We enrolled 128 pediatric patients with depression who were treated in Binzhou City Center Hospital between February, 2014 and February, 2016. Patients were aged between 7 and 16 years. The cohort had 55 males and 73 females. All pediatric patients were diagnosed with depression by experts specializing in diagnosis and treatment. Fifty healthy children were enrolled into the control group, with ages between 6 and 15 years, and containing 25 males and 25 females. All enrolled subjects were cleared of other chronic diseases. This study was approved by the Ethics Committee of Binzhou City Center Hospital. Signed written informed consents were obtained from the guardians. The research protocol was approved by the Ethics Committee of the hospital. All the patients received follow-up care for one year by the hospital. Accurate clinical history, pathological material, and treatment protocol were conserved through the study.

After total RNA was purified from peripheral blood from pediatric patients and healthy children using the TRIzol kit (Invitrogen, Garlsbad, CA, USA), the integrity of RNA was confirmed by agarose gel electrophoresis. The electrophoresis revealed that 28S, 18S and 5S bands were clear, and the 28S band was twice as strong as the 5S band, suggesting that the RNA was intact for the following experiments. cDNA was prepared by reverse transcription using the reverse transcription kit (Invitrogen). Using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal reference, we performed semi-quantitative PCR (ABI, Foster City, CA, USA) to detect the mRNA levels of BDNF. The reaction conditions were: initial denaturation at 95°C for 5 min; 95°C for 30 sec, 64°C for 25 sec and 72°C for 30 sec (35 cycles). Extension at 72°C for 5 min. Primers were synthesized by Tiangen Biotech (Beijing, China) and the sequences are shown in Table I. At the end of the reaction, the results were observed in ultraviolet imaging system (Biometra, Göttingen, Germany) after agarose gel electrophoresis.

Peripheral blood samples from patients and healthy subjects was concentrated, lysed in radioimmunoprecipitation assay (RIPA) in a 1:1 ratio (volume:volume), and centrifuged at 8,500 × g for 5 min. Then, supernatant was removed and the pellet contained the total protein that was quantified using the bicinchoninic acid (BCA) assay kit. The total protein was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We loaded 10 µl of sample per well for electrophoresis at 80 V followed by transfer onto the polyvinylidene fluoride (PVDF) membrane at 100 V for 90 min. The membranes were blocked for 2 h and the relevant bands were cut for incubation using rabbit anti-BDNF and anti-β-catenin antibodies (Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight. Membranes were washed using Tris-buffered saline and Tween-20 (TBST) 3 times for 5 min. Secondary antibody was incubated for 2 h at room temperature followed by membrane wash in TBST 3 times. Thereafter, ECL solution (A and B solutions were mixed 1:1) (Invitrogen) in appropriate volume was added onto the membrane in a dark room, and exposure was performed according to the fluorescent strength of the bands. Then, images were developed and fixed followed by scanning. Gray scanning was carried out using ImageJ software.

Peripheral blood was centrifuged at 8,500 × g for 5 min at 4°C. Supernatant was collected and added into the wells followed by rinsing using cleaning solution (400 µl in each well). Then, 50 µl rabbit anti-BDNF (Cell Signaling Technology) was added into each well, and the plate was incubated on a vertical shaker for 1 h. Next, the mix was discarded followed by rinsing 4 times. In each sampling well, 100 µl serum sample prepared in advance was added. In the well for the standard, 100 µl standard was added followed by 2 h of reaction on a shaker at room temperature followed by rinsing 4 times. In each well, 200 µl of color development reagent was added for 30 min in a dark room at room temperature. A hundred microliters stopping solution was added in each well to terminate the reaction, and the plate was placed in a plate reader at 450 nm to detect the optical density. Accordingly, the expression level of BDNF was calculated. ELISA kit for BDNF (R&D Systems, Minneapolis, MN, USA).

Pediatric patients with depression usually suffer from loneliness, anxiety and other depressing feelings, which can lead to the refusal of treatment. Thus, by understanding the psychological changes from all aspects and providing the necessary recognition education for patients, we inform the patients of the key role of the treatment to lessen the depression. In addition, we ameliorate the anxiety and resistance to treatment, relieve negative feelings such as pessimistic, lonely, and depressing feelings, correct the previous misconceptions and establish the right cognitive outlook, thereby prompting them to cooperate with the treatment. Regular nursing care was provided by a general ward nursing. Comprehensive nursing care included the general ward nursing care and psychological, bowel, and dietary nursing care, guidance for drug administration, and nursing care of exercise and family.

Symptoms like anorexia usually appear in pediatric patients with depression. Thus, during the treatment, understanding the dietary habits and preferences of patients helped selecting the food that could satisfy the dietary habit and nutritious requirement of each patient.

As pediatric patients with depression usually resist drug administration, education for drug administration was critical during the treatment. This education could guarantee the correct pattern of drug administration, thereby ameliorating the side effect and increasing the compliance of drug administration.

Due to the psychological states in pediatric patients with depression, such as eccentricity and unwillingness to exercise, we stipulated a detailed work-out plan, and prompted the patients to fulfill their daily assignment, thereby encouraging the patients to have more outdoor exercise and raising their interest in exercise.

Care and encouragement from the family are critical to depressed child patients. Thus, nursing care was carried out by deploying the living condition that conformed to the interest of patients, and providing convenience and conformity for patients. Patients should experience the warmth from a close relationship with parents and family members. In addition, patients were encouraged to overcome the bad feelings and psychological obstacles bravely.

We adopted the HAMD scale to evaluate depression. Scores were given to each question and depression condition was divided into four categories. The severity of depression was evaluated according to the scores.

Comprehensive evaluation was performed according to the cooperation of patients to treatment and compliance to drug administration. Complete compliance referred to patients that cooperated with the treatment and received medication strictly in accordance with the medical orders. Incomplete compliance referred to patients that cooperated with the treatment, but received the medication not strictly in accordance with the medical order. Non-compliance referred to patients that did not cooperate with the treatment or did not receive the medication in accordance with the orders. Finally, the cases of complete compliance and non-compliance were used to calculate the total compliance rate.

Through assessment of quality of life, we evaluated the progress of pediatric patients with depression. Scores were given to each question and the life quality was divided into 5 categories, with the high scores representing a better quality of life.

Data are presented as mean ± standard deviation. SPSS 19.0 (Statistic Package for Social Science; SPSS, Inc., Chicago, IL, USA) was used for data processing. Analysis by t-test was performed for comparison between two groups, and analysis of variance (ANOVA) for comparisons among groups. Through homogeneity test of variance, Bonferroni method was used for comparison between either two groups that had equal variance, Welch method for comparison between either two groups that had different variance, and Dunnett's T3 method for multiple comparisons. p<0.05 suggested that the difference had statistical significance.

We first compared the levels of BDNF in blood from children with depression and healthy children. Semi-quantitative PCR showed that the expression level of BDNF mRNA was lower in pediatric patients with depression compared with healthy children (p<0.01) (Fig. 1). To confirm this reduction in BDNF mRNA expression, we next examined the levels of BDNF protein. Compared with healthy children, BDNF protein level was low (p<0.01) (Fig. 2). Using the BDNF level in healthy children as standard, we selected 60 children with depression and low levels of BDNF. We found no statistically significant difference in the levels of BDNF within this group (p>0.05). Then, these 60 patients were divided in two treatments: regular nursing (n=30) and comprehensive nursing (n=30) groups. The two groups received the same medication, but patients in the comprehensive nursing care received additional physical, educational and emotional support. After 3 months of treatment, we examined BDNF expression by ELISA. Compared with the regular nursing group, patients subjected to comprehensive nursing care experienced an increase in BDNF expression with a statistically significant difference (p<0.05) (Fig. 3).

The HAMD scale was used to evaluate depression degree in three study groups: regular nursing (n=30), the comprehensive nursing (n=30), and healthy children (n=50). Before the treatment, the HAMD score of pediatric patients with depression was significantly higher than that in the healthy children (p<0.01) (Table II). However, there was no statistically significant difference in the HAMD score between the regular nursing and the comprehensive nursing groups (p>0.05) (Table II). After 3 months of treatment, patients that received comprehensive nursing care showed a significant decreased in depression scores (p<0.05) (Table III).

Compliance to treatment of pediatric patients was evaluated in the comprehensive nursing and regular nursing groups. Before medication and nursing care, compliance was similar in both groups (p>0.05) (Table IV). After 3 months of treatment, compliance in patients that received comprehensive nursing care was significant higher than in those receiving regular nursing care (p<0.05) (Table V).

We used a questionnaire to evaluate quality of life in pediatric patients with depression. Before the treatment, the quality of life in the regular nursing and comprehensive nursing groups were significantly lower than that in healthy children (p<0.05) (Table VI). However, there was no statistically significant difference in quality of life between the two nursing groups (p>0.05) (Table VI). After 3 months of treatment, the quality of life in patients that received comprehensive nursing care was elevated compared with those receiving regular nursing care (p<0.05) (Table VII).