RESUMO
Ocular inflammation is a common pathological condition of a series of retinal degenerative diseases. Tetramethylpyrazine (TMP), a Chinese herbal extraction, is widely used in the treatment of several ocular diseases in Eastern countries. However, the exact mechanisms correlating the vision protective effects of TMP have not been elucidated. Thus, this study aimed to investigate TMP's molecular targets in anti-inflammatory activity in endotoxin lipopolysaccharide (LPS)-induced retinal inflammation both in vitro and in vivo. The primary cultured retinal microglial cells were pretreated with TMP and then activated by LPS. We found pretreatment with TMP significantly inhibited LPS-induced upregulation of CD68, a marker of mononuclear microglia activation. The morphological changes induced by LPS were also inhibited by the TMP pretreatment. Moreover, Toll like receptor 4 (TLR4), phosphorylation of inhibitor of NF-κB alpha (p-IκB-α) and the translocation of nuclear factor kappa B p65 (NF-κB p65) were significantly downregulated in retinal microglial cells with TMP pretreatment, which indicated that TMP might suppress LPS-induced retinal microglial activation through TLR4/NF-κB signalling pathway. And these results were confirmed in vivo. Pretreatment with TMP inhibited microglial activation, migration and regeneration, especially in ganglion cell layer (GCL). In addition to the inhibition of TLR4, TMP significantly inhibited the translocation of NF-κB p-65 to the nucleus in vivo. The downstream genes of NF-κB, such as the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß), were significantly downregulated by TMP pretreatment in the retina. Accordingly, the increased expression of cleaved caspase-3 and the decreased ratio of B-cell lymphoma-2 (Bcl-2) to Bcl-2 associated X Protein (Bax) were significantly attenuated by TMP. TUNEL assay also demonstrated that TMP exerted neuroprotective effects in the retina. Therefore, this study elucidated a novel mechanism that TMP inhibits retinal inflammation by inhibiting microglial activation via a TLR4/NF-κB signalling pathway.
Assuntos
Anti-Inflamatórios/farmacologia , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , Pirazinas/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Uveíte/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Microglia/metabolismo , Microglia/patologia , Ratos Sprague-Dawley , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Transdução de Sinais , Uveíte/induzido quimicamente , Uveíte/metabolismo , Uveíte/patologiaRESUMO
Tetramethylpyrazine (TMP; an extract of the Chinese herbal medicine, Chuanxiong) has been shown to exert remarkable antiretinoblastoma (RB) effects. Based on our previous study, the target gene was found to be CXC chemokine receptor type 4 (CXCR4). CXCR4 is a prognostic marker in various types of cancer, but the exact mechanisms underlying the regulation of CXCR4 expression by TMP in WERIRb1 cells have yet to be fully elucidated. In the present study, it was revealed that TMP significantly downregulated CXCR4 expression and inhibited CXCR4 promoter activity in WERIRb1 cells, indicating that TMP inhibits CXCR4 expression in WERIRb1 cells through transcriptional regulatory mechanisms. Among the numerous transcription factors involved in CXCR4 function, including Yin Yang 1 (YY1), nuclear respiratory factor1 (Nrf1), Krüppellike Factor 2 (KLF2), specificity protein 1 (SP1), and nuclear factorκB subunit 1 (NFκB1), only TMP led to a significant downregulation of Nrf1 expression. Chromatin immunoprecipitation assays further indicated that Nrf1 directly binds to the promoter region of CXCR4, and silencing Nrf1 via siRNA transfection notably inhibited CXCR4 expression in WERIRb1 cells. In addition, the expression levels of both Nrf1 and CXCR4 increased concomitantly with WERIRb1 cell density. Furthermore, the downregulation of Nrf1 and CXCR4 expression in RB by TMP was confirmed in vivo. Taken together, the results of the present study have uncovered a novel mechanism in which CXCR4 expression is regulated by Nrf1 in WERIRb1 cells, thereby identifying novel potential targets for the treatment of RB, and providing evidence for the clinical application of TMP in adjuvant retinoblastoma therapy.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 1 Nuclear Respiratório/metabolismo , Pirazinas/farmacologia , Receptores CXCR4/genética , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Transcrição Gênica/efeitos dos fármacos , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Nus , Fator 1 Nuclear Respiratório/genética , Receptores CXCR4/metabolismo , Neoplasias da Retina/tratamento farmacológico , Neoplasias da Retina/genética , Retinoblastoma/tratamento farmacológico , Retinoblastoma/genética , Células Tumorais Cultivadas , Vasodilatadores/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Retinoblastoma is the most common ocular tumor in children, and it causes extensive damage. Current treatment options for retinoblastoma include surgery, chemotherapy, radiotherapy and cryotherapy. However, the majority of chemotherapy medicines cause complications and side effects that lead to severe impairment of patient health. Previous studies have reported that tetramethylpyrazine (TMP), which is an extract of the Chinese herbal medicine Chuanxiong, reduces the risk of multidrug resistance in chemotherapy and inhibits the proliferation and metastasis of various types of cancer cells. However, the underlying molecular mechanism of TMP in retinoblastoma remains unclear. The current study demonstrated that C-X-C chemokine receptor type 4 (CXCR4) was expressed in WERIRb1 cells and in retinoblastoma. Using reverse transcriptionquantitative polymerase chain reaction and western blotting techniques, the current study demonstrated that TMP significantly downregulated the expression of CXCR4 in WERIRb1 cells cultured at high density, whereas it had a minor effect in lowdensity WERIRb1 cells; additionally, this effect occurred in a timedependent manner. TMP inhibited the proliferation of WERIRb1 cells as effectively as a CXCR4 antagonist, AMD3100, consistent with a role of CXCR4 in cancer development. Notably, TMP did not affect the cell cycle of cells cultured at low density (1x105 cells/ml), whereas it induced G1phase arrest in highdensity cells (7.5x105 cells/ml; P<0.05). In addition, the expression of CXCR4 in primary rat retinal neurocytes was significantly downregulated by TMP treatment, and this treatment protected primary rat retinal neurocytes from H2O2induced damage. Thus, the results of this study indicate that TMP is a potential candidate for use in treatment of retinoblastoma, and also provides novel insights into the mechanisms of the anticancer and neuroprotective effects of this extract.