Protective effects of isorhamnetin on apoptosis and inflammation in TNF-α-induced HUVECs injury

Little is known about the role of isorhamnetin on endothelial cell apoptosis and inflammation when insulted by TNF-α injury. In our study, HUVECs were treated with TNF-α for 6 hours. HUVECs apoptosis were detected using flow cytometry. The expressions of ICAM-1, VCAM-1, E-selectin, NF-κB, AP-1 and eNOS were determined with western blotting or flow cytometry. The results showed TNF-α increased of apoptosis and the expression of ICAM-1, VCAM-1 and E-selectin in HUVECs, accompanied by significant augmentation of NF-κB and AP-1 expression. Pretreatment with isorhamnetin significantly reduced apoptosis in TNF-α-treated HUVECs. Moreover, isorhamnetin significantly attenuated TNF-α-induced upregulation of ICAM-1, VCAM-1, AP-1, E-selectin and NF-κB expression. Meanwhile, isorhamnetin also increased the expression of eNOS. So, isorhamnetin could suppress TNF-α-induced apoptosis and inflammation by blocking NF-κB and AP-1 signaling in HUVECs, which might be one of the underlying mechanisms for treatment of coronary heart disease.     

Squamosamide derivative FLZ inhibits TNF-α-induced ICAM-1 expression via down-regulation of the NF-κB signaling pathway in ARPE-19 cells

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). It has been reported that tumor necrosis factor-α (TNF-α) could induce intercellular adhesion molecule-1 (ICAM-1) expression in RPE cells. FLZ, a novel synthetic squamosamide derivative from a Chinese herb, Annona glabra, has displayed significant anti-inflammatory activity. However, the effects of FLZ on TNF-α-induced ICAM-1 expression in RPE cells remain unknown. Therefore, in the present study, we evaluated the effects of FLZ on TNF-α-induced ICAM-1 expression in RPE cells. We found that FLZ prevented TNF-α-induced ICAM-1 expression and the ability of monocytes to adhere to ARPE-19 cells induced by TNF-α. Furthermore, FLZ inhibited TNF-α-induced NF-κB p65 expression, as well as phosphorylation of IκBα in ARPE-19 cells. Taken together, these results suggest that FLZ inhibited TNF-α-induced ICAM-1 expression through blocking NF-κB signaling pathway in ARPE-19 cells. Thus, FLZ could be used for designing novel therapeutic agents against AMD.     

Heme oxygenase 1 plays role of neuron-protection by regulating Nrf2-ARE signaling post intracerebral hemorrhage

The NF-E2 related factor 2 (Nrf2) could be activated in intracerebral hemorrhage (ICH), and trigger the expression of ARE regulated heme oxygenase 1 (HO-1) subsequently. This study aims to explore neuroprotection of HO-1 protein in regulating the Nrf2-ARE signaling pathway in ICH. In this study, the femoral artery injection method was used to establish the ICH model. The zinc porphyrin-9 (ZPP-IX) was used to inhibit the HO-1 expression in ICH rats. The ICH rats were randomly divided into 3 groups, ICH group, ZPP-IX (10 mg/kg) + ICH group and DMSO (10 mg/kg) + ICH group. Neurological scores were evaluated for the 3 groups. Double immunofluorescence staining method was employed to observe the co-expression of HO-1, Nrf2, NF-κB and TNF-α and CD11b in glia cells. Western blot and RT-PCR assay were used to detect the total Nrf2, binding Nrf2, HO-1, NF-κB and TNF-α expression. The results indicated that ZPP-IX could aggravate the neurological dyafunstions of ICH rats. The HO-1 level in ZPP-IX group was significantly decreased compared to the ICH group (P < 0.05). The binding-Nrf2 protein was significantly increased in ZPP-IX group compared to ICH group (P < 0.05). The NF-κB and TNF-α level were significantly increased in ZPP-IX group compared to ICH group (P < 0.05). The ZPP-IX significantly inhibited the HO-1 and Nrf2, and enhanced NF-κB and TNF-α co-expressing with the CD11b compared to the ICH group (P < 0.05). In conclusion, HO-1 protein regulates the Nrf2-ARE pathway in ICH model by inhibiting the Nrf2 entering nucleus and activating the NF-κB and TNF-α expression.     

Effects of NF-κB and hypoxia on the biological behavior of Y79 retinoblastoma cells

We aimed to investigate the influence of nuclear factor-κB (NF-κB) on the biological behavior of Y79 retinoblastoma cells exposed to hypoxia and its possible mechanism. The cells were administrated with hypoxia, and/or 5 μM pyrrolidine dithiocarbamate (PDTC) (a selective NF-κB inhibitor) to inhibit the NF-κB activity, expressions of NF-κB was measured by western blot, and the translocation of NF-κB was detected. To examine the proliferation of Y79 cells, MTT assay was applied. Transwell assay was used to detect the invasion and migration ability of cells. The expressions of molecules involved in invasion was analyzed including HIF-1α, MMP-2, 9, and VEGF. We found that hypoxia significantly activated NF-κB activity. While once the NF-κB was inhibited, the proliferation, invasion and migration ability of Y79 cells were also blocked. Interestingly, the expressions of invasion-involved molecules elevated by hypoxia induction were also decreased when NF-κB was inhibited. Hypoxia could significantly change the adhesive and invasive ability of Y79 retinoblastoma cells, NF-κB signal might be one of the main mediators for these hypoxia induced cell changes of biological behavior via downregulation of HIF-1α and the invasion related molecules, and the mechanism still needs further investigation.     

Protective effects of bifidobacterial adhesin on intestinal mucosa of stressed male rats via modulation of inflammation

This study aimed to assess BA impact on inflammation markers and repair of intestinal mucosa. Forty-eight rats were randomly divided into stress (n = 24) and BA (n = 24) groups. Stress was induced by fettering in all animals, fed enterally with 125.4 kJ/kg/d and 0.2 g/kg/d nitrogen. Then, rats were treated for 8 days with 5 mg/kg/d BA (BA group) or 5 mg/kg/d saline (Stress group). Levels of NF-κB, IL-10, TNF-α, and IFN-γ were measured at different time points, in plasma and intestinal mucosa samples. Changes in intestinal mucosa morphology were observed by electron microscopy. Plasma and/or mucosal levels of NF-κB, TNF-α, and IFN-γ were significantly higher in both groups after stress induction (P < 0.05). These high levels persisted in control animals throughout the experiment, and were significantly reduced in the BA group, 3 and 8 days after stress induction (P < 0.05). Interestingly, IL-10 levels were increased after BA treatment (P < 0.05). At day 8, ileal mucosal villi and crypt structure were significantly restored in the BA group. Bifidobacterial adhesin plays a role in repairing intestinal mucosa injury after stress by regulating the release of inflammatory mediators in the intestinal mucosa.     

Effects of that ATRA inhibits Nrf2-ARE pathway on glial cells activation after intracerebral hemorrhage

Previous studies indicate that the Nrf2-ARE signaling pathway plays a neruo-protective role in glia cell, however, the mechanism was also elusive. This study aims to explore the inhibitive function of all-trans-retinoic (ATRA) on Nrf2-ARE pathway in intracerebral hemorrhage (ICH), and investigate the mechanism. In this study, the femoral artery injection method was employed to establish ICH model. The model rats were randomly divided into four groups, including Sham group, ICH group, ATRA group and DMSO group. The neurological scores were evaluated for the four groups at different time points. Hematoxylin-Eosin staining was used to stain the CD11b positive glia cells. Double immunofluorescence staining method was utilized to observe the co-expression of HO-1, NF-κB, Nrf2 and TNF-α and CD11b marker in glia cells. Western blot assay was used to detect the Nrf2 protein (total and binding Nrf2), HO-1, NF-κB and TNF-α proteins in every group. The results indicated that neurologiclal scores were significantly decreased in ATRA group compared to ICH gorup (P < 0.05). The glia cells were significantly activated and accumulated in ICH rats. ATRA significantly decreased co-expression of Nrf2, HO-1 and CD11b, and increased co-expression of NF-κB, TNF-α and CD11b of glia cells. ATRA significantly decreased total Nrf2 expression and increased binding Nrf2 expression in ATRA group compared to ICH group (P < 0.05). ATRA decreased anti-oxygen protein Nrf2 and HO-1, and increases inflammatory factors NF-κB and TNF-α. In conclusion, the application of ATRA could inhibit the neuro-protective function effectively by blocking the Nrf2-ARE pathway in glia cells.     

Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P < 0·05) compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4⁺ T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses.     

Putative role of ischemic postconditioning in a rat model of limb ischemia and reperfusion: involvement of hypoxia-inducible factor-1α expression

Hypoxia-inducible factor-1α (HIF-1α) is one of the most potent angiogenic growth factors. It improves angiogenesis and tissue perfusion in ischemic skeletal muscle. In the present study, we tested the hypothesis that ischemic postconditioning is effective for salvaging ischemic skeletal muscle resulting from limb ischemia-reperfusion injury, and that the mechanism involves expression of HIF-1α. Wistar rats were randomly divided into three groups (n=36 each): sham-operated (group S), hindlimb ischemia-reperfusion (group IR), and ischemic postconditioning (group IPO). Each group was divided into subgroups (n=6) according to reperfusion time: immediate (0 h, T₀), 1 h (T₁), 3 h (T₃), 6 h (T₆), 12 h (T₁₂), and 24 h (T₂₄). In the IPO group, three cycles of 30-s reperfusion and 30-s femoral aortic reocclusion were carried out before reperfusion. At all reperfusion times (T₀-T₂₄), serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities, as well as interleukin (IL)-6, IL-10, and tumor necrosis factor-α (TNF-α) concentrations, were measured in rats after they were killed. Histological and immunohistochemical methods were used to assess the skeletal muscle damage and HIF-1α expression in skeletal muscle ischemia. In groups IR and IPO, serum LDH and CK activities and TNF-α, IL-6, and IL-10 concentrations were all significantly increased compared to group S, and HIF-1α expression was up-regulated (P<0.05 or P<0.01). In group IPO, serum LDH and CK activities and TNF-α and IL-6 concentrations were significantly decreased, IL-10 concentration was increased, HlF-1α expression was down-regulated (P<0.05 or P<0.01), and the pathological changes were reduced compared to group IR. The present study suggests that ischemic postconditioning can reduce skeletal muscle damage caused by limb ischemia-reperfusion and that its mechanisms may be related to the involvement of HlF-1α in the limb ischemia-reperfusion injury-triggered inflammatory response.     

Continuous hemodiafiltration therapy reduces damage of multi-organs by ameliorating of HMGB1/TLR4/NFκB in a dog sepsis model

In the present study, we investigated whether CVVH can reduce HMGB1, TLR4, NF-κB and other serum cytokine levels, preventing organ injury in a dog sepsis model. A total of 10 dogs were injected with LPS and treated with either CVVH group (n = 5) or nothing (Control, n = 5) for 24 h. EILSA was used for examining the concentration of TNF-α, IL-6, HMGB 1 and TLR4. The histological change of lung, liver and kidney tissues was determined. The mRNA expression of HMGB1, TLR4 and NF-κB was examined by RT-PCR. The protein of HMGB1 and phosphated NF-κB was examined by Western-blot. The levels of serum HMGB1 came to the peak at 8 h, 16 h and then declined. The LPS-induced increase in HMGB1 level was suppressed by CVVH compared with Control. Likewise, serum TNF-α and IL-6 levels decreased with CVVH along with a significant improvement in the function of main organs. Histologic examination revealed significant reduction in inflammation in lung; liver and kidney tissues harvested 24 h after CVVH compared with Control. The mRNA of HMGB1, TLR4 and NF-κB in the kidney was expressed at high level after LPS administration, which was significantly decreased by CVVH. The increased protein expression of HMGB1 and phosphated NF-κB was reduced after CVVH compared with control. CVVH by reducing the level of HMGB1, TLR4, NF-κB and other cytokines could weaken the cascade of cytokines and restore the immune system, and reduce the damage of important organs in sepsis.     

Pterostilbene impact on retinal endothelial cells under high glucose environment

Diabetic retinopathy (DR) has complicated pathogenic factors. Studies showed that DR belongs to chronic inflammatory disease, and retinal endothelial cells oxidation by free radicals is one of its mechanisms. Pterostilbene, as the homologous derivative of resveratrol, has obvious antioxidant effect. Its influence on the DR has not been studied. This study intended to investigate the effect and mechanism of pterostilbene on human retinal endothelial cells (hRECs) under high glucose environment to illustrate pterostilbene impact on DR and provide basis for DR clinical treatment. hRECs cultured in high glucose environment were treated by 1.0 mmol/L pterostilbene. MTT assay was applied to test cell proliferation. ELISA was used to detect inflammatory factor TNF-α and IL-1β content. Real time PCR and Western blot were performed to examine NF-κB mRNA and protein expression. ROS and SOD activities were analyzed. Under high glucose environment, hRECs proliferation increased, TNF-α and IL-1β expression elevated, and NF-κB protein level upregulated significantly. On the other side, ROS production increased and SOD activity decreased obviously (P < 0.05). Pterostilbene can suppress hRECs over proliferation, decrease TNF-α and IL-1β, inhibit NF-κB protein expression, reduce ROS production, and increase SOD activity markedly compared with high glucose group (P < 0.05). Pterostilbene may delay DR progress through alleviating inflammation and antioxidation to suppress hRECs over proliferation.     

Effects of propofol on lipopolysaccharide-induced expression and release of HMGB1 in macrophages

This study aimed to determine the effects of different concentrations of propofol (2,6-diisopropylphenol) on lipopolysaccharide (LPS)-induced expression and release of high-mobility group box 1 protein (HMGB1) in mouse macrophages. Mouse macrophage cell line RAW264.7 cells were randomly divided into 5 treatment groups. Expression levels of HMGB1 mRNA were detected using RT-PCR, and cell culture supernatant HMGB1 protein levels were detected using enzyme-linked immunosorbent assay (ELISA). Translocation of HMGB1 from the nucleus to the cytoplasm in macrophages was observed by Western blotting and activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus was detected using ELISA. HMGB1 mRNA expression levels increased significantly in the cell culture supernatant and in cells after 24 h of stimulating RAW264.7 cells with LPS (500 ng/mL). However, HMGB1 mRNA expression levels in the P2 and P3 groups, which received 500 ng/mL LPS with 25 or 50 μmol/mL propofol, respectively, were significantly lower than those in the group receiving LPS stimulation (P<0.05). After stimulation by LPS, HMGB1 protein levels were reduced significantly in the nucleus but were increased in the cytoplasm (P<0.05). Simultaneously, the activity of NF-κB was enhanced significantly (P<0.05). After propofol intervention, HMGB1 translocation from the nucleus to the cytoplasm and NF-κB activity were inhibited significantly (each P<0.05). Thus, propofol can inhibit the LPS-induced expression and release of HMGB1 by inhibiting HMGB1 translocation and NF-κB activity in RAW264.7 cells, suggesting propofol may be protective in patients with sepsis.     

Blocking HMGB1 signal pathway protects early radiation-induced lung injury

It has been reported that HMGB1 participated in various types of lung injury. In this study, we explored whether blocking HMGB1 has a preventive effect on the early radiation-induced lung injury and investigate the mechanism. Mice model of radiation-induced lung injury were accomplished by a single dose irradiation (15 Gy) to the whole thorax. Irradiated mice were treated with HMGB1-neutralizing antibody intraperitoneally dosed 10 μg, 50 μg, 100 μg/mouse respectively and were sacrificed after one week post-irradiation. Lung tissue slices were stained by H&E, and alveolitis was quantified by Szapiel scoring system. The level of cytokines TNF-γ in bronchoalveolar lavage fluid was detected by ELISA method. And p65NF-κB, p50NF-κB protein expression in mice lung tissues was detected by Western blot analysis. The results showed that blocking HMGB1 inhibited the inflammatory response, and thereby decreased the degree of alveolitis of irradiated lung tissue. In addition, HMGB1 antagonist can restrain the expression of type Th2 or Th17 derived inflammatory cytokines TNF-α, IL-6 and IL-17A, promote the expression of Th1 type cytokines INF-γ, and inhibit p65 NF-κB but promote p50 NF-κB activation, which promoted the resolution of the radiation-induced inflammatory response. In conclusion, blocking HMGB1 can reduce the degree of early radiation-induced lung injury, and its mechanism may be related to the promotion of p50NF-κB activation and its downstream molecules expression. Inhibiting HMGB1 may be a new target to deal with early radiation-induced lung injury.     

Electroacupuncture modulated the inflammatory reaction in MCAO rats via inhibiting the TLR4/NF-κB signaling pathway in microglia

In this study, we aim to investigate the effects of electroacupuncture on the TLR4/NF-κB signaling pathway in microglia. Male Wistar rat of SPF grade (weighing 200±20 g) were randomly divided into (i): sham control group, which was subjected to sham operation (ii) vehicle group, which underwent the occlusion of middle cerebral artery; (iii-v): acupuncture groups, which were subjected to the occlusion of middle cerebral artery and treated with acupuncture on the Neiguan acupoint (P6), Quchi acupoint (LI11), and Diji acupoint (SP8), respectively. HE staining was performed to detect the necrotic rate of neurons. Mediators of inflammation were measured using ELISA. Immunofluorescence was performed to measure the expression of TLR4, HMGB1, TRAF6, IKKβ and NF-κB p65 in microglia. Severe decrease was noticed in the neurological score, necrotic rates of neuron, expression of IL-1β, IL-6, TLR4, HMGB1, TRAF6, IKKβ and NF-κB p65 in microglia. Compared with the vehicle group, significant decrease was revealed in the neurological score, necrotic rate, IL-1β, TLR4, TRAF6, IKKβ and NF-κB p65 in Neiguan group and Quchi group, respectively. In addition, remarkable decrease was observed in the expression of TNF-α and IL-6 in Quchi group. Compared with the Diji group, the necrotic rate of neurons in hippocampus region was significantly decreased in the Quchi group (P < 0.05). In Neiguan group, the expression of TLR4 and IKKβ was significantly attenuated (P < 0.05). The expression of TRAF6 was remarkably decreased in the Neiguan group and Quchi group, respectively. Electroacupuncture on Neiguan and Quchi could improve the neurological injury, attenuate the inflammation, and inhibit the activity of TLR4/NF-κB signaling pathway in microglia.     

Interleukin-13 inhibits cytokines synthesis by blocking nuclear factor-κB and c-Jun N-terminal kinase in human mesangial cells

Objective

Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin (IL) -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells (HMCs).

Methods

The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay (RPA). Activity of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) was examined by electrophoretic mobility shift assay (EMSA). NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase (JNK) activity were assayed by immunoblot.

Results

Recombinant IL-13 inhibited tumor necrosis factor-α (TNF-α), IL-1α, IL-1β, monocyte chemoattractant protein-1 (MCP-1), IL-8, and transforming growth factor-β1 (TGF-β1) mRNA expressions in a dose-dependent manner. Lipopolysacchorides (LPS) dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner.

Conclusion

IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.     

IL-1β promotes cervical cancer through activating NF-κB/CCL-2

Cervical cancer is a malignancy with high morbidity and mortality among women. Interleukin (IL)-1β, chemokine (C-C motif) ligand 2 (CCL-2), and activation of NF-κB have been proven to be closely related to the progression of various tumors. However, their role in cervical cancer remains unclear. Cell proliferation, migration, and invasion were detected using MTT, wound healing, and transwell assays. Western blotting and qRT-PCR were used to measure expression of target genes. IL-1β greatly promoted the release of CCL-2 from HeLa cells. Activation of NF-κB and phosphorylated NF-κB (p65) nuclear translocation were accelerated by IL-1β. TPCA-1, a blocker of NF-κB, significantly inhibited the release of CCL-2 from HeLa cells. TPCA-1 markedly reversed the promotional effect of IL-1β on viability of HeLa cells. IL-1β increased the cell migration, proliferation, and invasion of HeLa cells through targeting the NF-κB/CCL-2 pathway. IL-1β/NF-κB/CCL-2 might be a promising treatment target for cervical cancer treatment and prevention.     

NF-kappaB activation during acute Helicobacter pylori infection in mice

Nuclear factor κB (NF-κB) plays a key regulatory role in host cell responses to Helicobacter pylori infection in humans. Although mice are routinely used as a model to study H. pylori pathogenesis, the role of NF-κB in murine cell responses to helicobacters has not been studied in detail. We thus investigated the abilities of different Helicobacter isolates to induce NF-κB-dependent responses in murine gastric epithelial cells (GECs) and in transgenic mice harboring an NF-κB-responsive lacZ reporter gene. H. pylori and Helicobacter felis strains up-regulated the synthesis in mouse GECs of the NF-κB-dependent chemokines KC (CXCL1) and MIP-2 (CXCL2). These responses were cag pathogenicity island (cagPAI) independent and could be abolished by pretreatment with a pharmacological inhibitor of NF-κB. Consistent with the in vitro data, experimental Helicobacter infection of transgenic mice resulted in increased numbers of GECs with nuclear β-galactosidase activity, which is indicative of specific NF-κB activation. The numbers of β-galactosidase-positive cells in mice were significantly increased at day 1 postinoculation with wild-type H. pylori strains harboring or not harboring a functional cagPAI, compared to naive animals (P = 0.007 and P = 0.04, respectively). Strikingly, however, no differences were observed in the levels of gastric NF-κB activation at day 1 postinoculation with H. felis or at day 30 or 135 postinoculation with H. pylori. This work demonstrates for the first time the induction of NF-κB activation within gastric mucosal cells during acute H. pylori infection. Furthermore, the data suggest that helicobacters may be able to regulate NF-κB signaling during chronic infection.     

Dihydroartemisinin inhibits activation of the AIM2 inflammasome pathway and NF-κB/HIF-1α/VEGF pathway by inducing autophagy in A431 human cutaneous squamous cell carcinoma cells

The therapeutic effect of dihydroartemisinin (DHA) against cutaneous squamous cell carcinoma (cSCC) has been previously demonstrated; however, the underlying mechanism remains unclear. This study sought to verify the therapeutic effect of DHA against cSCC and explore its underlying mechanism in A431 cSCC cells. This study reported that DHA inhibited A431 cells proliferation in a time- and concentration-dependent manner and promoted A431 cells apoptosis. Moreover, DHA inhibited the invasion and migration of A431 cells. Mechanistically, DHA promoted autophagy and inhibited activation of the absent in melanoma 2 (AIM2) inflammasome pathway and NF-κB/HIF-1α/VEGF pathway. Treatment of A431 cells with the mTOR inhibitor, and autophagy promoter, rapamycin also inhibited these two pathways. In conclusion, DHA inhibited activation of the AIM2 inflammasome pathway and NF-κB/HIF-1α/VEGF pathway by promoting autophagy in A431 cells, thus accounting for its therapeutic effect. Induction of autophagy by DHA may be mediated by inhibiting the mTOR pathway and promoting reactive oxygen species production.     

Toll-like receptor 2 recognition of the microsporidia Encephalitozoon spp. induces nuclear translocation of NF-kappaB and subsequent inflammatory responses

Microsporidia are obligate intracellular parasites that are ubiquitous in nature and have been recognized as causing an important emerging disease among immunocompromised individuals. Limited knowledge exists about the immune response against these organisms, and virtually nothing is known about the receptors involved in host recognition. Toll-like receptors (TLR) are pattern recognition receptors that bind to specific molecules found on pathogens and signal a variety of inflammatory responses. In this study, we show that both Encephalitozoon cuniculi and Encephalitozoon intestinalis are preferentially recognized by TLR2 and not by TLR4 in primary human macrophages. This is the first demonstration of host receptor recognition of any microsporidian species. TLR2 ligation is known to activate NF-κB, resulting in inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8). We found that the infection of primary human macrophages leads to the nuclear translocation of NF-κB in as early as 1 h and the subsequent production of TNF-α and IL-8. To verify the direct role of TLR2 parasite recognition in the production of these cytokines, the receptor was knocked down in primary human macrophages using small interfering RNA. This knockdown resulted in decreases in both the nuclear translocation of NF-κB and the levels of TNF-α and IL-8 after challenge with spores. Taken together, these experiments directly link the initial inflammatory response induced by Encephalitozoon spp. to TLR2 stimulation in human macrophages.