The hemolytic activity of TE was dose-dependent between 25 and 375 μg protein/mL in 0.45% rat erythrocyte suspension, and the hemolysin unit (HU₅₀, is defined as the concentration of protein that causes 50% lysis of erythrocytes) was 149.5 μg protein/mL (Figure 1).

In the presence of Ca²⁺ from 0.25 to 2 mM, the hemolytic activity of TE (150 μg protein/mL) increased significantly compared with TE treatment alone without extracellular Ca²⁺ (Figure 2). The Ca²⁺ channel blockers, Diltiazem, Verapamil or Nifedipine, produced a slight hemolysis themselves (<5%), however, all significantly inhibited the hemolytic toxicity of TE compared with TE treatment alone, with Diltiazem having the strongest inhibitory effect (79.6% at 160 μM vs. TE treatment value) (Figure 3).

Ca²⁺ fluorescence in erythrocytes significantly increased from 7.52 ± 1.68 without TE intervention to 164.25 ± 12.87 (n = 8) after TE treatment at a concentration of 75 μg protein/mL which would not have led to many erthrocytes lysing rapidly, as shown in Figure 4A,B. When the erythrocytes were pre-incubated with Diltiazem at 37 °C for 30 min, the amplitude of Ca²⁺ fluorescence enhancement by TE treatment was attenuated, (the Ca²⁺ fluorescence increasing from 8.19 ± 2.75 to 13.25 ± 3.19 (n = 8), as shown in Figure 4C,D).

Hemolysis of TE was significantly reduced in the polyethylenglycol (PEG) series with molecular weight (MW) between 4000 Da and 10,000 Da (Figure 5). After pre-incubation with 25 mM PEG6000 for 5 min, the 0.45% erythrocyte suspension was centrifuged at 1000× g for 10 min and the supernatant was removed, such a process was repeated two more times to wash out PEG with PBS, the hemolysis of TE was restored to the level without PEG treatment (Figure 6).

When incubated with erythrocytes, both Vc (5 mM) and GSH (50 μM) produced a significant attenuation (71.2% and 32.3%, respectively) of the hemolysis of TE at 300 μg protein/mL (n = 5, * P < 0.05 vs. TE treatment alone; one-way ANOVA followed by Dunnett’s test). Vc and GSH did not cause hemolysis themselves in the absence of TE aliquots at the concentration chosen for the experiments.

When exposed to increasing concentrations of TE between 50 and 375 μg protein/mL, rat erythrocytes showed a dose-dependent increase of lipid peroxidation, with the MDA level increasing from 2.52 ± 0.35 μM to 12.81 ± 1.15 μM at the highest dose of TE (Figure 7).

After TE administration (1.25 mg protein/kg), the optical density (OD) of rat serum at 415 nm increased sharply and reached a peak after 10 min, then gradually returned to the baseline level in the following 60 min, while Vc (250, 500 mg/kg) treatment evidently attenuated the peak of OD (Figure 8). The electrolytes Na⁺, K⁺, Ca²⁺, Cl⁻ and lactic acid (Lac) were all significantly influenced by TE with Na⁺, Ca²⁺ and Cl⁻ decreasing and K⁺ and Lac increasing 10 min after TE administration. The amplitude of K⁺ and Lac increase was also significantly attenuated by Vc (250 mg/kg) (Table 1).

Specimens of C. capillata were collected in June 2010 in the Sanmenwan offing of the East China Sea in Zhejiang Province, China, and identified by Professor Huixin Hong from the Fisheries College of Jimei University, Xiamen, China. The removed tentacles were preserved in plastic bags on dry ice and immediately shipped to Shanghai, where the samples were frozen at −70 °C until use. The TE was prepared following the method described as in previous reports [25,35]. Briefly, frozen tentacles were thawed at 4 °C in filtered seawater at a mass/volume ratio of 1:1 to allow autolysis of the tissues for 4 days. The mixture was then stirred for 30 min twice daily. The autolyzed mixture was centrifuged at 10,000× g for 15 min three times. The resultant supernatant was the TE. All procedures were performed at 4 °C or in an ice bath. Before being used for injection, TE was centrifuged at 10,000× g for 15 min to remove sediment, followed by dialysis against PBS (0.01 M, pH 7.4) at 4 °C for over 8 h. Protein concentrations were determined in all samples by the method of Bradford (1976), with fetal bovine serum albumin (high purity, Sigma, St. Louis, MO, USA) as a standard [36].

Hemolytic activity of TE was tested in 0.45% male rat erythrocyte suspension. In brief, 100 μL of fresh heparinized blood samples were made up to 10 mL in phosphate buffered saline (PBS, self prepared, with the following composition in mM: 135 NaCl, 4.7 KCl, 10 Na₂HPO₄, 2 NaH₂PO₄, pH 7.4), and then centrifuged at 1000× g for 10 min at 4 °C. The supernatant and buffy coat were removed by gentle aspiration, and the above process was repeated two more times. Erythrocytes were finally resuspended in the same buffer to a final concentration of 0.45% (v/v). Aliquots of 0.45% erythrocyte suspension were incubated at 37 °C for 30 min in the presence of TE with the concentrations ranging from 0 to 375 μg protein/mL (50% v/v), and then centrifuged at 1000× g for 10 min at 4 °C to precipitate both the intact erythrocytes and ghosts. The 200 μL supernatants were transferred into 96-well microplates and the absorbance at 415 nm was determined by using a spectrophotometric microplate reader (BIO-RAD iMark™, Hercules, CA, USA) to measure the extent of red blood cell lysis. The hemolytic activity of TE was expressed as % absorbance compared with that observed after maximal lysis induced by sodium dodecyl sulphate (40 μg/mL). The supernatant of untreated 0.45% erythrocyte suspension was taken as the background and subtracted (n = 6). Male Sprague-Dawley (SD) rats (220–240 g) were provided by the Laboratory Animal Center of the Second Military Medical University, Shanghai. All were procured from the animal care facility at the university where they were housed in cages with 12/12 h light/dark cycle at 22 ± 2 °C and given standard diets plus water ad libitum. The investigation was carried out in conformity with the Ethics Committee of the Second Military Medical University and international guidelines for animal handling.

The effect of Ca²⁺ was studied by adding CaCl₂ into TE and then the hemolytic activity was assayed as described above. Ca²⁺ was employed at a final concentration of 0.25, 0.5, 0.75, 1.0 and 2.0 mM, respectively. To inspect the possible protective effect of Ca²⁺ channel blockers against hemolysis, Diltiazem, Verapamil and Nifedipine (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) were incubated with 0.45% erythrocyte suspension at 37 °C for 30 min before TE administration (150 μg protein/mL). Then the relative percentage of hemolytic activity was calculated as described above. All the Ca²⁺ channel blockers were employed at final concentrations of 10, 20, 40, 80 and 160 μM, respectively.

The intracellular Ca²⁺ indicator fluo-3 acetoxymethyl (AM) was used to measure the Ca²⁺ changes in the erythrocyte. Briefly, 1 mL of the 0.2% erythrocyte suspension was loaded with Fluo-3/AM (5 μM) and further incubated at 37 °C for 120 min in the dark. After this, the solution was centrifuged at 1000× g for 10 min and the supernatant was removed. Such a process was repeated two more times. The erythrocytes were finally resuspended in 1 mL Hank’s solution (Sangon Biotechnology, Shanghai, China, with the following composition in mM: 1.6 CaCl₂, 1.0 MgCl₂, 145 NaCl, 5.0 KCl, 0.5 NaH₂PO₄, 10 dextrose, and 10 Hepes, pH 7.4). To test the influence of Ca²⁺ channel blocker on the changes of intracellular Ca²⁺ induced by the TE, 0.2% erythrocyte suspension was pre-incubated with Diltiazem (100 μM) before exposure to the TE (75 μg protein/mL). For confocal laser scanning microscopy, 200 μL of such a solution with or without Diltiazem was replated onto special dishes. Intracellular Ca²⁺ changes were measured by monitoring the alteration of cell fluorescence.

To test the effect of osmotic protectants, 25 mM PEG with different molecular weights (2000, 4000, 6000 and 10,000 Da) was added to 1 mL 0.45% erythrocyte suspension. Then aliquots of TE (75, 150, 300 μg protein/mL) were added, and the hemolytic activity was detected as described above. In another separate experiment, 25 mM PEG6000 was pre-incubated with 0.45% erythrocyte suspension at 37 °C for 5 min, then the 0.45% erythrocyte was centrifuged at 1000× g for 10 min and the supernatant was removed. Such a process was repeated two more times to wash out PEG with PBS, and the erythrocytes were finally resuspended in PBS to a final concentration of 0.45% (v/v), and then aliquots of TE (75, 150, 300 μg protein/mL) were added to test the hemolytic activity.

In order to test the possible involvement of lipid peroxidation in the hemolysis, aliquots of 0.45% erythrocyte suspension were pre-treated with Vc and GSH at 37 °C for 30 min at a final concentration of 5 mM and 50 μM, respectively, before TE (300 μg protein/mL) administration, and hemolytic activity was detected as described in Section 4.2.

For lipid peroxidation assay, we used a commercial kit (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China) to quantify the generation of malondialdehyde (MDA) according to the manufacturer’s protocol. In brief, the 20% erythrocyte suspension aliquots were incubated with TE at 37 °C for 30 min and then centrifuged at 1000× g for 10 min, and the supernatant was subjected to measurement of MDA levels.

Thirty-two male Sprague-Dawley (SD) rats (220–240 g, provided by the Laboratory Animal Center of the Second Military Medical University, Shanghai, China) were randomized into four groups (n = 8) and anesthetized with urethane (1.2 g/kg i.p.). To inspect the possible protective effect of antioxidants against hemolysis of the TE in vivo, Vc at different doses (125, 250, 500 mg/kg i.p.) was adopted before TE administration (1.25 mg protein/kg i.v.). The blood sample was obtained through a glass capillary inserted into the retrobulbar venous plexus at 0 (pre-injection), 10, 30 and 60 min after TE administration, respectively. Similarly, the rats of the control group (n = 8), after intraperitoneal injection of PBS, were treated with TE through tail vein injection and blood samples were obtained at the corresponding time points. Aliquots of the blood samples were centrifuged at 1000× g for 10 min to precipitate both the intact erythrocytes and ghosts. The supernatants were collected as the serum and the ODs were spectrophotometrically measured at 415 nm as the marker of hemolytic activity in vivo.

To further observe the effects of antioxidants on the changes of electrolytes and Lac in vivo, eighteen male SD rats were randomized into three groups (n = 6) and pretreated with PBS or Vc (250 mg/kg i.p.), then TE at the dose of 1.25 mg protein/kg was chosen for tail vein injection and the serum was collected 10 min after TE administration. The sham-treated animals (n = 6) were administered with the same volume of PBS. Electrolytes (Na⁺, K⁺, Ca²⁺ and Cl⁻) and Lac in the serum collected as described above were analyzed by an arterial blood gas analyzer (GEM Premier 3000, International Laboratory, Bedford, MA, USA).

One-way analysis of variance (ANOVA) was used. In all cases, statistical significance was indicated by P < 0.05. All data were expressed as mean ± SD.