In total, 36 mice were randomly divided into three groups with 12 mice in each group, including a normal group that was given 0.5% CMC-Na (20 ml/kg/d), a positive control group which was treated with sertraline (5 mg/kg/d), and HLS group which was then treated with HLS (20 ml/kg/d, 2.7 g crude medicine per ml). The corresponding drugs were intragastrically administered for 7 days.

The TST was performed, as previously described (Cates et al., 2013). Mice were individually suspended by the tail with a clamp placed 1–2 cm from the tip of the end. After 1 min of adaptation, the immobility time of the animals was recorded in the subsequent 5 min.

The FST employed was similar to that described in a previous report (Hodosy et al., 2012). Mice were placed in a rectangular white tank (20 × 10 × 13 cm³) filled with water at 25 ± 1°C. After forced swimming for 6 min, immobility time was recorded in the subsequent 5 min. After each trial, the apparatus was cleaned.

Mice were placed in a ZZ-6 apparatus for measuring locomotor activity in a quiet environment. After 3 min of adaptation, the locomotor activity of the animals was recorded in the subsequent 5 min.

A total of 100 male Wistar rats underwent the open field test for behavioral scoring. The rats with scores between 30 and 120 were divided into four groups using a randomized block design: group 1 which was a normal group (normally reared rats, 20 ml/kg/d), group 2 which was a model group (depressive rats without treatment), group 3 which was a positive control group (depressive rats treated with sertraline, 5 mg/kg/d), and group 4 which was an HLS group (depressive rats treated with HLS, 20 ml/kg/d, and 2.7 g crude medicine per ml). After successful induction of depression by CUMS and separation for 21 days in groups 2, 3, and 4, the rats were intragastrically given the corresponding drugs for 21 days, except for the model group without treatment. During the treatment period, CUMS and separation were still administered. The experimental design can be seen in Figure 1.

The CUMS model was revised from a report on models of depression that allow investigation of key biological correlates of depression (Nollet et al., 2013). Except for group 1 rats, which were normally reared (5–6 rats/cage), the others were reared individually in a separate cage and given two different stimuli randomly daily; each stimulus was administered four times in 21 days. The stimuli included swimming in cold water at 4°C for 5 min, thermal stimulation (hot plate test, at 45°C, for 5 min), water deprivation (24 h; no fasting), fasting (24 h; no water deprivation), day/night reversal (24 h), moist bedding (10 h), restraint (1 h), and vibration (1 min).

OFT paradigm can be found in the previous report (Aboul-Fotouh, 2015). The rats were put into the central square in an apparatus (100 × 100 × 40 cm³) with a black inner side and white lines on the bottom (five in a row and five in line, equally). Then, they were allowed to move freely for 3 min, recording the number of crossings (at least three paws in one square) and rearings (standing upright on the hind legs) and finally calculating the summation.

The experimental indicators were measured 1 h after drug administration.

Open field tests and weight measurements were performed once a week after drug administration.

One hour after drug administration on day 21st, eight rats of each group were anesthetized (at 8:00–10:30) by intraperitoneal injection of 10% chloral hydrate (0.3 ml/100 g) and then killed by femoral artery bleeding. The bilateral hippocampus, adrenal glands, thymus, and spleen were removed and weighed to calculate the organ coefficients.

The blood samples were collected, as described earlier, standing for 1 h and centrifuged at 3000 r/min for 20 min, and the supernatants were collected for further use. Hippocampal samples were homogenized in PBS (1:1, w/v, mg/ml) and centrifuged at 3000 r/min for 20 min, and the supernatants were collected and preserved at −80°C for further use. The contents of 5-HT, NE, and DA in the hippocampus and serum were determined using commercial kits, according to the manufacturer’s instructions.

The serum contents of ACTH, CORT, and β-EP were determined using commercial kits, according to the manufacturer’s instructions.

The remaining eight rats of each group were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 ml/100 g), fixed in supine position, and perfused with 100 ml normal saline via the left ventricle; then, 4% paraformaldehyde was perfused after making an incision on the liver. The hippocampus and adrenal glands were then obtained and weighed. The samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. One section was selected from every 10 intermittent sections, and five sections were selected for each hippocampus or adrenal gland for hematoxylin and eosin (HE) staining. The TSView7 image analysis system (TUCSEN Imaging Technology, Inc.) was used to count the total number of neurons and the number of normal neurons in five grid squares in the CA3 area of the hippocampus (magnification, ×400) to calculate the proportion of normal neurons. The maximum thickness of the adrenal cortex was measured at a magnification of ×40.

A total of 1 ml of HLS was precisely weighed and placed in a 25 ml volumetric flask. Methanol was added to the 25 ml volume mark. About 2 ml of the obtained solution was sucked in a 10 ml volumetric flask, and methanol was added to the 10 ml volume mark to obtain the test solution of HLS. The contents of the volume bottle were filtered through a 0.45 μm microporous membrane. Methanol was added to 2,3,5,4′-tetrahydroxy stilbene-2-Ο-β-D-glucoside precisely weighed to obtain a standard solution in the concentration of 41.76 μg/ml. A negative sample of HLS without polygoni multiflora radix praeparata was prepared according to the treatment method of the test solution. HPLC was conducted using a Reloasil C18 column (150 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile water (22:78) at a velocity of 1 ml/min and a detection wavelength of 320 nm.

A total of 1 ml of HLS was precisely weighed and placed in a 25-ml volumetric flask. Methanol was added to the 25 ml volume mark to obtain the test solution. The contents of the volume bottle were filtered through a 0.45 μm microporous membrane. Methanol was added to icariin precisely weighed to obtain a standard solution in the concentration of 39.04 μg/ml. A negative sample of HLS without epimedii folium was prepared according to the treatment method of the test solution. HPLC was conducted by using a Reliasil C18 column (150 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile water (30:70) at a velocity of 1 ml/min and a detection wavelength of 270 nm.

A total of 1 ml of HLS was precisely measured, ultrasonically extracted in the 80% ethanol solution at 60°C, and the filtered solution was obtained and evaporated to dryness. The residue was dissolved in 1% sodium hydroxide and extracted with water-saturated n-butanol; then the organic solution was mixed together and evaporated to dryness, and the residue was dissolved in methanol to obtain the test solution (1 ml HLS was finally diluted in 10 ml methanol). Methanol was added to diosgenin precisely weighed to obtain a standard solution in the concentration of 39.04 μg/ml. The diosgenin standard solution was absorbed in perchloric acid to prepare a linear series solution of 7.2, 12.0, 16.8, 18, and 26.4 µg/ml; the solutions were placed in a water bath at 65°C for coloration and then placed in an ice bath for cooling. Using the blank solution as a control, absorbance at 410 nm was measured. Linear regression was performed with the absorbance as ordinate and diosgenin content as abscissa, and the regression equation was calculated.

A total of 50 ml of HLS was precisely weighed and extracted with n-butanol. The mixed extracted solution was washed with ammonia and evaporated to dryness. Methanol was added to the residue in a 10 ml volume mark to obtain the test solution. The contents of the volume bottle were filtered through a 0.45 μm microporous membrane. Methanol was added to astragaloside IV precisely weighed to obtain a standard solution in the concentration of 552 μg/ml. A negative sample of HLS without astragali radix was prepared according to the treatment method of the test solution. HPLC was conducted by using a Reloasil C18 column (250 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile water (35:65) at a velocity of 1 ml/min. The drift tube temperature of ELSD was set at 40°C, and the gas flow was 1.5 L/min.

A total of 50 ml of HLS was precisely weighed and extracted with ether. The mixed extract was evaporated to dryness. Methanol was added to the residue at a 5 ml volume mark. The contents of the volume bottle were filtered through a 0.45 μm microporous membrane. Methanol was added to tanshinone ⅡA precisely weighed to obtain a standard solution in the concentration of 0.6 μg/ml. A negative sample of HLS without salviae miltiorrhizae radix et rhizoma was prepared according to the treatment method of the test solution. HPLC was conducted by using a Reliasil C18 column (150 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile water (22:78) at a velocity of 1 ml/min and a detection wavelength of 320 nm.

As shown in Figure 2, HLS could significantly shorten the immobility time in TST and FST compared with the normal group (p < 0.05).

As shown in Figure 3, there was no significant difference in mouse locomotor activity in the HLS group compared with the normal group (p > 0.05).

Tables 1, 2 show the effects of HLS on the open field score (OFS) and body weight (BW) on depression-like behavior in rats, respectively. After inducing depression by CUMS and separation, the OFS and BW of the model group were significantly decreased compared with the normal group (p < 0.05). It hints that the behavior of rats in the model group is similar to depression largely. HLS or sertraline significantly enhanced OFS after 3-week treatment and increased BW after 1-week treatment compared with the model group (p < 0.05).

As shown in Figure 4 , compared with the normal group, monoamine neurotransmitter levels in the hippocampus were significantly decreased (the p < 0.05), but their levels in serum were significantly increased in the model group. Compared with the model group, 5-HT and DA levels in the hippocampus of the HLS group were all significantly increased (p < 0.05), but in serum, monoamine neurotransmitter levels were significantly decreased (p < 0.05).

As shown in Figure 5, ACTH, CORT, and β-EP levels in serum were all significantly higher in the model group than in the normal group (p < 0.05). The treatment with HLS significantly decreased ACTH, CORT, and β-EP levels in serum (p < 0.05).

As shown in Figure 6, the adrenal gland coefficient was significantly increased (p < 0.05), and the thymus and spleen coefficients were significantly decreased (p < 0.05) in the model group compared with the normal group. The treatment with HLS significantly increased thymus and spleen coefficients (p < 0.05).

The neurons in the CA3 area of the hippocampus of rats were orderly and densely arranged, with clearly visible nucleoli in the normal group. In contrast, the neurons in the CA3 area of the hippocampus of rats were sparsely arranged and showed increased intercellular space, nucleolus disappearance, and cytoplasm pyknosis in the model group (Figure 7). The total number of neurons and the proportion of normal neurons in the CA3 area of the hippocampus were significantly decreased in the model group compared with the normal group (p < 0.05). Compared with the model group, the total number of neurons and the proportion of normal neurons in the CA3 area of the hippocampus were significantly increased in the HLS groups (p < 0.05) (Figure 8).

The adrenal glands of the rats had a clear structure and can be divided into two parts: adrenal cortex and medulla, and the adrenal cortex can be further divided into the following zones: zona glomerulosa, zona fasciculata, and zona reticularis in the normal control group. The adrenal cortex was hypertrophic in the model group. The thickness of the adrenal cortex was significantly larger in the model group than that in the normal group (p < 0.05), while treatment with HLS significantly decreased the thickness of the adrenal cortex compared with the model group (p < 0.05) (Figure 9).

The content of 2,3,5,4′-tetrahydroxy stilbene-2-Ο-β-D-glucoside in HLS is 3.85 mg/ml based on HPLC (Figure 10).

The content of icariin in HLS is 1.04 mg/ml based on HPLC (Figure 11).

The content of Saponins of Rhizoma Polygonati in HLS is 1.22 mg/ml determined by colorimetry.

The content of astragaloside IV in HLS is 0.036 mg/ml based on HPLC (Figure 12).

The content of tanshinone ⅡA in HLS is 0.024 μg/ml based on HPLC (Figure 13).