RESUMO
Epilepsy is a syndrome involving chronic recurrent transient brain dysfunction. Activation and proliferation of microglia serve important roles in epilepsy pathogenesis and may be targets for treatment. Although osthole, an active constituent isolated from Cnidium monnieri (L.) Cusson, has been demonstrated to improve epilepsy in rats, its underlying mechanism remains to be elucidated. The present study investigated the effect of osthole on proliferation of kainic acid (KA)activated BV2 cells and explored the molecular mechanism by which it inhibited their proliferation. Using Cell Counting Kit8, enzymelinked immunosorbent assay, reverse transcriptionquantitative PCR, western blot analysis and immunofluorescence staining, it was identified that following exposure of KAactivated BV2 cells to 131.2 µM osthole for 24 h, cell proliferation and release of tumor necrosis factor α, interleukin 6 and nitric oxide synthase/induced nitric oxide synthase were significantly inhibited (P<0.05). Further experiments revealed that osthole significantly downregulated mRNA and protein levels of Notch signaling components in KAactivated BV2 cells (P<0.05). Therefore, it was hypothesized that osthole inhibited the proliferation of microglia by modulating the Notch signaling pathway, which may be useful for the treatment of epilepsy and other neurodegenerative diseases characterized by Notch upregulation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Cnidium/química , Cumarínicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Ácido Caínico/farmacologia , Microglia/efeitos dos fármacos , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Transformada , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Camundongos , Microglia/metabolismoRESUMO
CONTEXT: Osthole is a natural coumarin compound most frequently extracted from plants of the Apiaceae family such as Cnidium monnieri (L.) Cusson, Angelica pubescens Maxin.f., and Peucedanum ostruthium (L.). Osthole is considered to have potential therapeutic applications for the treatment of diseases including epilepsy. However, the mechanism of osthole induced-apoptosis in BV-2 microglia cells is not yet clear. OBJECTIVE: To investigate the molecular mechanisms underlying the effect of osthole on PI3K/AKt/mTOR expression in kainic acid (KA)-activated BV-2 microglia cells. MATERIALS AND METHODS: Optimal culture concentration and time of osthole were investigated by MTT assay. The concentration of osthole was tested from 10 to 400 µM and the culture time was tested from 2 to 72 h. Ultrastructure difference among control, KA and osthole group was analyzed under transmission electron microscope. The mRNA expression of PI3K/AKt/mTOR was investigated using reverse transcription (RT)-PCR and the protein expression was investigated using western blotting and immunofluorescence assay. Apoptosis rate of BV-2 cells between each group was measured by flow cytometry. RESULTS: IC50 for cell viability of BV-2 cells by osthole was 157.7 µM. Treated with osthole (140 µM) for 24 h significantly increased the inhibition rate. Pretreatment with osthole inhibited the KA-induced PI3K/AKt/mTOR mRNA and protein expression. The results of flow cytometry analysis showed that the apoptotic rate of osthole group was obviously higher than KA group. CONCLUSIONS: Date showed that osthole may be useful in the treatment of epilepsy and other neurodegenerative diseases that are characterized by over expression of PI3K/Akt/mTOR.
Assuntos
Cumarínicos/farmacologia , Microglia/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ácido Caínico/farmacologia , Camundongos , Microglia/citologia , Microglia/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
To study the antitumor effect of Xihuang pill (XHP) on the number of Treg cells in the tumor microenvironment of 4T1 breast tumor-bearing mice by PI3K/AKT/AP-1 pathway, a mouse model was established. Flow cytometry (FCM) and immunohistochemistry (IHC) were used to detect the number of Treg cells in the tumor microenvironment; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to detect the apoptosis of Treg cells in tumor microenvironment. Quantitative real-time PCR (RT-qPCR) was used to detect the mRNA expression of PI3K, AKT, and AP-1 in Treg cells in tumor microenvironment; immunofluorescence (IF) and Western Blot (WB) were used to detect the protein expression of PI3K, AKT, and AP-1 in Treg cells in tumor microenvironment. Compared with the naive control group, the tumor weight in XHP groups decreased significantly (P < 0.05); FCM and IHC results showed that the number of Treg cells in the tumor microenvironment decreased with the dose of XHP groups (P < 0.05); TUNEL staining showed that the number of Treg cells in tumor microenvironment increased with the dose of XHP groups (P < 0.05); RT-qPCR results showed that the mRNA expression of PI3K and AKT in Treg cells decreased with the dose of XHP groups, while RNA expression of AP-1 increased with the dose of XHP groups (P < 0.05); IF and WB results showed that the protein expression of PI3K and AKT in Treg cells decreased with the dose of XHP groups and the protein expression of AP-1 increased with the dose of XHP groups (P < 0.05). The results suggested that XHP decreased the number of Treg cells via inhibiting PI3K and AKT expression and upregulating AP-1 expression in Treg cells and then promoting the apoptosis of Treg cells. Thus, XHP could improve the immunosuppressive state of tumor microenvironment and reverse the immune escape to inhibit tumor growth.
RESUMO
The purpose of the present study was to determine whether triptolide (T10) had any effect on major histocompatibility complex class II (MHC II) expression in kainic acid (KA)activated microglia, and to investigate the underlying molecular mechanism. BV2 microglia were pretreated with T10 prior to activation with KA. The expression level of MHC II and class II transactivator (CIITA) mRNA was determined via reverse transcriptionpolymerase chain reaction. The expression of MHC II, CIITA and the phosphorylation level of cJun and protooncogene cFos (cFos) was determined by western blotting. The protein expression level of MHC II was determined by immunocytochemistry. It was observed that the mRNA and protein levels of MHC II and CIITA were increased in KAactivated BV2 microglia, and that this increase was almost completely eliminated by T10. AP1 is a family of homodimers or heterodimers, composed of Jun family and Fos family proteins. Sequence analysis revealed an AP1 DNA binding site in the promoter of CIITA. The phosphorylation of cJun and cFos was increased in KAactivated microglia, while T10 was able to suppress the phosphorylation of cJun and cFos in KAactivated microglia. These data suggested that T10 may exert suppressive effects on MHC II expression in KAactivated microglia, and that the mechanism may involve the regulation of AP1 activity.
Assuntos
Diterpenos/farmacologia , Epilepsia/etiologia , Expressão Gênica , Antígenos de Histocompatibilidade Classe II/genética , Ácido Caínico/efeitos adversos , Microglia/metabolismo , Fenantrenos/farmacologia , Animais , Epilepsia/metabolismo , Compostos de Epóxi/farmacologia , Imuno-Histoquímica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/genética , Ratos , Transativadores/genética , Transativadores/metabolismoRESUMO
OBJECTIVE: To discuss the anti-tumor effect of Xihuang pill on tumor-bearing rats and its effect on the immune clearance function of tumor-bearing organisms. METHOD: Walker256 tumor cells were adopted to establish the tumor-bearing rat model. The rats were randomly divided into five groups: the normal control group, the model control group, the lentinan group and Xihuang pill low dose, middle dose and high dose groups, with 10 rats in each group, and continuously treated and given drugs for 14 d after modeling. Blood and tumors were collected from abdominal aorta to calculate the tumor inhibition rate. The content of CD3+, CD4+, CD8+ T cells and adhesion molecule B7-1 (CD80) in peripheral blood were detected by flow cytometry (FCM). The expressions of IL-2 and IFN-gamma in were determined by ELISA. RESULT: The tumor inhibition rate of the Xihuang pill high dose group was 33. 1 percent. Compared with the model group, the Xihuang pill large dose group showed significantly low IL-2, IFN-gamma, CD3+, CD4+, B7-1 in peripheral blood, with statistical significance in their differences (P < 0.05). CONCLUSION: Xihuang pill could show its anti-tumor effect by enhancing the immune clearance function and increasing IL-2, IFN-gamma, CD3+ T, CD4+ T, B7-1 in peripheral blood.