BACKGROUND: Colorectal cancer is the third most common cancer worldwide and still lack of effective therapy so far. Petasin, a natural product found in plants of the genus Petasites, has been reported to possess anticancer activity. The present study aimed to investigate the anticolon cancer activity of petasin both in vitro and in vivo. The molecular mechanism of petasin was also further explored. METHODS: Caco-2, LoVo, SW-620, and HT-29 cell lines were used to detect the inhibitory effect of petasin on colon cancer proliferation. Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was analyzed by flow cytometry. Hoechst 33258 staining was used to visualize morphological changes. Cell migration was assessed using a wound-healing migration assay, and cell invasion was investigated using Transwell chambers. Western blotting assays were employed to evaluate the expression levels of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway. Finally, in vivo activity of petasin was evaluated using the SW-620 subcutaneous tumor model established in Balb/c nude mice. Twelve rats were randomly divided into control group and 10 mg/kg petasin group. The tumor volume was calculated every 7 days for 28 days. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to assess the apoptotic effect of petasin. Differences between two groups were assessed by analysis of independent-sample t tests. RESULTS: Petasin significantly inhibited the proliferation of human colon carcinoma cell lines, induced apoptosis, and suppressed migration and invasion in SW-620 cells. Western blotting results showed that petasin decreased the phosphorylation of Akt (1.01 ± 0.16 vs. 0.74 ± 0.06, P = 0.042), mTOR (0.71 ± 0.12 vs. 0.32 ± 0.11, P = 0.013), and P70S6K (1.23 ± 0.21 vs. 0.85 ± 0.14, P = 0.008), elevated the expression of caspase-3 (0.41 ± 0.09 vs. 0.74 ± 0.12, P = 0.018) and caspase-9 (1.10 ± 0.27 vs. 1.98 ± 0.22, P = 0.009), decreased the Bcl-2 protein (2.75 ± 0.47 vs. 1.51 ± 0.36, P = 0.008), downregulated the expression of matrix metalloproteinase (MMP)-3 (1.51 ± 0.31 vs. 0.82 ± 0.11, P = 0.021) and MMP-9 (1.56 ± 0.32 vs. 0.94 ± 0.15, P = 0.039) in SW-620 cell. In vivo, 10 mg/kg petasin inhibited tumor growth in Balb/c nude mice (924.18 ± 101.23 vs. 577.67 ± 75.12 mm at day 28, P = 0.001) and induced apoptosis (3.6 ± 0.7% vs. 36.0 ± 4.9%, P = 0.001) in tumor tissues. CONCLUSIONS Petasin inhibits the proliferation of colon cancer SW-620 cells via inactivating the Akt/mTOR pathway. Our findings suggest petasin as a potential candidate for colon cancer therapy.
Animals ; Antineoplastic Agents ; therapeutic use ; Apoptosis ; drug effects ; Caco-2 Cells ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HT29 Cells ; Humans ; In Situ Nick-End Labeling ; Matrix Metalloproteinase 3 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Sesquiterpenes ; therapeutic use ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; genetics ; metabolism

OBJECTIVE: To determine the correlation of phosphorylated ribosomal S6 protein (P-S6) content in blood and brain tissue in mice and rats with seizure. METHODS: Seizure models were induced by intraperitoric injection of kainic acid (KA) in C57BL/mice and SD rats. Flow cytometry was used to detect the content of P-S6 in blood; Western blot was used to detect the expression of P-S6 in brain tissues. The correlation between P-S6 expression in blood and in brain tissue was examine by Pearson analysis, and the correlation between P-S6 expression in blood and the severity of seizure was also observed. RESULTS: Western blotting analysis showed that the expression of P-S6 was significantly increased in peripheral blood and brain tissue in mice 1 h after KA-induced seizure,and the expression levels increased to (1.49±0.45) times (<0.05) and (2.55±0.66) times ( <0.01) of the control group, respectively. Flow cytometry showed that the positive percentage and average fluorescence intensity of P-S6 in the blood of mice increased significantly 1 h after KA-induced seizures (<0.01), which was consistent with the expression of P-S6 in brain tissue (=0.8474, <0.01). Flow cytometry showed that the average fluorescence intensity of P-S6 in blood increased from 14.89±9.75 to 52.35±21.72 (<0.01) in rats with seizure, which was consistent with the change of P-S6 in brain tissue (=0.9385, <0.01). Rats with higher levels of seizure were of higher levels of P-S6 in peripheral blood. CONCLUSIONS Consistent correlation of P-S6 expression is demonstrated in peripheral blood and in brain tissue after KA-induced seizure, suggesting that the expression of P-S6 in blood can accurately reflect the changes of mTOR signaling pathway in brain tissue.
Animals ; Brain ; drug effects ; physiopathology ; Gene Expression Regulation ; drug effects ; Kainic Acid ; Mice ; Mice, Inbred C57BL ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; Seizures ; blood ; chemically induced ; physiopathology

OBJECTIVE: To investigate the effects of ethanol extract of Patrinia scabiosaefolia (EEPS) on chemo-resistance of colorectal cancer cells (CRC) and explore the possible molecular mechanisms. METHODS: 5-fluorouracil (5-FU)-resistant human colorectal carcinoma cell line (HCT-8/5-FU) and its parental cells HCT-8 were treated with EEPS (0, 0.25, 0.50, 1 or 2 mg/mL), or 5-FU (0, 100, 200, 400, 800 or 1600 μmol/L). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the cell viability. Cell density was observed by phase-contrast microscope, cell counting and colony formation assay were used to determine the cell proliferation of HCT-8/5-FU cells treated with 0, 0.5, 1 or 2 mg/mL EEPS. Cell apoptosis was determined by Hoechst staining. Western-blot was performed to detect the phosphorylation of AKT as well as the protein expression level of B-cell CLL/lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax). RESULTS: Compared with HCT-8 cells, MTT assay results indicated that HCT-8/5-FU cells were resistant to 5-FU treatment (P<0.05), and sensitive to EEPS treatment (P>0.05). Moreover, compared with untreated HCT-8/5-FU cells, 1 and 2 mg/mL of EEPS treatment significantly reduced cell density, cell number, inhibited cell survival (P<0.05), and induced apoptosis in HCT-8/5-FU cells. Furthermore, 1 and 2 mg/mL of EEPS significantly decreased the phosphorylation level of p-AKT and Bcl-2 protein expression, and increased the expression of Bax protein (P<0.05). CONCLUSION EEPS is a promising therapeutic agent that may overcome chemo-resistance in cancer cells, likely through suppression of the AKT pathway and promotion of cancer cell apoptosis.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Colorectal Neoplasms ; drug therapy ; pathology ; Drug Resistance, Neoplasm ; drug effects ; Fluorouracil ; pharmacology ; therapeutic use ; Humans ; Patrinia ; chemistry ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects ; Tumor Stem Cell Assay ; bcl-2-Associated X Protein ; metabolism

Atractylenolide III (ATL-III), a sesquiterpene compound isolated from Rhizoma Atractylodis Macrocephalae, has revealed a number of pharmacological properties including anti-inflammatory, anti-cancer activity, and neuroprotective effect. This study aimed to evaluate the cytoprotective efficiency and potential mechanisms of ATL-III on corticosterone injured rat phaeochromocytoma (PC12) cells. Our results demonstrate that ATL-III increases cell viability and reduces the release of lactate dehydrogenase (LDH). The results suggest that ATL-III protects PC12 cells from corticosterone-induced injury by inhibiting the intracellular Ca overloading, inhibiting the mitochondrial apoptotic pathway and modulating the MAPK/NF-ΚB inflammatory pathways. These findings provide a novel insight into the molecular mechanism by which ATL-III protected the PC12 cells against corticosterone-induced injury for the first time. Our results provide the evidence that ATL-III may serve as a therapeutic agent in the treatment of depression.
Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Cell Survival ; drug effects ; Corticosterone ; toxicity ; Inflammation Mediators ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lactones ; pharmacology ; Mitochondria ; drug effects ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; NF-kappa B ; metabolism ; Neuroprotective Agents ; pharmacology ; PC12 Cells ; Phosphorylation ; drug effects ; Rats ; Sesquiterpenes ; pharmacology ; Signal Transduction ; drug effects

Macrophages show significant heterogeneity in function and phenotype, which could shift into different populations of cells in response to exposure to various micro-environmental signals. These changes, also termed as macrophage polarization, of which play an important role in the pathogenesis of many diseases. Numerous studies have proved that Hesperidin (HDN), a traditional Chinese medicine, extracted from fruit peels of the genus citrus, play key roles in anti-inflammation, anti-tumor, anti-oxidant and so on. However, the role of HDN in macrophage polarization has never been reported. Additional, because of its poor water solubility and bioavailability. Our laboratory had synthesized many hesperidin derivatives. Among them, hesperidin derivatives-12 (HDND-12) has better water solubility and bioavailability. So, we evaluated the role of HDND-12 in macrophage polarization in the present study. The results showed that the expression of Arginase-1 (Arg-1), interleukin-10 (IL-10), transforming growth factor β (TGF-β) were up-regulated by HDND-12, whereas the expression of inducible Nitric Oxide Synthase (iNOS) was down-regulated in LPS- and IFN-γ-treated (M1) RAW264.7 cells. Moreover, the expression of p-JAK2 and p-STAT3 were significantly decreased after stimulation with HDND-12 in M1-like macrophages. More importantly, when we taken AG490 (inhibitor of JAK2/STAT3 signaling), the protein levels of iNOS were significantly reduced in AG490 stimulation group compare with control in LPS, IFN-γ and HDND-12 stimulation cells. Taken together, these findings indicated that HDND-12 could prevent polarization toward M1-like macrophages, at least in part, through modulating JAK2/STAT3 pathway.
Animals ; Cytokines ; genetics ; metabolism ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; drug effects ; Hesperidin ; chemistry ; pharmacology ; Inflammation ; genetics ; metabolism ; Janus Kinase 2 ; antagonists & inhibitors ; metabolism ; Macrophages ; drug effects ; immunology ; metabolism ; Medicine, Chinese Traditional ; Mice ; Molecular Structure ; Phosphorylation ; drug effects ; RAW 264.7 Cells ; STAT3 Transcription Factor ; antagonists & inhibitors ; metabolism ; Signal Transduction ; drug effects

Objective To evaluate the antagonistic effects of N-acetylcysteine (NAC) on mitogen-activated protein kinases (MAPK) pathway activation, oxidative stress and inflammatory responses in rats with lung injury induced by fine particulate matter (PM_2.5). Methods Forty eight male Wistar rats were randomly divided into six groups: blank control group (C1), water drip control group (C2), PM_2.5 exposed group (P), low-dose NAC treated and PM_2.5 exposed group (L), middle-dose NAC treated and PM_2.5 exposed group (M), and high-dose NAC treated and PM_2.5 exposed group (H). PM_2.5 suspension (7.5 mg/kg) was administered tracheally once a week for four times. NAC of 125 mg/kg, 250 mg/kg and 500 mg/kg was delivered intragastrically to L, M and H group respectively by gavage (10 ml/kg) for six days before PM_2.5 exposure. The histopathological changes and human mucin 5 subtype AC (MUC5AC) content in lung tissue of rats were evaluated. We investigated IL-6 in serum and bronchoalveolar lavage fluid (BALF) by Enzyme-linked immunosorbent assay (ELISA), MUC5AC in lung tissue homogenate by ELISA, glutathione peroxidase (GSH-PX) in serum and BALF by spectrophotometry, and the expression of p-ERK1/2, p-JNK1/2 and p-p38 proteins by Western blot. All the measurements were analyzed and compared statistically. Results Lung tissue of rats exposed to PM_2.5 showed histological destruction and increased mucus secretion of bronchial epithelial cells. Rats receiving NAC treatment showed less histological destruction and mucus secretion. Of P, L, M and H group, MUC5AC in lung tissue, IL-6 in serum and BALF were higher than controls (C1 and C2) (all P<0.05), with the highest levels found in the P group and a decreasing trend with increase of NAC dose. The activity of GSH-PX in serum and BALF of PM_2.5 exposed rats (P, L, M and H) was lower than that of controls (all P<0.05), with higher activities found in NAC treated rats (L, M, and H), and an increasing trend with increase of NAC dose. The expressions of p-ERK1/2, p-JNK1/2 and p-p38 proteins in PM_2.5 exposed lung tissue (P, L, M and H) was higher than controls (all P<0.05), with decreased levels and dose dependent downregulation found in NAC treated rats. Conclusion NAC can antagonize major MAPK pathway activation, lung oxidative stress and inflammatory injury induced by PM_2.5 in rats.
Acetylcysteine/pharmacology* ; Animals ; Bronchoalveolar Lavage Fluid ; Enzyme Activation/drug effects* ; Glutathione Peroxidase/metabolism* ; Inflammation/pathology* ; Interleukin-6/metabolism* ; Lung/pathology* ; Lung Injury/pathology* ; Male ; Mitogen-Activated Protein Kinases/metabolism* ; Mucin 5AC/metabolism* ; Mucus/metabolism* ; Oxidative Stress/drug effects* ; Particle Size ; Particulate Matter/toxicity* ; Phosphorylation/drug effects* ; Rats, Wistar

Prostate inflammation (PI) is closely related to the development and progression of chronic prostatic diseases: benign prostatic hyperplasia and prostate cancer. Toll-like receptor (TLR) 2 has been reported to be associated with inflammatory diseases, such as infections, autoimmune diseases, and cancers. Meanwhile, TLR10, which can form heterodimers with TLR2, has been considered an orphan receptor without an exact function. The present study therefore aims to examine the effects of TLR2 and TLR10 on PI. Prostate samples and clinical data were obtained from the patients diagnosed with benign prostatic hyperplasia. The inflammatory cell model was established by adding lipopolysaccharide to RWPE-1 cells. Prostate tissues/cells were examined by histological, molecular, and biochemical approaches. Both TLR2 and TLR10 were found to be expressed in prostate tissues and RWPE-1 cells. mRNA/protein expression levels of TLR2 and TLR10 were both positively correlated with prostate tissue inflammatory grades. Lipopolysaccharide-stimulated RWPE-1 cells expressed higher levels of TLR2, TLR10, high mobility group box 1 (HMGB1), phospho-nuclear factor kappa-light-chain-enhancer of activated B-cells P65 (phospho-NF-κB P65), interleukin (IL)-6, and IL-8 than control cells. Moreover, HMGB1, phospho-NF-κB P65, IL-6, and IL-8 were downregulated after TLR2 knockdown and upregulated after TLR10 knockdown in RWPE-1 cells. TLR2 stimulation can activate the inflammatory signaling cascade in prostate epithelial cells. Conversely, TLR10 exhibited suppressive effects on inflammation. With antagonistic functions, both TLR2 and TLR10 were involved in PI. TLR10 could be a novel target in modulating inflammatory signal transduction of prostate epithelial cells.
Aged ; Cell Line ; Cytokines/metabolism* ; Epithelial Cells/pathology* ; Humans ; Inflammation/pathology* ; Lipopolysaccharides/pharmacology* ; Male ; Middle Aged ; Phosphorylation/drug effects* ; Prostate/pathology* ; Prostatic Hyperplasia/pathology* ; Signal Transduction/drug effects* ; Toll-Like Receptor 10/metabolism* ; Toll-Like Receptor 2/metabolism* ; Up-Regulation

We aimed to determine whether combination of LIM-kinase 2 inhibitor (LIMK2i) and phosphodiesterase type-5 inhibitor (PDE5i) could restore erectile function through suppressing cavernous fibrosis and improving cavernous apoptosis in a rat model of cavernous nerve crush injury (CNCI). Seventy 12-week-old Sprague-Dawley rats were equally distributed into five groups as follows: (1) sham surgery (Group S), (2) CNCI (Group I), (3) CNCI treated with daily intraperitoneal administration of 10.0 mg kg^-1 LIMK2i (Group I + L), (4) daily oral administration of 20.0 mg kg^-1 udenafil, PDE5i (Group I + U), and (5) combined administration of 10.0 mg kg^-1 LIMK2i and 20.0 mg kg^-1 udenafil (Group I + L + U). Rats in Groups I + L, I + U, and I + L + U were treated with respective regimens for 2 weeks after CNCI. At 2 weeks after surgery, erectile response was assessed using electrostimulation. Penile tissues were processed for histological studies and western blot. Group I showed lower intracavernous pressure (ICP)/mean arterial pressure (MAP), lower area under the curve (AUC)/MAP, decreased immunohistochemical staining for alpha-smooth muscle (SM) actin, higher apoptotic index, lower SM/collagen ratio, increased phospho-LIMK2-positive fibroblasts, decreased protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) phosphorylation, increased LIMK2/cofilin phosphorylation, and increased protein expression of fibronectin, compared to Group S. In all three treatment groups, erectile responses, protein expression of fibronectin, and SM/collagen ratio were improved. Group I + L + U showed greater improvement in erectile response than Group I + L. SM content and apoptotic index in Groups I + U and I + L + U were improved compared to those in Group I. However, Group I + L did not show a significant improvement in SM content or apoptotic index. The number of phospho-LIMK2-positive fibroblasts was normalized in Groups I + L and I + L + U, but not in Group I + U. Akt/eNOS phosphorylation was improved in Groups I + U and I + L + U, but not in Group I + L. LIMK2/cofilin phosphorylation was improved in Groups I + L and I + L + U, but not in Group I + U. Our data indicate that combined treatment of LIMK2i and PDE5i immediate after CN injury could improve erectile function by improving cavernous apoptosis or eNOS phosphorylation and suppressing cavernous fibrosis. Rectification of Akt/eNOS and LIMK2/cofilin pathways appears to be involved in their improvement.
Animals ; Apoptosis/drug effects* ; Arterial Pressure ; Electric Stimulation ; Erectile Dysfunction/pathology* ; Lim Kinases/antagonists & inhibitors* ; Male ; Nerve Crush ; Nitric Oxide Synthase Type III/metabolism* ; Penis/pathology* ; Peripheral Nerve Injuries/pathology* ; Phosphodiesterase 5 Inhibitors/therapeutic use* ; Phosphorylation ; Pyrimidines/therapeutic use* ; Rats ; Rats, Sprague-Dawley ; Sulfonamides/therapeutic use*

ObjectiveTo explore the inhibitory effect of polyphyllin Ⅰ (PPⅠ) on the proliferation of castration-resistant prostate cancer PC3 cells and its molecular mechanism.

METHODSWe cultured human prostate cancer PC3 cells in vitro and treated them with PPⅠ at the concentrations of 0 (blank group), 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 μmol/L for 24, 48, and 72 hours, respectively. Then we detected the proliferation of the cells by MTT assay, measured their apoptosis by flow cytometry, and determined the expressions of p-ERK1/2, ERK1/2, NF-κB/p65 and DNMT1 proteins as well as the level of NF-κB/p65 in the cells additionally treated with the ERK1/2 inhibitor SP600125 by Western blot.

RESULTSCompared with the blank control group, the PPⅠ-treated PC3 cells showed a concentration- and time-dependent reduction of the survival rate (1.00 ± 0.00 vs 0.85 ± 0.05, P < 0.01) at 0.4 μmol/L after 48 hours of intervention, concentration-dependent early apoptosis at 0.8 μmol/L (4.83 ± 0.95 vs 13.83 ± 2.97, P < 0.01), time-dependent increase of the expressions of p-ERK1/2 (1.00 ± 0.00 vs 1.73 ± 0.17, P < 0.01) and ERK1/2 (1.00 ± 0.00 vs 1.36 ± 0.12, P < 0.01) at 2 hours, and concentration-dependent decrease of the expressions of NF-κB/p65 and DNMT1 at 1.2 μmol/L (1.00 ± 0.00 vs 0.78 ± 0.10 and 0.63 ± 0.06, P < 0.01) and 1.6 μmol/L (1.00 ± 0.00 vs 0.67 ± 0.11 and 0.52 ± 0.09, P<0.01). Inhibition of ERK1/2 phosphorylation with PD98059 markedly reversed PPⅠ-induced decrease of the NF-κB/p65 expression as compared with that in the PPⅠ group (0.86 ± 0.18 vs 0.43 ± 0.09, P < 0.05).

CONCLUSIONSPPⅠ induces the early apoptosis and suppresses the proliferation of PC3 cells, probably by activating the ERK1/2 pathway and inhibiting the expressions of the NF-κB/p65 and DNMT1 proteins.

Apoptosis ; Cell Proliferation ; drug effects ; DNA (Cytosine-5-)-Methyltransferase 1 ; metabolism ; Diosgenin ; analogs & derivatives ; pharmacology ; Flavonoids ; metabolism ; Humans ; MAP Kinase Signaling System ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; NF-kappa B ; metabolism ; PC-3 Cells ; Phosphorylation ; Prostatic Neoplasms, Castration-Resistant ; drug therapy ; metabolism ; pathology ; Signal Transduction ; Transcription Factor RelA ; metabolism

OBJECTIVETo observe the effect of puerarin on methyl-CpG binding protein 2 (MeCP2) phosphorylation (pMeCP2) in the hippocampus of a rat model of vascular dementia (VD).

METHODSThirty-six healthy Sprague-Dawley rats were randomly assigned to the sham-operated group, dementia group and puerarintreated group using a random number table (n=12 per group). The modifified permanent bilateral common carotid artery occlusion method was used to establish the VD model. The sham-operated and dementia groups were given 2 mL/d of saline, while the puerarin-treated group was given 100 mg/(kg•d) of puerarin for 17 days. The learning and memory abilities were evaluated by the Morris water maze test. Hematoxylin-eosin staining, immunohistochemical (IHC) staining and Western blot analysis were carried out to observe changes in neuron morphology and in level of pMeCP2 in the hippocampus, respectively.

RESULTSThe morphologies of rat hippocampal neurons in the puerarintreated group were markedly improved compared with the dementia group. The escape latency of the dementia group was significantly longer than the sham-operated group (P<0.05), while the puerarin-treated group was obviously shorter than the dementia group (P<0.05). Cross-platform times of the dementia group were signifificantly decreased compared with the sham-operated group (P<0.05), while the puerarin-treated group was obviously increased compared with the dementia group (P<0.05). IHC staining showed no significant difference in the number of MeCP2 positive cells among 3 groups (P>0.05). The number of pMeCP2 positive cells in the CA1 region of hippocampus in the dementia group was signifificantly increased compared with the sham-operated group, and the puerarin-treated group was signifificantly increased compared with the dementia group (both P<0.05). Western blot analysis showed no signifificant difference of MeCP2 expression among 3 groups (P>0.05). The expression of pMeCP2 in the dementia group was signifificantly increased compared with the sham-operated group, while it in the puerarin-treated group was signifificantly increased compared with the dementia group (P<0.05).

CONCLUSIONPuerarin could play a role in the protection of nerve cells through up-regulating pMeCP2 in the hippocampus, improving neuron morphologies, and enhancing learning and memory ablities in a rat model of VD.

Animals ; Dementia, Vascular ; drug therapy ; genetics ; physiopathology ; Hippocampus ; pathology ; Isoflavones ; chemistry ; pharmacology ; therapeutic use ; Memory ; drug effects ; Methyl-CpG-Binding Protein 2 ; metabolism ; Phosphorylation ; drug effects ; Rats, Sprague-Dawley ; Up-Regulation ; drug effects