OBJECTIVE: To examine the therapeutic effect of Fangji Fuling Decoction (FFD) on sepsis through network pharmacological analysis combined with in vitro and in vivo experiments. METHODS: A sepsis mouse model was constructed through intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS). RAW264.7 cells were stimulated by 250 ng/mL LPS to establish an in vitro cell model. Network pharmacology analysis identified the key molecular pathway associated with FFD in sepsis. Through ectopic expression and depletion experiments, the effect of FFD on multiple organ damage in septic mice, as well as on cell proliferation and apoptosis in relation to the mitogen-activated protein kinase 14/Forkhead Box O 3A (MAPK14/FOXO3A) signaling pathway, was analyzed. RESULTS: FFD reduced organ damage and inflammation in LPS-induced septic mice and suppressed LPS-induced macrophage apoptosis and inflammation in vitro (P<0.05). Network pharmacology analysis showed that FFD could regulate the MAPK14/FOXO signaling pathway during sepsis. As confirmed by in vitro cell experiments, FFD inhibited the MAPK14 signaling pathway or FOXO3A expression to relieve LPS-induced macrophage apoptosis and inflammation (P<0.05). Furthermore, FFD inhibited the MAPK14/FOXO3A signaling pathway to inhibit LPS-induced macrophage apoptosis in the lung tissue of septic mice (P<0.05). CONCLUSION FFD could ameliorate the LPS-induced inflammatory response in septic mice by inhibiting the MAPK14/FOXO3A signaling pathway.
Mice ; Animals ; Mitogen-Activated Protein Kinase 14/metabolism* ; Wolfiporia ; Lipopolysaccharides/pharmacology* ; Sepsis/complications* ; Signal Transduction ; Inflammation/drug therapy* ; Oxygen Radioisotopes

Objective: To investigate the effects and mechanism of interleukin-4-modified gold nanoparticle (IL-4-AuNP) on the wound healing of full-thickness skin defects in diabetic mice. Methods: Experimental research methods were adopted. Gold nanoparticle (AuNP) and IL-4-AuNP were synthesized by improving the methods described in published literature. The morphology of those two particles were photographed by transmission electron microscopy, and their particle sizes were calculated. The surface potential and hydration particle size of the two particles were detected by nanoparticle potentiometer and particle size analyzer, respectively. The clearance rate of IL-4-AuNP to hydrogen peroxide and superoxide anion was measured by hydrogen peroxide and superoxide anion kits, respectively. Mouse fibroblast line 3T3 cells were used and divided into the following groups by the random number table (the same below): blank control group, hydrogen peroxide alone group treated with hydrogen peroxide only, hydrogen peroxide+IL-4-AuNP group treated with IL-4-AuNP for 0.5 h and then treated with hydrogen peroxide. After 24 h of culture, the reactive oxygen species (ROS) levels of cells were detected by immunofluorescence method; cell count kit 8 was used to detect relative cell survival rate. The macrophage Raw264.7 mouse cells were then used and divided into blank control group and IL-4-AuNP group that treated with IL-4-AuNP. After 24 h of culture, the expression of arginase 1 (Arg-1) in cells was observed by immunofluorescence method. Twelve male BALB/c mice (mouse age, sex, and strain, the same below) aged 8 to 10 weeks were divided into IL-4-AuNP group and blank control group, treated accordingly. On the 16^th day of treatment, whole blood samples were collected from mice for analysis of white blood cell count (WBC), red blood cell count (RBC), hemoglobin level, or platelet count and the level of aspartate aminotransferase (AST), alanine transaminase (ALT), urea, or creatinine. The inflammation, bleeding, or necrosis in the heart, liver, spleen, lung, and kidney tissue of mice were detected by hematoxylin-eosin (HE). Another 36 mice were selected to make diabetic model, and the full-thickness skin defect wounds were made on the back of these mice. The wounds were divided into blank control group, AuNP alone group, and IL-4-AuNP group, with 12 mice in each group, and treated accordingly. On the 0 (immediately), 4^th, 9^th, and 15^th day of treatment, the wound condition was observed and the wound area was calculated. On the 9^th day of treatment, HE staining was used to detect the length of neonatal epithelium and the thickness of granulation tissue in the wound. On the 15^th day of treatment, immunofluorescence method was used to detect ROS level and the number of Arg-1 positive cells in the wound tissue. The number of samples was 6 in all cases. Data were statistically analyzed with independent sample t test, corrected t test, Tukey test, or Dunnett T3 test. Results: The size of prepared AuNP and IL-4-AuNP were uniform. The particle size, surface potential, and hydration particle size of AuNP and IL-4-AuNP were (13.0±2.1) and (13.9±2.5) nm, (-45.8±3.2) and (-20.3±2.2) mV, (14±3) and (16±4) nm, respectively. For IL-4-AuNP, the clearance rate to hydrogen peroxide and superoxide anion were (69±4)% and (52±5)%, respectively. After 24 h of culture, the ROS level of 3T3 in hydrogen peroxide alone group was significantly higher than that in blank control group (q=26.12, P<0.05); the ROS level of hydrogen peroxide+IL-4-AuNP group was significantly lower than that in hydrogen peroxide alone group (q=25.12, P<0.05) and close to that in blank control group (P>0.05). After 24 h of culture, the relative survival rate of 3T3 cells in hydrogen peroxide+IL-4-AuNP group was significantly higher than that in hydrogen peroxide alone group (t=51.44, P<0.05). After 24 h of culture, Arg-1 expression of Raw264.7 cells in IL-4-AuNP group was significantly higher than that in blank control group (t'=8.83, P<0.05).On the 16^th day of treatment, there were no significant statistically differences in WBC, RBC, hemoglobin level, or platelet count and the level of AST, ALT, urea, or creatinine of mice between blank control group and IL-4-AuNP group (P>0.05). No obvious inflammation, bleeding or necrosis was observed in the heart, liver, spleen, lung, and kidney of important organs in IL-4-AuNP group, and no significant changes were observed compared with blank control group. On the 0 and 4^th day of treatment, the wound area of diabetic mice in blank control group, AuNP alone group, and IL-4-AuNP group had no significant difference (P>0.05). On the 9^th day of treatment, the wound areas both in AuNP alone group and IL-4-AuNP group were significantly smaller than that in blank control group (with q values of 9.45 and 14.87, respectively, P<0.05), the wound area in IL-4-AuNP group was significantly smaller than that in AuNP alone group (q=5.42, P<0.05). On the 15^th day of treatment, the wound areas both in AuNP alone group and IL-4-AuNP group were significantly smaller than that in blank control group (with q values of 4.84 and 20.64, respectively, P<0.05), the wound area in IL-4-AuNP group was significantly smaller than that in AuNP alone group (q=15.80, P<0.05); moreover, inflammations such as redness and swelling were significantly reduced in IL-4-AuNP group compared with the other two groups. On the 9^th day of treatment, compared with blank control group and AuNP alone group, the length of neonatal epithelium in the wound of diabetic mice in IL-4-AuNP group was significantly longer (all P<0.05), and the thickness of the granulation tissue in the wound was significantly increased (with q values of 11.33 and 9.65, respectively, all P<0.05). On the 15^th day of treatment, compared with blank control group, ROS levels in wound tissue of diabetic mice in AuNP alone group and IL-4-AuNP group were significantly decreased (P<0.05). On the 15^th day of treatment, the number of Arg-1 positive cells in the wounds of diabetic mice in IL-4-AuNP group was significantly more than that in blank control group and AuNP alone group, respectively (all P<0.05). Conclusions: IL-4-AuNP is safe in vivo, and can improve the oxidative microenvironment by removing ROS and induce macrophage polarization towards M2 phenotype, thus promote efficient diabetic wound healing and regeneration of full-thickness skin defects in diabetic mice.
Mice ; Male ; Animals ; Interleukin-4 ; Gold/pharmacology* ; Diabetes Mellitus, Experimental ; Creatinine ; Hydrogen Peroxide ; Reactive Oxygen Species ; Superoxides ; Metal Nanoparticles ; Soft Tissue Injuries ; Antibodies ; Inflammation ; Necrosis ; Hemoglobins

OBJECTIVE: To evaluate the apoptosis and cycle arrest effects of Oldenlandia diffusa flavonoids on human gastric cancer cells, determine the action mechanisms in association with the mitochondrial dependent signal transduction pathway that controls production of reactive oxygen species (ROS), and evaluate the pharmacodynamics of a mouse xenotransplantation model to provide a reference for the use of flavonoids in prevention and treatment of gastric cancer. METHODS: Flavonoids were extracted by an enzymatic-ultrasonic assisted method and purified with D-101 resin. Bioactive components were characterized by high-performance liquid chromatography. Cell lines MKN-45, AGS, and GES-1 were treated with different concentrations of flavonoids (64, 96, 128, 160 µg/mL). The effect of flavonoids on cell viability was evaluated by MTT method, and cell nuclear morphology was observed by Hoechst staining. The apoptosis rate and cell cycle phases were measured by flow cytometry, the production of ROS was detected by laser confocal microscope, the mitochondrial membrane potential (MMP) were observed by fluorescence microscope, and the expression of apoptotic proteins related to activation of mitochondrial pathway were measured by immunoblotting. MKN-45 cells were transplanted into BALB/c nude mice to establish a xenograft tumor model. Hematoxylin and eosin staining was used to reveal the subcutaneous tumor tissue. The tumor volume and tumor weight were measured, the expression levels of proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by immunohistochemistry, and the expression levels of CA72-4 were measured by enzyme linked immunosorbent assay. RESULTS: Oldenlandia diffusa flavonoids inhibited proliferation of MKN-45 and AGS human gastric cancer cells, arrested the cell cycle in G₁/S phase, induced accumulation of ROS in the process of apoptosis, and altered MMP. In addition, flavonoids increased Apaf-1, Cleaved-Caspase-3, and Bax, and decreased Cyclin A, Cdk2, Bcl-2, Pro-Caspase-9, and Mitochondrial Cytochrome C (P<0.05). The MKN-45 cell mouse xenotransplantation model further clarified the growth inhibitory effect of flavonoids towards tumors. The expression levels of PCNA and Ki-67 decreased in each flavonoid dose group, the expression level of CA72-4 decreased (P<0.05). CONCLUSION Flavonoids derived from Oldenlandia diffusa can inhibit proliferation and induce apoptosis of human gastric cancer cells by activating the mitochondrial controlled signal transduction pathway.
Humans ; Animals ; Mice ; Oldenlandia/metabolism* ; Proliferating Cell Nuclear Antigen ; Stomach Neoplasms ; Flavonoids/pharmacology* ; Reactive Oxygen Species/metabolism* ; Mice, Nude ; Ki-67 Antigen ; Cell Line, Tumor ; Apoptosis ; Plant Extracts/pharmacology* ; Caspases ; Cell Proliferation

This study aimed to explore the mechanism of Cistanches Herba in the treatment of cancer-induced fatigue(CRF) by network pharmacology combined with in vivo and in vitro experiments to provide a theoretical basis for the clinical medication. The chemical constituents and targets of Cistanches Herba were searched from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). The targets of CRF were screened out by GeneCards and NCBI. The common targets of traditional Chinese medicine and disease were selected to construct a protein-protein interaction(PPI) network, followed by Gene Ontology(GO) functional and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses. A visual signal pathway rela-ted to Chinese medicine and disease targets was constructed. The CRF model was induced by paclitaxel(PTX) in mice. Mice were divided into a control group, a PTX model group, and low-and high-dose Cistanches Herba extract groups(250 and 500 mg·kg~(-1)). The anti-CRF effect in mice was evaluated by open field test, tail suspension test, and exhaustive swimming time, and the pathological morphology of skeletal muscle was evaluated by hematoxylin-eosin(HE) staining. The cancer cachexia model in C2C12 muscle cells was induced by C26 co-culture, and the cells were divided into a control group, a conditioned medium model group, and low-, medium-, and high-dose Cistanches Herba extract groups(62.5, 125, and 250 μg·mL~(-1)). The reactive oxygen species(ROS) content in each group was detected by flow cytometry, and the intracellular mitochondrial status was evaluated by transmission electron microscopy. The protein expression levels of hypoxia-inducible factor-1α(HIF-1α), BNIP3L, and Beclin-1 were detected by Western blot. Six effective constituents were screened out from Cistanches Herba. The core genes of Cistanches Herba in treating CRF were AKT1, IL-6, VEGFA, CASP3, JUN, EGFR, MYC, EGF, MAPK1, PTGS2, MMP9, IL-1B, FOS, and IL10, and the pathways related to CRF were AGE-RAGE and HIF-1α. Through GO enrichment analysis, it was found that the main biological functions involved were lipid peroxidation, nutrient deficiency, chemical stress, oxidative stress, oxygen content, and other biological processes. The results of the in vivo experiment showed that Cistanches Herba extract could significantly improve skeletal muscle atrophy in mice to relieve CRF. The in vitro experiment showed that Cistanches Herba extract could significantly reduce the content of intracellular ROS, the percentage of mitochondrial fragmentation, and the protein expression of Beclin-1 and increase the number of autophagosomes and the protein expression of HIF-1α and BNIP3L. Cistanches Herba showed a good anti-CRF effect, and its mechanism may be related to the key target proteins in the HIF-1α signaling pathway.
Animals ; Mice ; Cistanche ; Network Pharmacology ; Beclin-1 ; Reactive Oxygen Species ; Plant Extracts ; Drugs, Chinese Herbal/pharmacology* ; Molecular Docking Simulation ; Medicine, Chinese Traditional ; Neoplasms/genetics*

Objective: To investigate the effect of nuclear factor erythroid 2-related factor 2 (Nrf2) in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated matrix metalloproteinase 9 (MMP9) . Methods: In October 2020, immortalized rat choroid plexus epithelial cell line (Z310) cells were used as the blood-cerebrospinal fluid barrier in vitro, and were divided into control group and 0.125, 0.25, 0.5 mmol/L lanthanum chloride (LaCl(3)) treatment group. After treating Z310 cells with different concentrations of LaCl(3) for 24 hours, the morphological changes of Z310 cells were observed under inverted microscope, the protein expression levels of MMP9, occludin and zonula occludens-1 (ZO-1) were observed by cellular immunofluorescence method, and the protein expression levels of MMP9, tissue inhibitors of metalloproteinase1 (TIMP1) , occludin, ZO-1 and Nrf2 were detected by Western blotting. The level of reactive oxygen species (ROS) in cells was detected by flow cytometry. Results: Compared with the control group, Z310 cells in the LaCl(3) treatment group were smaller in size, with fewer intercellular junctions, and more dead cells and cell fragments. The expression level of MMP9 protein in cells treated with 0.25 and 0.5 mmol/L LaCl(3) was significantly higher than that in the control group (P<0.05) , and the expression level of TIMP1 and tight junction proteins occudin and ZO-1 was significantly lower than that in the control group (P<0.05) . Compared with the control group, the ROS production level in the 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly increased (P<0.05) , and the Nrf2 protein expression level in the 0.125, 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly decreased (P<0.05) . Conclusion: Lanthanum may increase the level of ROS in cells by down regulating the expression of Nrf2, thus activating MMP9 to reduce the expression level of intercellular tight junction proteins occludin and ZO-1.
Rats ; Animals ; Matrix Metalloproteinase 9/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Tight Junction Proteins/metabolism* ; Occludin/pharmacology* ; Choroid Plexus/metabolism* ; Reactive Oxygen Species/metabolism* ; Lanthanum/pharmacology* ; Epithelial Cells ; Zonula Occludens-1 Protein/metabolism* ; Phosphoproteins/pharmacology*

Xenogenic organ transplantation has been considered the most promising strategy in providing possible substitutes with the physiological function of the failing organs as well as solving the problem of insufficient donor sources. However, the xenograft, suffered from immune rejection and ischemia-reperfusion injury (IRI), causes massive reactive oxygen species (ROS) expression and the subsequent cell apoptosis, leading to the xenograft failure. Our previous study found a positive role of PPAR-γ in anti-inflammation through its immunomodulation effects, which inspires us to apply PPAR-γ agonist rosiglitazone (RSG) to address survival issue of xenograft with the potential to eliminate the excessive ROS. In this study, xenogenic bioroot was constructed by wrapping the dental follicle cells (DFC) with porcine extracellular matrix (pECM). The hydrogen peroxide (H₂O₂)-induced DFC was pretreated with RSG to observe its protection on the damaged biological function. Immunoflourescence staining and transmission electron microscope were used to detect the intracellular ROS level. SD rat orthotopic transplantation model and superoxide dismutase 1 (SOD1) knockout mice subcutaneous transplantation model were applied to explore the regenerative outcome of the xenograft. It showed that RSG pretreatment significantly reduced the adverse effects of H₂O₂ on DFC with decreased intracellular ROS expression and alleviated mitochondrial damage. In vivo results confirmed RSG administration substantially enhanced the host's antioxidant capacity with reduced osteoclasts formation and increased periodontal ligament-like tissue regeneration efficiency, maximumly maintaining the xenograft function. We considered that RSG preconditioning could preserve the biological properties of the transplanted stem cells under oxidative stress (OS) microenvironment and promote organ regeneration by attenuating the inflammatory reaction and OS injury.
Mice ; Humans ; Rats ; Animals ; Swine ; PPAR gamma/pharmacology* ; Reactive Oxygen Species/pharmacology* ; Heterografts ; Hydrogen Peroxide/pharmacology* ; Rats, Sprague-Dawley ; Rosiglitazone/pharmacology* ; Oxidative Stress

OBJECTIVE: The current study evaluated various new colchicine analogs for their anticancer activity and to study the primary mechanism of apoptosis and in vivo antitumor activity of the analogs with selective anticancer properties and minimal toxicity to normal cells. METHODS: Sulforhodamine B (SRB) assay was used to screen various colchicine analogs for their in vitro cytotoxicity. The effect of N-[(7S)-1,2,3-trimethoxy-9-oxo-10-(pyrrolidine-1-yl)5,6,7,9-tetrahydrobenzo[a] heptalene-7-yl] acetamide (IIIM-067) on clonogenicity, apoptotic induction, and invasiveness of A549 cells was determined using a clonogenic assay, scratch assay, and staining with 4',6-diamidino-2-phenylindole (DAPI) and annexin V/propidium iodide. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels were observed using fluorescence microscopy. Western blot analysis was used to quantify expression of proteins involved in apoptosis, cell cycle, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling. Pharmacokinetic and in vivo efficacy studies against Ehrlich ascites carcinoma (EAC) and Ehrlich solid tumor models were conducted using Swiss albino mice. RESULTS: IIIM-067 showed potent cytotoxicity and better selectivity than all other colchicine analogs screened in this study. The selective activity of IIIM-067 toward A549 cells was higher among other cancer cell lines, with a selectivity index (SI) value of 2.28. IIIM-067 demonstrated concentration- and time-dependent cytotoxicity against A549 cells with half-maximal inhibitory concentration values of 0.207, 0.150 and 0.106 μmol/L at 24, 48 and 72 h, respectively. It also had reduced toxicity to normal cells (SI > 1) than the parent compound colchicine (SI = 1). IIIM-067 reduced the clonogenic ability of A549 cells in a dose-dependent manner. IIIM-067 enhanced ROS production from 24.6% at 0.05 μmol/L to 82.1% at 0.4 μmol/L and substantially decreased the MMP (100% in control to 5.6% at 0.4 μmol/L). The annexin V-FITC assay demonstrated 78% apoptosis at 0.4 μmol/L. IIIM-067 significantly (P < 0.5) induced the expression of various intrinsic apoptotic pathway proteins, and it differentially regulated the PI3K/AKT/mTOR signaling pathway. Furthermore, IIIM-067 exhibited remarkable in vivo anticancer activity against the murine EAC model, with tumor growth inhibition (TGI) of 67.0% at a dose of 6 mg/kg (i.p.) and a reduced mortality compared to colchicine. IIIM-067 also effectively inhibited the tumor growth in the murine solid tumor model with TGI rates of 48.10%, 55.68% and 44.00% at doses of 5 mg/kg (i.p.), 6 mg/kg (i.p.) and 7 mg/kg (p.o.), respectively. CONCLUSION IIIM-067 exhibited significant anticancer activity with reduced toxicity both in vitro and in vivo and is a promising anticancer candidate. However, further studies are required in clinical settings to fully understand its potential.
Animals ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Antineoplastic Agents, Phytogenic/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Reactive Oxygen Species/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Colchicine/pharmacology* ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Mammals/metabolism*

OBJECTIVES: To investigate the effect of borneol on cutaneous toxicity of gilteritinib and to explore possible compounds that can intervene with the cutaneous toxicity. METHODS: C57BL/6J male mice were given gilteritinib by continuous gavage for 28 d and the damage to keratinocytes in the skin tissues was observed with hematoxylin and eosin (HE) staining, TUNEL assay and immunohistochemistry. Human keratinocytes HaCaT were treated with gilteritinib, and cell death and morphological changes were examined by SRB staining and microscopy; apoptosis of HaCaT cells was examined by Western blotting, flow cytometry with propidium iodide/AnnexinⅤ double staining and immunofluorescence; the accumulation of cellular reactive oxygen species (ROS) was examined by flow cytometry with DCFH-DA. Compounds that can effectively intervene the cutaneous toxicity of gilteritinib were screened from a natural compound library using SRB method, and the intervention effect of borneol on gilteritinib cutaneous toxicity was further investigated in HaCaT cells and C57BL/6J male mice. RESULTS: In vivo studies showed pathological changes in the skin with apoptosis of keratinocytes in the stratum spinosum and stratum granulosum in the modeling group. Invitro studies showed apoptosis of HaCaT cells, significant up-regulation of cleaved poly (ADP-ribose) polymerase (c-PARP) and gamma-H2A histone family member X (γ-H2AX) levels, and increased accumulation of ROS in gilteritinib-modeled skin keratinocytes compared with controls. Screening of the natural compound library revealed that borneol showed excellent intervention effects on the death of HaCaT cells. In vitro, cell apoptosis was significantly reduced in the borneol+gilteritinib group compared to the gilteritinib control group. The levels of c-PARP, γ-H2AX and ROS in cells were significantly decreased. In vivo, borneol alleviated gilteritinib-induced skin pathological changes and skin cell apoptosis in mice. CONCLUSIONS Gilteritinib induces keratinocytes apoptosis by causing intracellular ROS accumulation, resulting in cutaneous toxicity. Borneol can ameliorate the cutaneous toxicity of gilteritinib by reducing the accumulation of ROS and apoptosis of keratinocytes in the skin tissue.
Male ; Humans ; Animals ; Mice ; Reactive Oxygen Species/metabolism* ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology* ; Mice, Inbred C57BL ; Apoptosis ; Poly(ADP-ribose) Polymerases/metabolism*

OBJECTIVE: To investigate the effect of Baicalin on the proliferation and pyroptosis of diffuse large B-cell lymphoma cell line DB and its mechanism. METHODS: DB cells were treated with baicalin at different concentrations (0, 5, 10, 20, 40 μmol/L). Cell proliferation was detected by CCK-8 assay and half maximal inhibitory concentration (IC₅₀) was calculated. The morphology of pyroptosis was observed under an inverted microscope, the integrity of the cell membrane was verified by LDH content release assay, and the expressions of pyroptosis-related mRNA and protein (NLRP3, GSDMD, GSDME, N-GSDMD, N-GSDME) were detected by real-time fluorescence quantitative PCR and Western blot. In order to further clarify the relationship between baicalin-induced pyroptosis and ROS production in DB cells, DB cells were divided into control group, baicalin group, NAC group and NAC combined with baicalin group. DB cells in the NAC group were pretreated with ROS inhibitor N-acetylcysteine (NAC) 2 mmol/L for 2 h. Baicalin was added to the combined treatment group after pretreatment, and the content of reactive oxygen species (ROS) in the cells was detected by DCFH-DA method after 48 hours of culture. RESULTS: Baicalin inhibited the proliferation of DB cells in a dose-dependent manner (r=-0.99), and the IC₅₀ was 20.56 μmol/L at 48 h. The morphological changes of pyroptosis in DB cells were observed under inverted microscope. Compared with the control group, the release of LDH in the baicalin group was significantly increased (P<0.01), indicating the loss of cell membrane integrity. Baicalin dose-dependently increased the expression levels of NLRP3, N-GSDMD, and N-GSDME mRNA and protein in the pyroptosis pathway (P<0.05). Compared with the control group, the level of ROS in the baicalin group was significantly increased (P<0.05), and the content of ROS in the NAC group was significantly decreased (P<0.05). Compared with the NAC group, the content of ROS in the NAC + baicalin group was increased. Baicalin significantly attenuated the inhibitory effect of NAC on ROS production (P<0.05). Similarly, Western blot results showed that compared with the control group, the expression levels of pyroptosis-related proteins was increased in the baicalin group (P<0.05). NAC inhibited the expression of NLRP3 and reduced the cleavage of N-GSDMD and N-GSDME (P<0.05). Compared with the NAC group, the NAC + baicalin group had significantly increased expression of pyroptosis-related proteins. These results indicate that baicalin can effectively induce pyroptosis in DB cells and reverse the inhibitory effect of NAC on ROS production. CONCLUSION Baicalin can inhibit the proliferation of DLBCL cell line DB, and its mechanism may be through regulating ROS production to affect the pyroptosis pathway.
Humans ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Reactive Oxygen Species/pharmacology* ; Pyroptosis ; Cell Line ; RNA, Messenger ; Lymphoma, Large B-Cell, Diffuse

OBJECTIVE: To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages. METHODS: RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE₂) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O₂^-) radical and nitrite scavenging activity were also measured. RESULTS: The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE₂ production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O₂^- radical and nitrite scavenging activity. CONCLUSION EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.
Animals ; Mice ; Antioxidants/pharmacology* ; Lipopolysaccharides/pharmacology* ; Polygala ; Transcription Factor RelA/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Ethanol/chemistry* ; Interleukin-6/metabolism* ; Anti-Inflammatory Agents/chemistry* ; Reactive Oxygen Species/metabolism* ; Cyclooxygenase 2/metabolism* ; Nitrites/metabolism* ; NF-kappa B/metabolism* ; Nitric Oxide/metabolism* ; Superoxide Dismutase/metabolism* ; RNA, Messenger ; Nitric Oxide Synthase Type II/metabolism*