The effect of SHJKS on cytokines production and NF-kappaB activation in the peripheral blood mononuclear cells of patients with cerebral infarction

The Korean genuine medicine "Seonghyangjeongkisan" (SHJKS) has long been used for various cerebrovascular diseases. However, very little scientific investigation has been carried out. Cytokines involved in the regulation of inflammatory reactions and immune responses may play a role in the pathogenesis of cerebral infarction (CI). The aim of the present study is to elucidate how SHJKS modulates the inflammatory reaction in lipopolysaccaride (LPS) plus phytohaemagglutinin (PHA)-stimulated peripheral mononuclear cells (PBMCs) from CI patients. The amount of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and IL-8 in PBMC culture supernatant was significantly increased in the LPS plus PHA treated cells compared to unstimulated cells. SHJKS inhibited the TNF-alpha, IL-1beta, IL-6, and IL-8 production in dose dependent manner. Maximal inhibition rate of the TNF-alpha, IL-1beta, IL-6, and IL-8 by SHJGS (1.0 mg/ml) was 68.01 +/- 0.28% (P < 0.01), 52.11 +/- 0.56 % (P < 0.01), 53.42 +/- 0.46 % (P < 0.01), and 46.70 +/- 0.37% (P < 0.05), respectively. In addition, we show that SHJKS suppressed nuclear factor (NF)-kappaB activation induced by LPS plus PHA, leading to suppression of IkappaB-alpha phosphorylation and degradation. These results suggest that SHJKS might have regulatory effects on LPS plus PHA-induced cytokine production and NF-kappaB activation, which might explain its beneficial effect in the treatment of CI.     

Differential induction and activation of NF-kappaB transcription complexes in radiation-induced chromosomally unstable cell lines

Radiation-induced genomic instability is a delayed effect of ionizing radiation that may contribute to radiation carcinogenesis. Prior microarray studies investigating gene expression changes in genomically unstable cell lines isolated after radiation exposure uncovered the differential expression of the NF-kappaB p105 mRNA. In this study, the functionality of the NF-kappaB pathway was examined to determine its role in regulating gene expression changes after oxidative stress in chromosomally stable and unstable human-hamster hybrid clones. Basal DNA-binding activity assays showed no significant differences between the clones; however, further experiments established differences in NF-kappaB induction in three chromosomally unstable clones after acute hydrogen peroxide treatment. A second assay was used to confirm this differential activity in the chromosomally unstable clones by studying reporter gene activation after treatment with hydrogen peroxide. Yet an initial upstream analysis of the pathway revealed no significant increase in the level of IkappaBalpha inhibitor protein in the unstable clones. Downstream tests analyzing the induction of the antiapoptotic target protein Bcl-2 found variable induction among the stable and unstable clones. These differences did not translate to a reduction in clonogenic survival after acute exposure to oxidative stress, as the irradiated but chromosomally stable clone displayed the most sensitivity. Due to its role in regulating a diverse set of cellular functions, including responses to oxidative stress, alterations in the NF-kappaB pathway in chromosomally unstable clones may regulate the differential physiology of a subset of chromosomally unstable clones and could contribute to the perpetuation of the phenotype. However, a specific role for defective induction and activation of this pathway remains unidentified.     

Magnolol suppresses NF-kappaB activation and NF-kappaB regulated gene expression through inhibition of IkappaB kinase activation

The mis-regulation of nuclear factor-kappa B (NF-kappaB) signal pathway is involved in a variety of inflammatory diseases that leds to the production of inflammatory mediators. Our studies using human U937 promonocytes cells suggested that magnolol, a low molecular weight lignan isolated from the medicinal plant Magnolia officinalis, differentially down-regulated the pharmacologically induced expression of NF-kappaB-regulated inflammatory gene products MMP-9, IL-8, MCP-1, MIP-1alpha, TNF-alpha. Pre-treatment of magnolol blocked TNF-alpha-induced NF-kappaB activation in different cell types as evidenced by EMSA. Magnolol did not directly affect the binding of p65/p50 heterodimer to DNA. Immunoblot analysis demonstrated that magnolol inhibited the TNF-alpha-stimulated phosphorylation and degradation of the cytosolic NF-kappaB inhibitor IkappaBalpha and the effects were dose-dependent. Mechanistically, a non-radioactive IkappaB kinases (IKK) assay using immunoprecipitated IKKs protein demonstrated that magnolol inhibited both intrinsic and TNF-alpha-stimulated IKK activity, thus suggesting a critical role of magnolol in abrogating the phosphorylation and degradation of IkappaBalpha. The involvement of IKK was further verified in a HeLa cell NF-kappaB-dependent luciferase reporter system. In this system magnolol suppressed luciferase expression stimulated by TNF-alpha and by the transient transfection and expression of NIK (NF-kappaB-inducing kinase), wild type IKKbeta, constitutively active IKKalpha and IKKbeta, or the p65 subunit. Magnolol was also found to inhibit the nuclear translocation and phosphorylation of p65 subunit of NF-kappaB. In line with the observation that NF-kappaB activation may up-regulate anti-apoptotic genes, it was shown in U937 cells that magnolol enhanced TNF-alpha-induced apoptotic cell death. Our results suggest that magnolol or its derivatives may have potential anti-inflammatory actions through IKK inactivation.     

ZFP36L1 is regulated by growth factors and cytokines in keratinocytes and influences their VEGF production

Keratinocyte-derived growth factors and cytokines play an important role in epidermal homeostasis and particularly in cutaneous wound repair. Thus, we analyzed a potential role of the ZFP36/tristetraprolin family of zinc finger proteins, which are targets of these factors, but also regulate their production, in keratinocytes. We show that expression of ZFP36, ZFP36L1, and ZFP36L2 is induced by a broad variety of growth factors and cytokines, and by scratch wounding. Since ZFP36L1 is a modulator of vascular endothelium growth factor (VEGF) mRNA stability, we subsequently used siRNA technology to inhibit ZFP36L1 gene expression. Notably, this treatment resulted in prolonged maintenance of elevated VEGF levels in HaCaT keratinocytes upon epidermal growth factor stimulation of these cells. Taken together, our results suggest an important role of ZFP36L1 in wound healing.     

Organotypic culture of HPV-transformed keratinocytes: a model for testing lymphocyte infiltration of (pre)neoplastic lesions of the uterine cervix

 The aim of our study was to establish the relevance of an in vitro model for analysing the ability of human lymphocytes to infiltrate human papillomavirus (HPV)-associated (pre)neoplastic lesions of the uterine cervix. To mimic these lesions, we have used the organotypic raft culture of HPV-transformed keratinocytes (SiHa). The SiHa organotypic raft culture was co-cultured with resting or prestimulated (IL-2 or IL-2+anti-CD3 mAb) allogeneic peripheral blood mononuclear cells (PBMC) for 24 and 72 h. The majority of infiltrating cells were T lymphocytes. Occasional NK cells were also identified. The stimulation with IL-2+anti-CD3 mAb induced the highest number of infiltrating cells, with the maximum lymphocyte infiltration observed after 24 h of co-culture. The lymphocyte infiltration was associated with an increased number of apoptotic cells in the organotypic cultures. The ability of PBMC and purified T cell and NK cell populations to lyse HPV-transformed keratinocytes was also investigated on monolayer cultures. As expected in an allogenic model, the highest cytotoxicity was mediated by NK cells activated by IL-2 or IL-2+anti-CD3 mAb. The cytotoxic activity of T cells was weak but, interestingly, increased in the presence of phytohaemagglutinin A (PHA), assuming that T cells were able to kill HPV-infected keratinocytes when a bridge between T cells and keratinocytes was provided. In conclusion, the organotypic culture of HPV-transformed keratinocytes may provide an effective in vitro model for investigating novel T cell-based immunotherapy protocols for the treatment of HPV-associated lesions. Received: 1 September 1997 / Accepted: 9 December 1997     

Effect of betamethasone on neuropathic pain and cerebral expression of NF-kappaB and cytokines

Glucocorticoids have been used to treat neuropathic pain for many years, but the underlying mechanisms are still unknown. Recent studies indicate that pathological pain states may be mediated by cytokines. We, therefore, examined the effect of betamethasone on neuropathic pain and the relationship between pain behavior and the expression of cytokines in the brain. Rats were given epidural injections of betamethasone (Diprospan) after L5 spinal nerve transection. Mechanical allodynia and thermal hyperalgesia were evaluated on post-operative days 1, 3, 7, 14 and 21 with von Frey and Hargreaves tests. Cerebral expression of NF-kappaB, TNFalpha, IL-1beta and IL-10 was quantified using electrophoretic mobility shift assay (EMSA) or enzyme-linked immunosorbent assay (ELISA). We found that spinal nerve injury caused long-lasting mechanical and thermal hyperalgesia in the hind paw and stimulated the expression of NF-kappaB, TNFalpha, IL-1beta and IL-10 in the brain. A single epidural injection of betamethasone at the time of nerve injury partially inhibited the development of neuropathic hyperalgesia and reduced the subsequent elevated levels of pro-inflammatory cytokines in the brain, while stimulating the expression of the anti-inflammatory cytokine IL-10. These data support our hypothesis that pro-inflammatory cytokines in the brain may mediate the hyperalgesic effects of spinal nerve injury and that the long-acting anti-hyperalgesic effects of epidural glucocorticoid treatment are due to an inhibitory effect on pro-inflammatory cytokine levels and a stimulatory effect on anti-inflammatory cytokine levels in the brain.     

Regulation of constitutive and induced NF-kappaB activation in malignant melanoma cells by capsaicin modulates interleukin-8 production and cell proliferation.

In the present study, we demonstrate that upregulation of interleukin-1beta(IL-1beta)-mediated and tumor necrosis factor-alpha (TNF-alpha)-mediated IL-8 expression in human malignant melanoma cells is modulated by the activation of nuclear factor-kappaB (NF-kappaB). Addition of capsaicin (8-methyl-N-vanillyl-6-nonenamide), a known inhibitor of NF-kappaB, resulted in the inhibition of constitutive as well as IL-1beta-induced and TNF-alpha-induced IL-8 expression in melanoma cells. The inhibition of IL-8 expression was dependent on the concentration of capsaicin and duration of treatment. Further, electrophoretic mobility shift assay (EMSA) of nuclear extracts from melanoma cells showed a constitutive activation of NF-kappaB and activated protein 1 (AP-1), which was upregulated following treatment with IL-1beta. Treatment of melanoma cells with capsaicin inhibited activation of constitutive and IL-1beta-induced NF-kappaB, but not AP-1, leading to inhibition of IL-8 expression. Further, downregulation of IL-8 expression in capsaicin-treated melanoma cells resulted in inhibition of in vitro cell proliferation. These results demonstrate that constitutive and induced NF-kappaB activation regulates IL-8 expression in melanoma cells. Downregulation of constitutive and induced NF-kappaB activation in malignant melanoma cells leads to inhibition of IL-8 production and in vitro cell proliferation.