NF-κB P65 Protein Levels Research Articles

Knockout of RIG-I in HEK293 cells by CRISPR/Cas9

We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P < 0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability (P < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.

The role of ischaemia-modified albumin in the prognosis of acute pancreatitis and its correlation with the NF-κB-mediated inflammatory response.

To investigate the correlation between the serum levels of ischaemia-modified albumin (IMA) and disease severity in rats with acute pancreatitis (AP). A rat AP model was established and blood samples from each group were analysed at different time points. After the experiment, the pancreatic tissues of the rats were collected for pathological examination and the measurement of protein levels of NF-κB and NF-κB p65. Serum levels of amylase (α-AMY), tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8 were also compared between groups of rats. The serum IMA concentration in the severe acute pancreatitis (SAP) group was greater than that in the mild acute pancreatitis (MAP) group. The levels of the NF-κB and NF-κB p65 proteins were increased in the MAP and SAP groups in a time-dependent manner. α-AMY, TNF-α and IL-6 were increased at all time points in the MAP and SAP groups. The increases were greatest at 24 h in the SAP group. In terms of pathological changes in the pancreas, renal and lung tissues, the damage in the SAP group was more obvious than that in the MAP group. Serum IMA level was associated with inflammatory markers and NF-κB p65 in rats with AP.

Protopine-Type Alkaloids Alleviate Lipopolysaccharide-Induced Intestinal Inflammation and Modulate the Gut Microbiota in Mice.

In this study, we assessed the therapeutic effects of Macleaya cordata (Willd). R. Br.-derived protopine-type alkaloids (MPTAs) in a mouse model of lipopolysaccharide (LPS)-induced intestinal inflammation. The experimental design involved the allocation of mice into distinct groups, including a control group, a model group treated with 6 mg/kg LPS, a berberine group treated with 50 mg/kg berberine hydrochloride and low-, medium- and high-dose MPTA groups treated with 6, 12 and 24 mg/kg MPTAs, respectively. Histological analysis of the ileum, jejunum and duodenum was performed using Hematoxylin and Eosin (H&E) staining. Moreover, the quantification of intestinal goblet cells (GCs) was performed based on PAS staining. The serum levels of IL-1β, IL-6, IL-8 and TNF-α were quantified using an enzyme-linked immunosorbent assay (ELISA), while the mRNA levels of TLR4, NF-κB p65, NLRP3, IL-6 and IL-1β were assessed using quantitative PCR (qPCR). The protein levels of TLR4, Md-2, MyD88, NF-κB p65 and NLRP3 were determined using Western blotting. Furthermore, the 16S rDNA sequences of bacterial taxa were amplified and analysed to determine alterations in the gut microbiota of the mice following MPTA treatment. Different doses of MPTAs were found to elicit distinct therapeutic effects, leading to enhanced intestinal morphology and an increased abundance of intestinal GCs. A significant decrease was noted in the levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α). Additionally, the protein levels of TLR4, MyD88, NLRP3 and p-p65/p65 were markedly reduced by MPTA treatment. Furthermore, 16S rDNA sequencing analysis revealed that the administration of 24 mg/kg MPTAs facilitated the restoration of microbial composition.

2,5-Dihydroxyacetophenone attenuates acute kidney injury induced by intra-abdominal infection in rats.

As one of the most serious complications of sepsis, acute kidney injury (AKI) is pathologically associated with excessive inflammation. 2,5-Dihydroxyacetophenone (DHAP) is isolated from Radix rehmanniae praeparataand exhibit potent anti-inflammatory property. This research aimed at determining the role of DHAP in sepsis-associated AKI (SA-AKI) and the underlying mechanism. Plasma creatinine (Cre), blood urea nitrogen (BUN), tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels of SA-AKI patients were detected to evaluate their clinical characteristics. SA-AKI rat models were established by using caecum ligation puncture (CLP) surgery. CLP-induced rats were administered via oral gavage with 20 or 40 mg DHAP after 2 h of CLP surgery. Subsequently, survival rates, serum indexes, histopathological changes, inflammatory factors, renal function indexes and extracellular regulated protein kinases (ERK) and nuclear factor-κB (NF-κB) signalling pathways were detected. SA-AKI patients exhibited markedly higher levels of plasma Cre, BUN, TNF-α and IL-1β than healthy people. Compared with sham rats, CLP-induced septic rats showed significantly decreased survival rate, increased serum lactate dehydrogenase activity and serum lactate level, obvious renal histopathological injury, upregulated TNF-α, IL-1β and TGF-β1 levels, elevated serum creatinine, BUN and serum cystatin C concentrations, serum neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 levels and reduced renal artery blood flow. All the above CLP-induced changes in septic rats were mitigated after DHAP administration. Additionally, CLP-induced elevation in phosphorylated-ERK1/2 and nuclear NF-κB p65 protein levels was inhibited by DHAP treatment. DHAP hinders SA-AKI progression in rat models by inhibiting ERK and NF-κB signalling pathways.

Antiproliferative Effect of 7-Ketositosterol in Breast and Liver Cancer Cells: Possible Impact on Ceramide, Extracellular Signal-Regulated Kinases, and Nuclear Factor Kappa B Signaling Pathways.

Background: This study aimed to examine the effect of 7-Ketositosterol (7-KSS), on sphingomyelin/ceramide metabolites and apoptosis in human breast MCF-7 and human liver HepG2 cancer cells. Methods: Anti-proliferative effects of 7-KSS treatment were assessed at different concentrations and periods. Cell viability was assessed through MTT analysis, whereas the levels of sphingosine-1-phosphate (S1P), sphingomyelins (SMs), and ceramides (CERs) were measured using LC-MS/MS. Phosphorylated 44/42 ERK1/2 and NF-κB p65 (Ser536) protein levels were measured by Western blot analysis and immunofluorescence staining. Apoptosis was evaluated by TUNEL staining and flow cytometric assessment of annexin-V and propidium iodide (PI) labeling. Results: Treatment with 7-KSS significantly decreased cell survival and S1P, p-44/42 ERK1/2, and p-NF-κB p65 protein levels in cancer cells compared to controls. A substantial rise was detected in intracellular amounts of C16-C24 CERs and apoptosis in cancer cells incubated with 7-KSS. Conclusions: 7-KSS stimulated ceramide accumulation and apoptosis while decreasing cell proliferation via downregulating S1P, p-44/42 ERK1/2, and p-NF-κB p65 protein levels.

Integrated network pharmacology and pharmacological investigations to discover the active compounds of Toona sinensis pericarps against diabetic nephropathy

Ethnopharmacological relevanceToona sinensis (A. Juss.) Roem. Is a deciduous woody plant native to Eastern and Southeastern Asia. Different parts of this plant have a long history of being applied as traditional medicines to treat various diseases. The fruits have been used for antidiabetic, antidiabetic nephropathy (anti-DN), antioxidant, anti-inflammatory, and other activities. Aim of the studyThe purpose of this study was to investigate the effects of EtOAc (PEAE) and n-BuOH extracts (PNBE) from T. sinensis pericarps (TSP) on kidney injury in high-fat and high-glucose diet (HFD)/streptozotocin (STZ)-induced DN mice by network pharmacology and pharmacological investigations, as well as to further discover active compounds that could ameliorate oxidative stress and inflammation, thereby delaying DN progression by regulating the Nrf2/NF-κB pathway in high glucose (HG)-induced glomerular mesangial cells (GMCs). Materials and methodsThe targets of TSP 1–16 with DN were analyzed by network pharmacology. HFD/STZ-induced DN mouse models were established to evaluate the effects of PEAE and PNBE. Six groups were divided into normal, model, PEAE100, PEAE400, PNBE100, and PNBE400 groups. Fasting blood glucose (FBG) levels, organ indices, plasma MDA, SOD, TNF-α, and IL-6 levels, as well as renal tissue Nrf2, HO-1, NF-κB, TNF-α, and TGF-β1 levels were determined, along with hematoxylin-eosin (H&E) and immunohistochemical (IHC) analysis of kidney sections. Furthermore, GMC activity screening combined with molecular docking was utilized to discover active compounds targeting HO-1, TNF-α, and IL-6. Moreover, western blotting assays were performed to validate the mechanism of Nrf2 and NF-κB in HG-induced GMCs. ResultsNetwork pharmacology predicted that the main targets of PEAE and PNBE in the treatment of DN include IL-6, INS, TNF, ALB, GAPDH, IL-1β, TP53, EGFR, and CASP3. Additionally, major pathways include AGE-RAGE and IL-17. In vivo experiments, treatment with PEAE and PNBE effectively reduced FBG levels and organ indices, while plasma MDA, SOD, TNF-α, and IL-6 levels, renal tissue Nrf2, HO-1, NF-κB, TNF-α, and TGF-β1 levels, and renal function were significantly improved. PEAE and PNBE significantly improved glomerular and tubule injury, and inhibited the development of DN by regulating the levels of oxidative stress and inflammation-related factors. In vitro experiments, compound 11 strongly activated HO-1 and inhibited TNF-α and IL-6. The molecular docking results revealed that compound 11 exhibited a high binding affinity towards the targets HO-1, TNF-α, and IL-6 (<−6 kcal/mol). Western blotting results showed compound 11 effectively regulated Nrf2 and NF-κB p65 protein levels, and significantly improved oxidative stress damage and inflammatory responses in HG-induced GMCs. ConclusionPEAE, PNBE, and their compounds, especially compound 11, may have the potential to prevent and treat DN, and are promising natural nephroprotective agents.

Electroacupuncture improves low-grade duodenal inflammation in FD rats by reshaping intestinal flora through the NF-κB p65/NLRP3 pyroptosis pathway

Electroacupuncture (EA) is an effective alternative for the treatment of functional dyspepsia (FD). It reduces low-grade duodenal inflammation and improves the symptoms of FD by downregulating the expression of NF-κB p65 and NLRP3, but its mechanism needs to be elucidated. To examine the regulatory effect of electroacupuncture (EA) on intestinal flora and NF-κB p65/NLRP3 pyroptosis pathway in FD rats. The FD rat model was established via multi-factor stress intervention for two weeks. The rats were randomly divided into the NC group, model group, NF-kB inhibitor group (NF-κB inhibitor BAY 11–7082 was administered), EA group, and EA + NF-kB inhibitor group. After 14 days of treatment, the rats were sacrificed, and the protein and mRNA levels of NF-κB p65, IκB, and NLRP3 in the duodenum were evaluated by Western blotting assays and real-time fluorescent quantitative PCR. The Illumina MiSeq sequencing platform was used to analyze the V4 region of the 16S rRNA gene of intestinal flora and predict functional genes. The concentration of short-chain fatty acids (SCFAs) in feces was assessed by metabolomics. EA can decrease low-grade duodenal inflammation and promote gastrointestinal motility in FD rats. This effect is mediated by inhibition of the NF-κB p65/NLRP3 pyroptosis pathway, an increase in the alpha and beta diversity of gut microbiota in the duodenum, an increase in the abundance of beneficial bacteria at the phylum and genus levels, and an increase in the content of SCFAs. The protective effect of EA against FD might involve multiple hierarchy and pathways. EA may remodel intestinal flora by inhibiting the NF-κB p65/NLRP3 pyroptosis pathway, thereby improving low-grade duodenal inflammation in FD rats.

Nephroprotective effect of Ginsenoside Rg1 in lipopolysaccharide-induced sepsis in mice through the SIRT1/NF-κB signaling.

During sepsis, the kidney is one of the most vulnerable organs. Sepsis-associated acute kidney injury (S-AKI) is hallmarked by renal inflammation, apoptosis, and oxidative injury. Ginsenoside Rg1 (Rg1) is a natural product that possesses abundant pharmacological actions and protects against many sepsis-related diseases. Nevertheless, its role and related mechanism in S-AKI remain to be determined. S-AKI was induced using lipopolysaccharide (LPS, 10 mg/kg) via a single intraperitoneal injection. Rg1 (200 mg/kg) was intraperitoneally administered for 3 consecutive days before LPS treatment. For histopathological examination, murine kidney tissues were stained with hematoxylin and eosin. Tubular injury score was calculated to evaluate kidney injury. Serum creatinine and BUN levels were measured for assessing renal dysfunction. The levels and activities of oxidative stress markers (MDA, 4-HNE, PC, GSH, SOD, and CAT) in renal tissue were measured by corresponding kits. Renal cell apoptosis was detected by TUNEL staining. The protein levels of apoptosis-related markers (Bcl-2, Bax, and Cleaved caspase-3), proinflammatory factors, SIRT1, IκBα, p-NF-κB p65, and NF-κB p65 in kidneys were determined using western blotting. Immunofluorescence staining was employed to assess p-NF-κB p65 expression in renal tissues. LPS-induced injury of kidneys and renal dysfunction in mice were ameliorated by Rg1. Rg1 also impeded LPS-evoked renal cell apoptosis in kidneys. Moreover, Rg1 attenuated LPS-triggered inflammation and oxidative stress in kidneys by inhibiting proinflammatory cytokine release, enhancing antioxidant levels and activities, and reducing lipid peroxidation. However, all these protective effects of Rg1 in LPS-induced AKI mice were reversed by EX527, an inhibitor of sirtuin 1 (SIRT1). Mechanistically, Rg1 upregulated SIRT1 protein expression, increased SIRT1 activity, and inactivated NF-κB signaling in the kidney of LPS-induced AKI mice, which was also reversed by EX527. Rg1 ameliorates LPS-induced kidney injury and suppresses renal inflammation, apoptosis, and oxidative stress in mice via regulating the SIRT1/NF-κB signaling.

Mmu-miR-374b-5p modulated inflammatory factors via downregulation of C/EBP β/NF-κB signaling in Kupffer cells during Echinococcus multilocularis infection

BackgroundAlveolar echinococcosis (AE) is an important infectious disease caused by the metacestode larvae of Echinococcus multilocularis, seriously threatening global public health security. Kupffer cells (KCs) play important roles in liver inflammatory response. However, their role in hepatic alveolar echinococcosis has not yet been fully elucidated.MethodsIn this study, qRT-PCR was used to detect the expression level of miR-374b-5p in KCs. The target gene of miR-374b-5p was identified through luciferase reporter assays and loss of function and gains. Critical genes involved in NFκB signaling pathway were analyzed by qRT-PCR and western blot.ResultsThis study reported that miR-374b-5p was significantly upregulated in KCs during E. multilocularis infection and further showed that miR-374b-5p was able to bind to the 3'-UTR of the C/EBP β gene and suppressed its expression. The expression levels of NF-κBp65, p-NF-κBp65 and pro-inflammatory factors including iNOS, TNFα and IL6 were attenuated after overexpression of miR-374b-5p while enhanced after suppression of miR-374b-5p. However, the Arg1 expression level was promoted after overexpression of miR-374b-5p while suppressed after downregulation of miR-374b-5p. Additionally, increased protein levels of NF-κBp65 and p-NF-κBp65 were found in the C/EBP β-overexpressed KCs.ConclusionsThese results demonstrated that miR-374b-5p probably regulated the expression of inflammatory factors via C/EBP β/NF-κB signaling. This finding is helpful to explore the mechanism of inflammation regulation during E. multilocularis infection.Graphical

SAMHD1 dysfunction induces IL-34 expression via NF-κB p65 in neuronal SH-SY5Y cells

Dysfunctional mutations in SAMHD1 cause Aicardi-Goutières Syndrome, an autoinflammatory encephalopathy with elevated interferon-α levels in the cerebrospinal fluid. Whether loss of function mutations in SAMHD1 trigger the expression of other cytokines apart from type I interferons in Aicardi-Goutières Syndrome is largely unclear. This study aimed to explore whether SAMHD1 dysfunction regulated the expression of IL-34, a key cytokine controlling the development and maintenance of microglia, in SH-SY5Y neural cells. We found that downregulation of SAMHD1 in SH-SY5Y cells resulted in the upregulation of IL-34 expression. The protein and mRNA levels of NF-κB p65, the transactivating subunit of a transcription factor NF-κB, were also upregulated in SAMHD1-knockdown SH-SY5Y cells. It was further found SAMHD1 knockdown in SH-SY5Y cells induced an upregulation of IL-34 expression through the canonical NF-κB-dependent pathway in which NF-κB p65, IKKα/β and the NF-κB inhibitor IκBα were phosphorylated. Moreover, knockdown of SAMHD1 in SH-SY5Y cells led to the translocation of NF-κB p65 into the nucleus and promoted NF-κB transcriptional activity. In conclusion, we found SAMHD1 dysfunction induced IL-34 expression via NF-κB p65 in neuronal SH-SY5Y cells. This finding could lay the foundation for exploring the role of IL-34-targeting microglia in the pathogenesis of Aicardi-Goutières Syndrome.

Ligusticum cycloprolactam inhibits IL-1β-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1β(IL-1β) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1β, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1β-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

Acupoint application therapy alleviates pain by regulating immune function through inhibiting TLR4/MyD88/NF-κB p65 signaling in a primary dysmenorrhea rat model.

To investigate the effects of graphene-based warm uterus acupoint paste on uterine Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear transcription factor-kappa B p65 (NF-κB p65) signaling pathway and Th1/Th2 immune balance in primary dysmenorrhea ( PD ) model rats, so as to reveal its immunological mechanisms of relieving dysmenorrhea. Thirty SD female rats were randomly divided into 3 groups：normal group, model group and acupoint paste group, with 10 rats in each group. PD rat model was established by subcutaneous injection of estradiol benzoate for 10 consecutive days. At the same time of modeling, graphene-based warm uterus acupoint paste was applied to the acupoints of "Guanyuan" (CV4), bilateral "Zigong" (EX-CA1) and "Sanyinjiao" (SP6) of rats in the acupoint paste group. The application was continuously applied once daily for 10 d, 5 h each time. On the 11th day, oxytocin was injected intraperitoneally to observe the writhing latency, writhing times within 30 min and writhing score of rats in each group. The spleen and thymus indexes were calculated. The pathological changes of spleen and thymus tissue were observed after HE staining. The contents of serum immunoglobulin (Ig) A, IgG, tumor necrosis factor-α (TNF-α), interleukin (IL)-2, interferon-γ (IFN-γ), IL-4 and IL-10 were detected by ELISA . The protein and mRNA expression levels of TLR4, MyD88 and NF-κB p65 in rat uterine tissue were detected by Western blot and real-time quantitative PCR, respectively. Compared with the normal group, the writhing times and writhing scores within 30 min of rats in the model group were significantly increased(P<0.001), and the rats showed writhing reaction (P<0.01). The spleen index and thymus index were significantly decreased(P<0.01, P<0.05). The spleen and thymus had obvious pathological changes. The contents of IgA, IgG, TNF-α, IL-2 and IFN-γ in serum were significantly increased, while the contents of serum IL-4 and IL-10 were significantly decreased(P<0.001, P<0.01). The expression levels of TLR4, MyD88, NF-κB p65 protein and corresponding mRNA in uterine tissue were significantly increased(P<0.001). Following intervention, compared with the model group, the writhing latency time of rats in the acupoint paste group was prolonged, and the writhing times and writhing scores within 30 min were significantly decreased (P<0.001). The spleen index and thymus index were significantly increased(P<0.01, P<0.05). The pathological changes of spleen and thymus were improved. The contents of serum IgA, IgG, TNF-α, IL-2 and IFN-γ were significantly decreased, while the contents of IL-4 and IL-10 were significantly increased(P<0.001, P<0.05, P<0.01). The expression of TLR4, MyD88, NF-κB p65 protein and the corresponding mRNA levels in uterine tissue were decreased(P<0.001, P<0.01). Graphene-based warm uterus acupoint paste can regulate the immune balance of Th1/ Th2 by regulating TLR4/ MyD88/ NF-κB p65 signaling pathway, repair the pathological damage of immune tissue, improve immune function, and effectively relieve the pain symptoms of PD rats.

Maternal Western diet programs cardiometabolic dysfunction and hypothalamic inflammation via epigenetic mechanisms predominantly in the male offspring

ObjectiveMaternal exposure during pregnancy is a strong determinant of offspring health outcomes. Such exposure induces changes in the offspring epigenome resulting in gene expression and functional changes. In this study, we investigated the effect of maternal Western hypercaloric diet (HCD) programming during the perinatal period on neuronal plasticity and cardiometabolic health in adult offspring. MethodsC57BL/6J dams were fed HCD for 1 month prior to mating with regular diet (RD) sires and kept on the same diet throughout pregnancy and lactation. At weaning, offspring were maintained on either HCD or RD for 3 months resulting in 4 treatment groups that underwent cardiometabolic assessments. DNA and RNA were extracted from the hypothalamus to perform whole genome methylation, mRNA, and miRNA sequencing followed by bioinformatic analyses. ResultsMaternal programming resulted in male-specific hypertension and hyperglycemia, with both males and females showing increased sympathetic tone to the vasculature. Surprisingly, programmed male offspring fed HCD in adulthood exhibited lower glucose levels, less insulin resistance, and leptin levels compared to non-programmed HCD-fed male mice. Hypothalamic genes involved in inflammation and type 2 diabetes were targeted by differentially expressed miRNA, while genes involved in glial and astrocytic differentiation were differentially methylated in programmed male offspring. These data were supported by our findings of astrogliosis, microgliosis and increased microglial activation in programmed males in the paraventricular nucleus (PVN). Programming induced a protective effect in male mice fed HCD in adulthood, resulting in lower protein levels of hypothalamic TGFβ2, NF-κB2, NF-κBp65, Ser-pIRS1, and GLP1R compared to non-programmed HCD-fed males. Although TGFβ2 was upregulated in male mice exposed to HCD pre- or post-natally, only blockade of the brain TGFβ receptor in RD-HCD mice improved glucose tolerance and a trend to weight loss. ConclusionsOur study shows that maternal HCD programs neuronal plasticity in the offspring and results in male-specific hypertension and hyperglycemia associated with hypothalamic inflammation in mechanisms and pathways distinct from post-natal HCD exposure. Together, our data unmask a compensatory role of HCD programming, likely via priming of metabolic pathways to handle excess nutrients in a more efficient way.

Diosmetin alleviates neuropathic pain by regulating the Keap1/Nrf2/NF-κB signaling pathway

BackgroundNeuropathic pain, a chronic condition with a high incidence, imposes psychological burdens on both patients and society. It is urgent to improve pain management and develop new analgesic drugs. Traditional Chinese medicine has gained popularity as a method for pain relief. Diosmetin (Dio) is mainly found in Chinese herbal medicines with effective antioxidant, anti-cancer, and anti-inflammatory properties. There are few known mechanisms underlying the effectiveness of Dio in treating neuropathic pain. However, the complete understanding of its therapeutic effect is missing. PurposeThis study aimed to evaluate Dio's therapeutic effects on neuropathic pain models and determine its possible mechanism of action. We hypothesized that Dio may activate antioxidants and reduce inflammation, inhibit the activation of Kelch-like epichlorohydrin-associated protein 1 (Keap1) and nuclear factor-k-gene binding (NF-κB), promote the metastasis of nuclear factor erythroid 2-related factor 2 (Nrf2) and the expression of heme oxygenase 1 (HO-1), thus alleviating the neuropathic pain caused by spinal nerve ligation. MethodsChronic nociceptive pain mouse models were established in vivo by L4 spinal nerve ligation (SNL). Different dosages of Dio (10, 50, 100 mg/kg) were intragastrically administered daily from the third day after the establishment of the SNL model. Allodynia, caused by mechanical stimuli, and hyperalgesia, caused by heat, were assessed using the paw withdrawal response frequency (PWF) and paw withdrawal latency (PWL), respectively. Cold allodynia were assessd by acetone test. RT-PCR was used to detect the content of interleukin-(IL)− 1β, IL-6 and tumor necrosis factor (TNF)-a. Immunofluorescence and western blotting were employed to assess the expression levels of Glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule (Iba1), Keap1, Nrf2, HO-1, and NF-κB p-p65 protein. ResultsDio administration relieved SNL-induced transient mechanical and thermal allodynia in mice. The protective effect of Dio in the SNL model was associated with its anti-inflammatory and anti-glial responses in the spinal cord. Dio inhibited both inflammatory factors and macrophage activation in the DRG. Furthermore, Dio regulated the Keap1/Nrf2/NF-κB signaling pathway. HO-1 and Nrf2 were upregulated following Dio administration, which also decreased the levels of Keap1 and NF-κB p65 protein. ConclusionMice with SNL-induced neuropathic pain were therapeutically treated with Dio. Dio may protect against pain by inhibiting inflammatory responses and improved Keap1/Nrf2/NF-κB pathway. These results highlight the potential therapeutic effect of Dio for the development of new analgesic drugs.

Ginsenoside Rg1 Attenuates Eccentric Exercise-Induced Muscle Damage via the Modulation of Lipid Peroxidation and Inflammation

Eccentric exercise (EE) may lead to skeletal muscle injury, including oxidative stress and inflammation induction. Ginsenoside Rg1, a glycosylated triterpene present in the traditional Chinese medicine ginseng, was previously shown to prevent the development of multiple diseases through the attenuation of oxidative stress and inflammation. Therefore, this article hopes to investigate whether Rg1 exhibits anti-oxidant and anti-inflammatory effects in eccentric exercise-induced muscle damage (EEIMD). Additionally, Adult male Wistar rats were intraperitoneally injected with Rg1 (20 or 40 mg/kg) every day before EE for 5 consecutive days. The impact of Rg1 administration on levels of serum creatine kinase was evaluated, followed by observation of histological muscle damage through H&amp;E staining. To assess protein nitrotyrosylation, lipid peroxidation and leukocyte infiltration in rat skeletal muscles, the levels of nitrotyrosine, MDA and MPO protein were analysed through western blotting analysis. The inflammatory response was evaluated by detecting iNOS, COX-2, IL-1β, IL-6, MCP-1 and TNF-α mRNA and protein levels in rat skeletal muscles. The regulation of Rg1 on the NF-κB pathway was examined through the analysis of phosphorylated NF-κB p65 and IκBα protein levels. Result display, EE resulted in elevated serum creatine kinase levels, widespread leukocyte infiltration, and notable muscle cell vacuolization and fragmentation in muscles. Furthermore, EE increased nitrotyrosine, MDA, MPO, iNOS, COX-2, IL-1β, IL-6, MCP-1, and TNF-α levels in rats. However, these changes were reversed by Rg1 treatment. Furthermore, EE-induced upregulation in phosphorylated NF-κB p65 and IκBα levels was counteracted by Rg1. Overall, ginsenoside Rg1 plays an anti-oxidant and anti-inflammatory role in EEIMD through suppressing this NF-κB signaling pathway.

Effect of acupuncture on arthritic pathological injury in rats with type Ⅱ collagen-induced rheumatoid arthritis.

To observe the effect of acupuncture stimulation of "Yanglingquan"(GB34), "Zusanli"(ST36) and "Xuanzhong" (GB39) on arthritis index (AI), joint synovial membrane pathology, serum-related immunoinflammatory factors, and expressions of tumor suppressor gene mt-p53, nuclear factor kappa B (NF-κB) and peroxisome proliferator activated receptor gamma (PPARγ) in knee joint synovial tissue of rats with type Ⅱ collagen-induced arthritis (CIA), so as to explore its possible mechanisms underlying improvement of rheumatoid arthritis (RA). Male SD rats were used in the present study. The CIA model was established by subcutaneous injection of collagen emulsion (200 μL/rat) in the tail root region on the first day and repeat (100 μL/rat) once on the 9th day. Eighteen successful CIA rats were randomized into model, medication and acupuncture groups, with 6 rats in each group. Other 6 normal rats were used as the normal control group. For rats of the medication group, leflunomide (1.9 mg/kg) was administrated by gavage, once a day, and for rats of the acupuncture group, manual acupuncture stimulation was applied to bilateral GB34, ST36, GB39 for 30 min, once a day, for 12 weeks. The arthritis index (AI) score (0-4 points) was evaluated once every week. The contents of IL-6, IL-17 and TNF-α in the serum were determined by ELISA. Histopathological changes of the ankle joint were observed by H.E. staining. The protein and mRNA expression levels of mt-p53, NF-κB p65, and PPARγ in the knee joint synovial tissue were determined by Western blot and quantitative real time PCR, separately. Compared with the normal control group, the AI scores at different time-points after modeling, contents of serum TNF-α, IL-6 and IL-17, expression levels of mt-p53, NF-κB p65, PPARγ proteins and mRNAs were significantly increased in the model group (P<0.01, P<0.05). In comparison with the model group, the AI scores at the 10th week in the medication group and at the 3rd, 9th and 10th week in the acupuncture group, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of mt-p53 and NF-κB p65 proteins in both medication and acupuncture groups, as well as mt-p53 and NF-κB p65 mRNAs in the medication group were apparently decreased (P<0.01, P<0.05), while the expression levels of PPARγ protein in both medication and acupuncture group and PPARγ mRNA in the medication group were significantly up-regulated (P<0.05, P<0.01). No significant differences were found between the acupuncture and medication groups in down-regulating the AI score and serum TNF-α, IL-6 and IL-17 contents. The effect of acupuncture was weaker than that of medication in down-regulating the expression of mt-p53 and NF-κB p65 proteins and mRNAs and in up-regulating PPARγ mRNA (P<0.01). H.E. results showed ankle cartilage hyperplasia, reduced joint cavity, mild fibroproliferation and inflammatory cell infiltration in the surrounding soft tissue of the ankle joint in rats of the model group, which was milder in both medication and acupuncture groups. Acupuncture stimulation can improve the degree of joint inflammation and swelling in CIA rats, which may be related to its effects in inhibiting the overexpression of immunoinflammatory factors in serum and regulating expression of mt-p53, NF-κB p65, PPARγ mRNAs and proteins in the synovial tissue.

Immunomodulation of potassium diformate in juvenile grass carp (Ctenopharyngodon idella) after Aeromonas hydrophila infection: T-cell differentiation and cytokine production

Potassium diformate (KDF) is a green and safe feed additive to replace antibiotics in animal. Although a few studies have found that KDF improved disease resistance, the specific mechanisms have not been explored in depth. Therefore, the study aims to examine the effects of KDF on immune defense substances, inflammatory process, T cell differentiation, and related molecular expression in fish. The juvenile grass carp were selected from six groups after feeding graded levels of KDF (0.0, 1.5, 3.0, 4.5, 6.0 and 7.5 g/kg) for 70 days. Then 6-day challenge experiment was performed by intraperitoneal injection Aeromonas hydrophila. Appropriate levels of KDF increased the lysozyme and acid phosphatase activities, the complement 3 and complement 4 contents in the head kidney and spleen of juvenile grass carp. Meanwhile, Appropriate addition levels of KDF down-regulated the abundances of TNF-α, IL-2, IL-6, IL-8, IL-1β, IL-12, IL-22, IL-17D, T-bet and RORγt (RORγ2) mRNA and up-regulated the abundances of TGF-β, IL-4, IL-10, GATA3, FOXP3, CD4, MHCIIα and MHCIIβ mRNA in the head kidney and spleen. Besides, appropriate levels of KDF down-regulated the mRNA levels of IKKγ (not IKKα), IKKβ, NF-κB p65 (not p52), STAT1, STAT4, and the protein level of NF-κB p65, up-regulated the mRNA levels of IκBα, JAK1, JAK2, JAK3, STAT3b1, STAT3b2 (not STAT3a), STAT5b, STAT6, and the protein level of p-STAT3Tyr705. The appropriate levels of KDF enhanced the immune defense and mediated the inflammatory process that might be related to T cell differentiation probably via JAK-STATs and NF-κB signalings in fish.

Protective effect of Liujing Toutong Tablets on rats with permanent cerebral ischemia via NF-κB signaling pathway

This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1β, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(β-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1β, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and β-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1β, IL-6, CGRP, and NO in rat serum, increased VEGF and β-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and β-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1β. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing β-EP levels.

Electroacupuncture pretreatment alleviates spasticity after stroke in rats by inducing the NF-κB/NLRP3 signaling pathway and the gut-brain axis

ObjectiveSpasticity is one of the most prevalent ischemic stroke sequelae and the leading cause of disability after stroke. Although electroacupuncture pretreatment has been shown to be effective in the treatment of ischemic stroke, its therapeutic effect and mechanism on post-stroke spasm remain unknown. The purpose of this study was to look into the potential mechanism of electroacupuncture pretreatment in inducing the NF-κB/NLRP3 signaling pathway and the gut-brain axis in the therapy of spasm after stroke. MethodsAfter electroacupuncture treatment at Baihui (DU20) and Qubin (G87), the rat model of middle cerebral artery occlusion (MCAO) was first established. HE, Nissl, and TUNEL staining were used to detect pathological alterations in the rat brain. The relative levels of IL-4, IL-6, TNF-α, and TMAO were determined by ELISA. qRT-PCR and Western blot were used to evaluate the mRNA and protein levels of NF-κB p65, NLRP3, caspase3 and caspase9. Gas chromatography-mass spectrometry (GC–MS) was used to determine the levels of short-chain fatty acids (SCFAs) in rat gut. ResultsHippocampal cells from rats with spasticity following stroke in the MCAO group were chaotic and loosely distributed with an unclear border, a blurred nucleolus, and vanished cytoplasm when compared to those from the sham operation group. Furthermore, the number of surviving neurons decreased while the number of apoptotic cells increased. In the I/R group, relative levels of IL-6, TNF-α, and TMAO increased considerably, while NF-κB p65, NLRP3, caspase3, and caspase9 were dramatically downregulated. The intestinal contents of n-propyl acetate and propyl butyrate were lowered in rats with spasticity following stroke. Electroacupuncture treatments miraculously remedied all of the foregoing pathogenic alterations. ConclusionPretreatment with electroacupuncture relieves spasticity after stroke by decreasing the inflammatory response, suppressing the NF-κB/NLRP3 signaling pathway, and modulating the gut-brain axis by increasing n-propyl acetate and propyl butyrate levels in the bowel. Our findings establish a new molecular mechanism and theoretical foundation for electroacupuncture therapy of ischemic stroke.

Lead exposure induces neuronal apoptosis via NFκB p65/RBBP4/Survivin signaling pathway

Lead (Pb), as a heavy metal that is easily exposed in daily life, can cause damage to various systems of body. Apoptosis is an autonomous cell death process regulated by genes in order to maintain the stability of internal environment, which plays an important role in the development of nervous system. RB binding protein 4 (RBBP4) is one of the core histone binding subunits and is closely related to the apoptosis process of nervous system cells. However, it is not known whether RBBP4 can regulate neuronal apoptosis in lead-exposed environments. We exposed PC12 cells to 0 μM (control group), 1 μM, and 100 μM PbAc for 24 h to obtain cell samples. The female rats ingested drinking water containing 0, 0.5 g/L, and 2.0 g/L PbAc from the first day of pregnancy to three weeks after delivery to obtain hippocampal tissue samples from mammary rats. The results of TUNEL showed that lead exposure promoted the onset of apoptosis in cells and hippocampus. The mRNA and protein levels of the apoptosis-related protein Survivin were significantly reduced in the lead-exposed group compared to the control group. In addition, we found that lead exposure reduces the mRNA and protein levels of RBBP4 in PC12 cells and hippocampus, and increases the mRNA and protein levels of NFκB p65. Moreover, inhibiting NFκB p65 can reverse the decrease in RBBP4 expression in the lead exposure model. Overexpression of RBBP4 increased Survivin expression and reduced apoptosis induced by lead exposure. This suggests that lead exposure induces apoptosis through the NFκB p65/RBBP4/Survivin signaling pathway.