil (NEU) and white blood cell (WBC), the level of myeloperoxidase (MPO), and the release of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 increased by KP or FM1 infection, which were reversed by anemoside B4. In addition, anemoside B4 significantly suppressed the FM1-induced expression of toll-like receptor 4 (TLR4), myeloid differential protein-88 (MyD88), and myeloid differentiation protein-2 (MD-2), which were further validated by molecular docking data that anemoside B4 bound to bioactive sites of TLR4. Therefore, anemoside B4 exhibited a significant therapeutic effect on pneumonia via the TLR4/MyD88 pathway. CONCLUSION: Our findings demonstrated that anemoside B4 attenuates pneumonia via the TLR4/Myd88 signaling pathway, suggesting that anemoside B4 is a promising therapeutic candidate for bacterial-infected or viral-infected pneumonia.