Sample
The study was approved by the Ethics Committee of Yunnan Mental Hospital Institution and all the experimental procedures were conducted in accordance with the Helsinki Declaration of 1975, as revised in 1983. Studies suggested that gender differences influenced the BDNF level [23-25], so we included only male patients in our study.
Male patients (n=30) with alcohol dependence were recruited from Yunnan Mental Hospital detoxification treatment unit in China from September 2010 to May 2011. Patients were enrolled to participate in the study after they had signed the informed consents for participation. The Chinese version of the Mini International Neuropsychiatric Interview (MINI) and the alcohol use disorders identification test (AUDIT) were used to screen their psychiatric and drinking conditions to confirm that they met the diagnosis of alcohol dependence according to the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10) criteria by senior psychiatrists. Once the patients were in hospital, they stopped drinking completely. Excluded criteria were those: who had comorbidity with other current non-nicotine substance abuse or dependence, including sedatives; who had significant physical illnesses; who had other psychiatric disorders, such as schizophrenia, bipolar disorder, or major depressive disorder and suffered from severe cognitive impairment with difficulty in understanding the study content. All the alcohol dependence patients were hospitalized, and all received alcohol detoxification treatment with oral Oxazepam 30 - 60 mg for one to two doses with gradual tapering thereafter to prevent severe withdrawal manifestations. Multivitamins were also given. All smoking patients simultaneously received aided nicotine patches, with dose administration adjusted by the individual’s average smoking amount, for smoking cessation program. (The same group of patients we recruited also participated our previous research at the same time [18].)
Blood sampling
After having obtained the permission, the coagulant and anti-coagulant vacuum blood collection tubes with Complete Proteinase Inhibitor Cocktail (cat.#04693116001, Roche Diagnostics, Indianapolis, IN, USA), plasminogen activator inhibitor 1 (PAI-1) ( cat. A8111, Sigma-Aldrich) and the furin inhibitor I (Lot.#D00078005, Calbiochem) were used to get 2-5 ml peripheral blood immediately before starting the detoxification treatment (baseline) and on the eighth day after the detoxification treatments (alcohol withdrawal after 7 days).
Isolation of serum
Human blood obtained from the antecubital vein was collected in coagulant tubes and kept on ice for 4 hours, and then centrifuged for 15 min at 3000 rpm, 4°C. The top layer with light yellow color was collected and kept at -80°C before further experiment.
Isolation of lymphocytes
Blood collected in anti-coagulant tubes were kept at 4°C. The human lymphocytes separation medium (cat.# P8610-200, Solarbio, China) was used to isolate the lymphocytes from the blood. The isolated lymphocytes were kept in -80°C before protein extraction.
Protein isolation from lymphocytes
The protein isolation method was described in our previously paper (Zhou et al., 2013). Briefly, the lymphocytes were homogenized in RIPA buffer (containing 50mM Tris, 150mM NaCl, 1.0% Triton x-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with Complete Proteinase Inhibitor Cocktail (cat.#04693116001, Roche, USA), Plasminogen Activator Inhibitor 1 (PAI-1) (cat. A8111, Sigma-Aldrich, USA) and furin inhibitor I (Lot.#D00078005, Calbiochem, USA). After centrifugation at 13,000 rpm for 10 min, the supernatant was collected and the total protein was quantified using BCA Protein Assay kit (cat. CW0014, Cwbiotech, China).
The western blotting with lymphocytes protein
The western blotting with lymphocytes protein methods were interpreted in detail in our previous paper [26]. Briefly, 50μg lymphocyte protein was separated by running an SDS-PAGE gel, and transferred onto a PVDF membrane (400mA, 2 hours). The membrane was blocked with 5% skimmed milk dissolved in 0.1% Tween-20/Tris-buffered saline (TBST) at room temperature for 1 hour. Followed by incubation of primary antibodies (sheep anti-human proBDNF 1:200, from professor Xin-Fu Zhou’s laboratory; goat anti-human p75NTR 1:250, #sc-6188, Santa Cruz, USA; rabbit anti-human sortilin 1:1000, cat.ab16640, Abcam, USA; rabbit anti-human TrkB 1:1000, cat.#07-225, Millipore, USA; mouse anti-human beta-tubulin 1:1000, #T5168, Sigma, USA) at 4 °C, overnight. After washed in TBST, the membranes were further incubated with horseradish peroxidase-conjugated anti-rabbit/mouse/sheep/goat specie-specific secondary antibodies (cat.sc-2020, Santa Cruz, USA /cat.CW0102, Cwbiotech, China / cat.CW0240, Cwbiotech, China /cat.CW0103, Cwbiotech, China) at room temperature for 1 hour. After washed again in TBST, enhanced chemiluminescent (ECL) substrate (cat. cw0049, Cwbiotech, China) was applied to the membrane before imaging using the ChemiDocTM XRS+ System (Bio-Rad, USA). For quantify the protein level in lymphocytes, beta-tubulin was blotted as loading control. The blots were analyzed using ImageJ software (NIH, USA).
Determination of mBDNF concentration with ELISA
The ELISA method was reported first in our previous paper [26] and proved by Lim et al. [27]. The ELISA steps were as following: 2 ug/ml of the capture antibody (protein G-purified mouse anti-mBDNF monoclonal antibody, B34D10) is diluted in coating buffer (50 mM Carbonate) and 100 ul coating antibody is immediately added into every well in the microplate, incubated for 1 hour at 37 °C. Then the plate is washed for 3 times with PBS. Next, 150 ul of blocking buffer (PBS-BSA) is added into per well and incubated for 1 hour at 37 °C. Dilutions of standard mBDNF are made in a range from 2 ng/ml - 0.125 ng/ml. 100 ul of diluted serum is applied per well and the plate is incubated at 37 °C for 1 hour. Then the plate is washed for 4 times with wash buffer. The next step, 2.5 ug/ml of biotin-labelled detecting antibody is diluted in sample diluent and 100 ul is applied to every well. Then the plate is incubated at 37 °C for 1 hour. Side by side, the plate is washed 4 times with wash buffer and incubated with streptavidin-horse radish peroxidase at 37 °C for 1 hour. After washed for 4 times with wash buffer, 100 ul of freshly prepared TMB substrate is applied to every well and develop for 10 -15 minutes. At last, 1 N sulfuric acid is used to stop the color reaction. The absorbance at 450 nm was measured with a microplate reader (Model Sunrise, TECAN, Germany) to determine BDNF concentration according to the standard curve.
Determination of serum level of TrkB with ELISA
Serum level of TrkB was determined by the commercial human TrkB kit (Sino Biological, Inc., Beijing, China) according to the manufacturer’s instructions.
Statistical Analyses
All the analyses were carried out using SPSS, version 19. The age, average lifetime drinking and average daily amount of ethanol consumption were expressed as mean ± standard deviation (SD). The non-normal distribution data were presented as median + Interquartile range (IQR), others were expressed as mean ± standard error (SE). Comparisons between baseline and one week’s withdrawal were made using a Paired Sample t-test or the Wilcoxon test, depending on the normality of the distribution. A P value which is smaller than 0.05 (2-tailed) was set as statistically significant.