Ketogenic diet alleviates cognitive dysfunction and neuroinflammation in APP/PS1 mice via the Nrf2/HO-1 and NF-κB signaling pathways

Alzheimer's disease is a progressive neurological disorder characterized by cognitive decline and chronic inflammation within the brain.The ketogenic diet,a widely recognized therapeutic intervention for refractory epilepsy,has recently been proposed as a potential treatment for a variety of neurological diseases,including Alzheimer's disease.However,the efficacy of ketogenic diet in treating Alzheimer's disease and the underlying mechanism remains unclear.The current investigation aimed to explore the effect of ketogenic diet on cognitive function and the underlying biological mechanisms in a mouse model of Alzheimer's disease.Male amyloid precursor protein/presenilin 1(APP/PS1)mice were randomly assigned to either a ketogenic diet or control diet group,and received their respective diets for a duration of 3 months.The findings show that ketogenic diet administration enhanced cognitive function,attenuated amyloid plaque formation and proinflammatory cytokine levels in APP/PS1 mice,and augmented the nuclear factor-erythroid 2-p45 derived factor 2/heme oxygenase-1 signaling pathway while suppressing the nuclear factor-kappa B pathway.Collectively,these data suggest that ketogenic diet may have a therapeutic potential in treating Alzheimer's disease by ameliorating the neurotoxicity associated with Aβ-induced inflammation.This study highlights the urgent need for further research into the use of ketogenic diet as a potential therapy for Alzheimer's disease.

Atsttrin reduces lipopolysaccharide-induced neuroinflammation by inhibiting the nuclear factor kappa B signaling pathway

Progranulin is closely related to neuronal survival in a neuroinflammatory mouse model and attenuates inflammatory reactions. Atsttrin is an engineered protein composed of three progranulin fragments and has been shown to have an effect similar to that of progranulin. Atsttrin has anti-inflammatory actions in multiple arthritis mouse models, and it protects against further arthritis development. However, whether Atsttrin has a role in neuroinflammation remains to be elucidated. In this study, we produced a neuroinflammatory mouse model by intracerebroventricular injection of 1 μL lipopolysaccharide(10 μg/μL). Atsttrin(2.5 mg/kg) was administered via intraperitoneal injection every 3 days over a period of 7 days before intracerebroventricular injection of 1 μL lipopolysaccharide(10 μg/μL). In addition, astrocyte cultures were treated with 0, 100 or 300 ng/mL lipopolysaccharide, with 200 ng/mL Atsttrin simultaneously. Immunohistochemistry, enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction were performed to examine the protein and mRNA levels of inflammatory mediators and to assess activation of the nuclear factor kappa B signaling pathway. Progranulin expression in the brain of wild-type mice and in astrocyte cultures was increased after lipopolysaccharide administration. The protein and mRNA expression levels of tumor necrosis factor-α, interleukin-1β and inducible nitric oxide synthase were increased in the brain of progranulin knockout mice after lipopolysaccharide administration. Atsttrin treatment reduced the lipopolysaccharide-induced increase in the protein and mRNA levels of tumor necrosis factor-α, interleukin-1β, matrix metalloproteinase-3 and inducible nitric oxide synthase in the brain of progranulin knockout mice. Atsttrin also reduced the expression of cyclooxygenase-2, inducible nitric oxide synthase and matrix metalloproteinase 3 mRNA in lipopolysaccharide-treated astrocytes in vitro, and decreased the concentration of tumor necrosis factor α and interleukin-1β in the supernatant. Furthermore, Atsttrin significantly reduced the levels of phospho-nuclear factor kappa B inhibitor α in the brain of lipopolysaccharide-treated progranulin knockout mice and astrocytes, and it decreased the expression of nuclear factor kappa B2 in astrocytes. Collectively, our findings show that the anti-neuroinflammatory effect of Atsttrin involves inhibiton of the nuclear factor kappa B signaling pathway, and they suggest that Atsttrin may have clinical potential in neuroinflammatory therapy.

Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney,and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

BACKGROUND Jianpi Gushen Huayu Decoction(JPGS)has been used to clinically treat diabetic nephropathy(DN)for many years.However,the protective mechanism of JPGS in treating DN remains unclear.AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN.METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model.We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics.Furthermore,we examined the effects of JPGS on c-Jun N-terminal kinase(JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3).RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress.Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice.A total of 51 differential metabolites were screened.Pathway analysis results indicated that nine pathways significantly changed between the control and model groups,while six pathways significantly altered between the model and JPGS groups.Pathways related to cysteine and methionine metabolism;alanine,tryptophan metabolism;aspartate and glutamate metabolism;and riboflavin metabolism were identified as the key pathways through which JPGS affects DN.Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-κB/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors.CONCLUSION JPGS could markedly treat mice with streptozotocin(STZ)-induced DN,which is possibly related to the regulation of several metabolic pathways found in kidneys.Furthermore,JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-κB/NLRP3 pathway and inhibit JNK/P38 pathwaymediated apoptosis in DN mice.

Acacetin protects against cerebral ischemia-reperfusion injury via the NLRP3 signaling pathway

Acacetin(5,7-dihydroxy-4′-methoxyflavone), a potential neuroprotective agent, has an inhibitory effect on lipopolysaccharide-induced neuroinflammatory reactions. However, whether acacetin has an effect on inflammatory corpuscle 3(NLRP3) after cerebral ischemia-reperfusion injury has not been fully determined. This study used an improved suture method to establish a cerebral ischemia-reperfusion injury model in C57BL/6 mice. After ischemia with middle cerebral artery occlusion for 1 hour, reperfusion with intraperitoneal injection of 25 mg/kg of acacetin(acacetin group) or an equal volume of saline(0.1 mL/10 g, middle cerebral artery occlusion group) was used to investigate the effect of acacetin on cerebral ischemia-reperfusion injury. Infarct volume and neurological function scores were determined by 2,3,5-triphenyltetrazolium chloride staining and the Zea-Longa scoring method. Compared with the middle cerebral artery occlusion group, neurological function scores and cerebral infarction volumes were significantly reduced in the acacetin group. To understand the effect of acacetin on microglia-mediated inflammatory response after cerebral ischemia-reperfusion injury, immunohistochemistry for the microglia marker calcium adapter protein ionized calcium-binding adaptor molecule 1(Iba1) was examined in the hippocampus of ischemic brain tissue. In addition, tumor necrosis factor-α, interleukin-1β, and interleukin-6 expression in ischemic brain tissue of mice was quantified by enzyme-linked immunosorbent assay. Expression of Iba1, tumor necrosis factor-α, interleukin-1β and interleukin-6 was significantly lower in the acacetin group compared with the middle cerebral artery occlusion group. Western blot assay results showed that expression of Toll-like receptor 4, nuclear factor kappa B, NLRP3, procaspase-1, caspase-1, pro-interleukin-1β, and interleukin-1β were significantly lower in the acacetin group compared with the middle cerebral artery occlusion group. Our findings indicate that acacetin has a protective effect on cerebral ischemia-reperfusion injury, and its mechanism of action is associated with inhibition of microglia-mediated inflammation and the NLRP3 signaling pathway.

Neuroprotection mediated by the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage

The Wnt/Frizzled signaling pathway participates in many inflammation-linked diseases. However, the inflammatory response mediated by the Wnt/Frizzled signaling pathway in experimental subarachnoid hemorrhage has not been thoroughly investigated. Consequently, in this study, we examined the potential role of the Wnt/Frizzled signaling pathway in early brain injury in rat models of subarachnoid hemorrhage.Simultaneously, possible neuroprotective mechanisms were also investigated. Experimental subarachnoid hemorrhage rat models were induced by injecting autologous blood into the prechiasmatic cistern. Experiment 1 was designed to examine expression of the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. In total, 42 adult rats were divided into sham(injection of equivalent volume of saline), 6-, 12-, 24-, 48-, 72-hour, and 1-week subarachnoid hemorrhage groups. Experiment 2 was designed to examine neuroprotective mechanisms of the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. Rats were treated with recombinant human Wnt1(rhwnt1), small interfering Wnt1(siwnt1) RNA, and monoclonal antibody of Frizzled1(anti-Frizzled1) at 48 hours after subarachnoid hemorrhage. Expression levels of Wnt1, Frizzled1, β-catenin, peroxisome proliferator-activated receptor-γ, CD36, and active nuclear factor-κB were examined by western blot assay and immunofluorescence staining. Microglia type conversion and inflammatory cytokine levels in brain tissue were examined by immunofluorescence staining and enzyme-linked immunosorbent assay. Our results show that compared with the sham group, expression levels of Wnt1, Frizzled1, and β-catenin were low and reduced to a minimum at 48 hours, gradually returning to baseline at 1 week after subarachnoid hemorrhage. rhwnt1 treatment markedly increased Wnt1 expression and alleviated subarachnoid hemorrhage-induced early brain injury(within 72 hours), including cortical cell apoptosis, brain edema, and neurobehavioral deficits, accompanied by increasing protein levels of β-catenin, CD36, and peroxisome proliferator-activated receptor-γ and decreasing protein levels of nuclear factor-κB. Of note, rhwnt1 promoted M2-type microglia conversion and inhibited release of inflammatory cytokines(interleukin-1β, interleukin-6, and tumor necrosis factor-α). In contrast, siwnt1 RNA and anti-Frizzled1 treatment both resulted in an opposite effect. In conclusion, the Wnt/Frizzled1 signaling pathway may participate in subarachnoid hemorrhage-induced early brain injury via inhibiting the inflammatory response, including regulating microglia type conversion and decreasing inflammatory cytokine release. The study was approved by the Animal Ethics Committee of Anhui Medical University and First Affiliated Hospital of USTC,Division of Life Sciences and Medicine, University of Science and Technology of China(approval No. LLSC-20180202) in May 2017.

Testosterone alleviates tumor necrosis factor-alpha-mediated tissue factor pathway inhibitor downregulation via suppression of nuclear factor-kappa B in endothelial cells

We have observed earlier that testosterone at physiological concentrations can stimulate tissue factor pathway inhibitor(TFPI)gene expression through the androgen receptor in endothelial cells.This study further investigated the impact of testosterone on TFPI levels in response to inflammatory cytokine tumor necrosis factor-alpha(TNF-α).Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-α.TFPI protein and mRNA levels were assessed by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction.To study the cellular mechanism of testosterone’s action,nuclear factor-kappa B(NF-κB)translocation was confirmed by electrophoretic mobility shift assays.We found that after NF-κB was activated by TNF-α,TFPI protein levels declined significantly by 37.3%compared with controls(P<0.001),and the mRNA levels of TFPI also decreased greatly(P<0.001).A concentration of 30 nmol L-1 testosterone increased the secretion of TFPI compared with the TNF-α-treated group.NF-κB DNA-binding activity was significantly suppressed by testosterone(P<0.05).This suggests that physiological testosterone concentrations may exert their antithrombotic effects on TFPI expression during inflammation by downregulating NF-κB activity.

Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway

Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae （Gegen）, has been reported to inhibit neuronal apoptosis and play an anti-inflammatory role in focal cerebral ischemia model rats. Recent findings regarding stroke pathophysiology have recognized that anti-inflammation is an important target for the treatment of ischemic stroke. The cholinergic anti-inflammatory pathway is a highly robust neural-immune mechanism for inflammation control. This study was to investigate whether activating the cholinergic anti-inflammatory pathway can be involved in the mechanism of inhibiting the inflammatory response during puerarin-induced cerebral ischemia/reperfusion in rats. Results showed that puerarin pretreatment （intravenous injection） re- duced the ischemic infarct volume, improved neurological deficit after cerebral ischemia/reperfusion and decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-a in brain tissue. Pretreatment with puerarin （intravenous injection） attenuated the inflammatory response in rats, which was accompanied by janus-activated kinase 2 （JAK2） and signal transducers and activators of transcription 3 （STAT3） activation and nuclear factor kappa B （NF-KB） inhibition. These observa- tions were inhibited by the alpha7 nicotinic acetylcholine receptor （a7nAchR） antagonist a-bungarotoxin （a-BGT）. In addition, puerarin pretreatment increased the expression of a7nAchR mRNA in ischemic cerebral tissue. These data demonstrate that puerarin pretreatment strongly protects the brain against cerebral ischemia/reperfusion injury and inhibits the inflammatory re- sponse. Our results also indicated that the anti-inflammatory effect of puerarin may partly be medi- ated through the activation of the cholinergic anti-inflammatory pathway.

Electroacupuncture Delays Cartilage Degeneration by Modulating Nuclear Factor-κB Signaling Pathway

Objective: To illustrate the molecular mechanisms underlying the therapeutic effects of electroacupuncture(EA) on knee osteoarthritis(OA). Methods: Twenty-seven six-month-old New Zealand white rabbits were allocated into three groups in accordance with a random number table: normal group(no surgery-induced OA;without treatment), model group(surgery-induced OA;without treatment) and EA group [surgery-induced OA;received treatment with EA at acupoints Dubi(ST 35) and Neixiyan(EX-LE 5), 30 min twice a day]. After eight consecutive weeks of treatment, the histopathological alterations in cartilage were observed using optical microscopy and transmission electron microscopy, cartilage degeneration was evaluated by modified Mankin’s score principles, the synovial fluid concentration of interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α) and matrix metalloproteinase-3(MMP-3) were evaluated by enzyme-linked immunosorbent assay, and the protein expression levels of IL-1β, IL-6, TNF-α, MMP-3, IκB kinase-β(IKK-β), nuclear factor of α light polypeptide gene enhancer in B-cells inhibitor α(IκB-α) and nuclear factor-κB(NF-κB) p65 were quantified by Western blot analysis. Results: EA treatment significantly improved cartilage structure arrangement and reduced cellular degeneration. The IL-1β, IL-6, TNF-α and MMP-3 of synovial fluid in the EA-treated group were significantly decreased compared with the model group(all P<0.01). Compared with the model group, the IL-1β, IL-6, TNF-α, MMP-3, IKK-β and NF-κB p65 protein expressions in cartilage of EA-treated group were significantly decreased(all P<0.01), whereas IκB-α expression was significantly up-regulated(P<0.01). Conclusion: EA treatment may delay cartilage degeneration by down-regulating inflammatory factors through NF-κB signaling pathway, which may, in part, explain its clinical efficacy in the treatment of knee OA.

Medicinal plant activity on Helicobacter pylori related diseases

More than 50% of the world population is infected with Helicobacter pylori (H. pylori). The bacterium highly links to peptic ulcer diseases and duodenal ulcer, which was classified as a group&#x02005;I&#x02005;carcinogen in 1994 by the WHO. The pathogenesis of H. pylori is contributed by its virulence factors including urease, flagella, vacuolating cytotoxin A (VacA), cytotoxin-associated gene antigen (Cag A), and others. Of those virulence factors, VacA and CagA play the key roles. Infection with H. pylori vacA-positive strains can lead to vacuolation and apoptosis, whereas infection with cagA-positive strains might result in severe gastric inflammation and gastric cancer. Numerous medicinal plants have been reported for their anti-H. pylori activity, and the relevant active compounds including polyphenols, flavonoids, quinones, coumarins, terpenoids, and alkaloids have been studied. The anti-H. pylori action mechanisms, including inhibition of enzymatic (urease, DNA gyrase, dihydrofolate reductase, N-acetyltransferase, and myeloperoxidase) and adhesive activities, high redox potential, and hydrophilic/hydrophobic natures of compounds, have also been discussed in detail. H. pylori-induced gastric inflammation may progress to superficial gastritis, atrophic gastritis, and finally gastric cancer. Many natural products have anti-H. pylori-induced inflammation activity and the relevant mechanisms include suppression of nuclear factor-&#x003ba;B and mitogen-activated protein kinase pathway activation and inhibition of oxidative stress. Anti-H. pylori induced gastric inflammatory effects of plant products, including quercetin, apigenin, carotenoids-rich algae, tea product, garlic extract, apple peel polyphenol, and finger-root extract, have been documented. In conclusion, many medicinal plant products possess anti-H. pylori activity as well as an anti-H. pylori-induced gastric inflammatory effect. Those plant products have showed great potential as pharmaceutical candidates for H. pylori eradication and H. pylori induced related gastric disease prevention.

The Regulatory Effects of Polyporus Polysaccharide on the Nuclear Factor Kappa B Signal Pathway of Bladder Cancer Cells Stimulated by Bacillus Calmette-Guerin

Objective：To detect the effects of Polyporus polysaccharide（PPS）,Bacillus Calmette-Guerin （BCG）,and their combination on the nuclear factor kappa B（NF-κB）signaling pathway associated-gene expression and investigate the molecular mechanisms of the toxic-reducing effect of PPS in coordination with BCG against bladder cancer.Methods：After T739 cells were treated with PPS,BCG and their combination, the changes in mRNA and protein expression of inhibitor of kappa B kinase beta（IKKβ）,NF-κB subunit p65 （NF-κB p65）,intracellular adhesion molecule 1（ICAM1）and chemokine（C-c motif）ligand 2（CCL2）in bladder cancer cell line T739 were determined by relative quantitative real-time PCR,Western blot,and flow cytometry （FCM）.NF-κB p65 DNA-binding activity in T739 cell was detected by biotinylated probe-ELISA,and NF-κB p65 nuclear expression in T739 cell was observed by immunohistochemistry.Results：Compared with the T739 control group,the mRNA expression of IKBKB（IKKβ）,Rel A（NF-κB p65）,ICAM1 and CCL2 in T739 cells treated with BCG were increased obviously（Ratio2.0）,as well as the expression of IKKβ,CCL2 and ICAM1 proteins.Meanwhile,NF-κB p65 DNA-binding activity and NF-κB p65 nuclear expression in T739 cells treated with BCG were up-regulated significantly（P0.05）.Compared with the control,the increased expression in T739 cells were simultaneously down-regulated after PPS treatment,except for ICAM1 protein expression.With cells treated with a combination of BCG and PPS,the expression of genes associated with the NF-κB signaling pathway,such as IKBKB,ICAM1 and CCL2,were all down-regulated compared to the BCG group,as well as Rel A mRNA expression,NF-κB p65 DNA-binding activity and NF-κB p65 nuclear expression.Conclusions： PPS could inhibit the over-activation of the NF-κB signaling pathway induced by BCG in bladder cancer cells and accordingly attenuate the adverse reactions to BCG therapy.

Role of Helicobacter pylori in gastric mucosa-associated lymphoid tissue lymphomas

Mucosa-associated lymphoid tissue (MALT) lymphoma is an indolent extranodal marginal zone B-cell lymphoma, originating in acquired MALT that is induced in mucosal barriers as part of a normal adaptive immune response to a chronic immunoinflammatory stimulus, most notably chronic infection by Helicobacter pylori (H. pylori). This antigenic stimulation initially leads to lymphoid hyperplasia; the acquisition of additional genetic aberrations culminates in the activation of intracellular survival pathways, with disease progression due to proliferation and resistance to apoptosis, and the emergence of a malignant clone. There are descriptions of MALT lymphomas affecting practically every organ and system, with a marked geographic variability partially attributable to the epidemiology of the underlying risk factors; nevertheless, the digestive system (and predominantly the stomach) is the most frequently involved location, reflecting the gastrointestinal tract’s unique characteristics of contact with foreign antigens, high mucosal permeability, large extension and intrinsic lymphoid system. While early-stage gastric MALT lymphoma can frequently regress after the therapeutic reversal of the chronic immune stimulus through antibiotic eradication of H. pylori infection, the presence of immortalizing genetic abnormalities, of advanced disease or of eradication-refractoriness requires a more aggressive approach which is, presently, not consensual. The fact that MALT lymphomas are rare neoplasms, with a worldwide incidence of 1-1.5 cases per 105 population, per year, limits the ease of accrual of representative series of patients for robust clinical trials that could sustain informed evidence-based therapeutic decisions to optimize the quality of patient care.

Temporal changes in inflammatory mitochondria-enriched microRNAs following traumatic brain injury and effects of miR-146a nanoparticle delivery

MicroRNAs(miRNAs)are small non-coding RNA molecules that regulate post-transcriptional gene expression and contribute to all aspects of cellular function.We previously reported that the activities of several mitochondria-enriched miRNAs regulating inflammation(i.e.,miR-142-3p,miR-142-5p,and miR-146a)are altered in the hippocampus at 3–12 hours following a severe traumatic brain injury.In the present study,we investigated the temporal expression profile of these inflammatory miRNAs in mitochondria and cytosol fractions at more chronic post-injury times following severe controlled cortical impact injury in rats.In addition,several inflammatory genes were analyzed in the cytosol fractions.The analysis showed that while elevated levels were observed in cytoplasm,the mitochondria-enriched miRNAs,miR-142-3p and miR-142-5p continued to be significantly reduced in mitochondria from injured hippocampi for at least 3 days and returned to near normal levels at 7 days post-injury.Although not statistically significant,miR-146a also remained at reduced levels for up to 3 days following controlled cortical impact injury,and recovered by 7 days.In contrast,miRNAs that are not enriched in mitochondria,including miR-124a,miR-150,miR-19b,miR-155,and miR-223 were either increased or demonstrated no change in their levels in mitochondrial fractions for 7 days.The one exception was that miR-223 levels were reduced in mitochondria at 1 day following injury.No major alterations were observed in sham operated animals.This temporal pattern was unique to mitochondria-enriched miRNAs and correlated with injury-induced changes in mitochondrial bioenergetics as well as expression levels of several inflammatory markers.These observations suggested a potential compartmental re-distribution of the mitochondria-enriched inflammatory miRNAs and may reflect an intracellular mechanism by which specific miRNAs regulate injury-induced inflammatory signaling.To test this,we utilized a novel peptide-based nanoparticle strategy for in vitro and in vivo delivery of a miR-146a mimic as a potential therapeutic strategy for targeting nuclear factor-kappa B inflammatory modulators in the injured brain.Nanoparticle delivery of miR-146a to BV-2 or SH-SY5Y cells significantly reduced expression of TNF receptor-associated factor 6(TRAF6)and interleukin-1 receptor-associated kinase 1(IRAK1),two important modulators of the nuclear factor-kappa B(NF-κB)pro-inflammatory pathway.Moreover,injections of miR-146a containing nanoparticles into the brain immediately following controlled cortical impact injury significantly reduced hippocampal TNF receptor-associated factor 6 and interleukin-1 receptor-associated kinase 1 levels.Taken together,our studies demonstrate the subcellular alteration of inflammatory miRNAs after traumatic brain injury and establish proof of principle that nanoparticle delivery of miR-146a has therapeutic potential for modulating pro-inflammatory effectors in the injured brain.All of the studies performed were approved by the University of Kentucky Institutional Animal Care and Usage Committee(IACUC protocol#2014-1300)on August 17,2017.

Cross-talk between microRNA-let7c and transforming growth factor-β2 during epithelial-to-mesenchymal transition of retinal pigment epithelial cells

AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P<0.05). In turn, overexpressed miR-let7 c significantly inhibited TGF-β2-induced EMT(P<0.05). However, miR-let7 c was unable to inhibit TGF-β2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P<0.01). CONCLUSION: The miR-let7 c regulates TGF-β2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.

DNA damage-induced nuclear factor-kappa B activation and its roles in cancer progression

DNA damage is a vital challenge to cell homeostasis.Cellular responses to DNA damage(DDR)play essential roles in maintaining genomic stability and survival,whose failure could lead to detrimental consequences such as cancer development and aging.Nuclear factor-kappa B(NF-kB)is a family of transcription factors that plays critical roles in cellular stress response.Along with p53,NF-kB modulates transactivation of a large number of genes which participate in various cellular processes involved in DDR.Here the authors summarize the recent progress in understanding DNA damage response and NF-kB signaling pathways.This study particularly focuses on DNA damage-induced NF-kB signaling cascade and its physiological and pathological significance in B cell development and cancer therapeutic resistance.The authors also discuss promising strategies for selectively targeting this genotoxic NF-kB signaling aiming to antagonize acquired resistance and resensitize refractory cancer cells to cytotoxic treatments。

Effect of Allicin on the Formation of Kidney Stones in Rats by Regulating the Expression of OPN and NF-κB Signaling Pathway

Objective:To investigate the effect of allicin on the formation of kidney stones in rats by regulating the expression of osteopontin(OPN)and nuclear factor-κB(NF-κB)signaling pathway.Methods:A total of 50 healthy adult male SD rats with SPF grade were selected and divided into five groups by random number and computer random combination,with 10 rats in each group.Except the blank group,the other four groups were given 2 m L/d mixed solution of 1%ethylene glycol+2%ammonium chloride to construct the nephrolith model.During the modeling process,the blank group and the model group were given normal saline by gavage.The positive group was given 600 mg/(kg·d)of potassium sodium hydrogen citrate granules by gavage,the low-dose group was given 7.5 mg/(kg·d)of allicin by gavage,and the high-dose group was given 15 mg/(kg·d)of allicin by gavage.After administration,renal function,urine related indicators,calcium oxalate crystallization score,OPN protein expression and NF-κB signaling pathwayrelated protein expression were observed and compared among the five groups of rats.Results:There were significant differences in kidney index,urea nitrogen(BUN)and blood creatinine(Cr)levels among the five groups(P<0.05).There were no differences in kidney index,BUN and Cr levels between the high-dose group and the positive group(P>0.05),and were all lower than those in the model group and low-dose group(P<0.05).There were significant differences in the levels of oxalic acid(OA),calcium(Ca),magnesium(Mg),and phosphorus(P)in the urine of five groups of rats(P<0.05).The high-dose group showed no difference in the levels of OA,Ca,Mg,and P compared to the positive control group(P>0.05),and all were lower than the model group and low-dose group(P<0.05).There were significant differences in the scores of calcium oxalate crystallization and the expression of OPN protein in the five groups(P<0.05).There was no difference in the scores of calcium oxalate crystallization between the high-dose group and the positive group(P>0.05).The expression of OPN protein was higher than that in the positive group(P<0.05),and both were lower than that in the model group and low-dose group(P<0.05).There were significant differences in the expression levels of NF-κB inhibitory protein-α(IκB-α)and NF-κB in five groups(P<0.05),and the expression levels of IκB-αand NF-κB in the high-dose group were lower than those in the model group,positive control group,and low-dose group(P<0.05).Conclusion:Allicin may inhibit the formation of kidney stones in rats by down-regulating the expression levels of OPN and NF-κB signaling pathway-related proteins,and a high dose of allicin can obtain a similar effect of kidney stones inhibition as that of potassium sodium hydrogen citrate granules.

Tea Polyphenols Reducing Lipopolysaccharide-induced Inflammatory Responses in RAW264.7 Macrophages via NF-κB Pathway

The anti-inflammatory activity of tea polyphenols(TPs)in RAW264.7 macrophages stimulated by lipopolysaccharide(LPS)was investigated in this paper.RAW264.7 macrophages were treated with different concentrations of TP(0,12.5,25,50,100,and 200μg/mL)and then stimulated by LPS.Another blank control group was set up.The expression of pro-inflammatory cytokines associated with the nuclear factor-kappa B(NF-κB)signaling pathway was investigated before and after TP treatment.Pretreatment of RAW264.7 cells with TP decreased the expression of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin 1 beta(IL-1β)pro-inflammatory cytokines.In addition,TP inhibited the phosphorylation of p65 and IκB by blocking the phosphorylation and the degradation of NF-κB inhibitor protein.In conclusion,TP exerts anti-inflammatory effects by regulating the release of inflammatory mediators via the NF-κB signaling pathway.

Clinical heterogeneity of NLRP12-associated autoinflammatory diseases

Nod-like receptor family pyrin domain-containing protein 12 (NLRP12) is one of the critical pattern recognition receptors which participates in the regulation of multiple inflammatory responses. Mutations in NLRP12 cause exceptionally rare NLRP12-associated autoinflammatory disease (NLRP12-AID). So far, very few patients with NLRP12-AID have been identified worldwide;therefore, data on the clinical phenotype and genetic profile are limited. In this study, we reported 10 patients who presented mainly with periodic fever syndrome or arthritis. Next-generation sequencing (NGS) identified 6 heterozygous mutations of NLRP12, including 2 novel null mutations. Of the patients, some with same mutations showed different clinical features. Compared to healthy controls, the increased levels of cytokines were revealed in the patients' plasmas, as well as in the supernatants of patients’ cells stimulated with lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). The missense mutations did not change the protein expression;but decreased level of NLRP12 protein was shown in the null mutations. And in vitro expression assay demonstrated a truncating protein induced by the frameshift mutation. Further functional studies revealed the deleterious effect of mutations on nuclear factor-kappa B (NF-κB) signaling. Both the null and missense mutations impaired their inhibition of NF-κB activation induced by p65. Collectively, this study reported a relatively large NLRP12-AID case series. Our findings expand the clinical spectrum, and reinforce the diversity of genetic mutations and clinical phenotypes. The NLRP12-associated disorder should be considered when autoinflammatory diseases are encountered in the clinical practice, especially for patients presenting with periodic fever but no other genetic cause identified.

Protective Effect of Total Alkaloids of Sophora Alopecuroides on Dextran Sulfate Sodium-Induced Chronic Colitis

Objective：To investigate the effect of total alkaloids of Sophora alopecuroides（TASA） on dextran sulfate sodium（DSS）-induced colitis in mice.Methods：Chronic experimental colitis was induced by administration of 4 cycles of 4%DSS.Fifty mice were randomly distributed into 4 groups（normal,DSS,DSS/high-dose TASA, and DSS/low-dose TASA groups） by a random number table with body weight stratification.Mice in the normal group（n=11） and DSS-induced colitis control group（n=15） received control treatment of 20 mL/kg distilled water; DSS plus TASA high- and low-dose groups（n=12 each） were treated with TASA solution（20 mL/kg） at the doses of 60 mg/kg and 30 mg/kg,respectively.The severity of colitis was assessed on the basis of clinical signs, colon length,and histology scores.Moreover,secretory immunoglobulin A（slgA） and haptoglobin（HP） were analyzed by enzyme linked immunosorbent assay;intercellular adhesion molecule 1（ICAM-1） and macrophage-migration inhibitory factor（MIF） gene expressions were analyzed by quantitative reverse transcriptase realtime polymerase chain reaction（qRT-PCR） using SYBA greenⅠ;and nuclear factorκB（NF-κB） expression and activation and p65 interaction with the promoter of ICAM-1 gene were assessed by Western blotting and chromatin immunoprecipitation assay.Results：TASA administration significantly attenuated the damage and substantially reduced HP elevation and maintained the level of cecum slgA.TASA inhibited the ICAM-1 gene expression and had no effect on MIF gene expression.Also,TASA was able to reduce phospho-lκBα（p-lκBα） protein expression;however,it had no effect on the activation of IκB kinaseα（IKKα） and inhibitor of NF-κBα（IκBα）.Moreover,TASA inhibited the p65 recruitment to the ICAM-1 gene promoter.Conclusions：TASA had a protective effect on DSS-induced colitis.Such effect may be associated with its inhibition of NF-κB activation and blockade of NF-κB-regulated transcription activation of proinflammatory mediator gene.