Immunostimulatory activity of protein hydrolysate from oviductus ranae on macrophage in vitro

Abstract

Oviductus Ranae is the dry oviduct of Rana chensinensis, which is also called R. chensinensis oil. Oviductus Ranae is a valuable Chinese crude drug and is recorded in the Pharmacopoeia of the People's Republic of China. The aim of this study was to investigate the immunostimulatory activity of protein hydrolysate of Oviductus Ranae (ORPH) and to assess its possible mechanism. Immunomodulatory activity of ORPH was examined in murine macrophage RAW 264.7 cells. The effect of ORPH on the phagocytic activity of macrophages was determined by the neutral red uptake assay. After treatment with ORPH, NO production levels in the culture supernatant were investigated by Griess assay. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS) were detected by RT-PCR and Western blotting. The production of TNF-α, IL-1β, and IL-6 after treatment with ORPH was measured using ELISA assay. In addition, NF-κB levels were also investigated by Western blot. The results showed that ORPH enhanced the phagocytosis of macrophage, increased productions of TNF-α, IL-1β, IL-6, and NO in RAW 264.7 cells, and upregulated the mRNA and protein expression of iNOS. Besides, NF-κB, levels in RAW 264.7 cells were elevated after ORPH treatment. These findings suggested that ORPH might stimulate macrophage activities by activating the NF-κB pathway.

Figure 1

Effect of protein hydrolysate of Oviductus Ranae on cell viability. RAW 264.7 cells were pretreated with test substances (0, 10, 100, 500, 1000, and 2000 μg/mL) for 48 h and were used to test the cytotoxicity by MTT assay. OR, Oviductus Ranae; ORP, Oviductus Ranae protein; ORPH-pa, Oviductus Ranae protein hydrolysate prepared with papain; ORPH-tp, Oviductus Ranae protein hydrolysate prepared with trypsin; ORPH-pe, Oviductus Ranae protein hydrolysate prepared with pepsin; ORPH-np, Oviductus Ranae protein hydrolysate prepared with neutral protease; ORPH-ap, Oviductus Ranae protein hydrolysate prepared with alkaline protease. Control, untreated cells.

Figure 2

Effect of protein hydrolysate of Oviductus Ranae on phagocytosis. (a) After treatment with test substances (500 μg/mL) or LPS 1 μg/mL for 48 h, RAW 264.7 cells were used to test the phagocytosis by neutral red uptake assay. Control, untreated cells. (b) RAW 264.7 cells were pretreated with ORPH-np (0, 10, 100, 500, and 1000 μg/mL) for 48 h and were used to test the phagocytosis by neutral red uptake assay. Control cells were pretreated with the medium that contained same levels of neutral protease with ORPH-np, the neutral protease was processed by the same procedure with ORPH-np. (c) RAW 264.7 cells were pretreated with ORPH-np (0, 10, 100, 500, and 1000 μg/mL) for 24, 48, and 72 h and were used to test the phagocytosis by neutral red uptake assay. Control, 0 μg/mL. Results were expressed as means ± S.D. of four separate experiments. Statistical significance test for comparison with untreated group was done by t-test. ^* P < 0.05; ^** P < 0.01.

Figure 3

Effect of protein hydrolysate of Oviductus Ranae on NO production. (a) RAW 264.7 cells were pretreated with test substances (500 μg/mL) or LPS (1 μg/mL) for 48 h. The supernatant nitrite levels were determined using Griess reagent. Control, untreated. (b) RAW 264.7 cells were pretreated with ORPH-np (0, 10, 100, 500, and 1000 μg/mL) for 48 h, the supernatant nitrite levels were determined using Griess reagent. Control cells were pretreated with the medium that contained same levels of neutral protease with ORPH-np, the neutral protease was processed by the same procedure with ORPH. (c) RAW 264.7 cells were pretreated with ORPH-np (0, 10, 100, 500, and 1000 μg/mL) for 24, 48, and 72 h and the supernatant nitrite levels were determined using Griess reagent. Control, 0 μg/mL. Results were expressed as means ± S.D. of four separate experiments. Statistical significance test for comparison with untreated group was done by t-test. ^* P < 0.05; ^** P < 0.01.

Figure 4

Effect of ORPH on expression of iNOS in RAW 264.7 cells. (a) Cells were incubated with ORPH (500 μg/mL) or LPS (1 μg/mL) for 24 h. Total RNA was prepared and mRNA levels encoding iNOS was measured by RT-PCR. β-actin was used as an internal control. (b) Corresponding quantification data of iNOS mRNA expressional levels. (c) The protein level of iNOS in ORPH-stimulated RAW 264.7 cells was analyzed by Western blotting. (d) Corresponding quantification data of iNOS protein levels. ORPH, Oviductus Ranae protein hydrolysate prepared with neutral protease. Control, untreated. The results were stated in iNOS versus β-actin; data are expressed as mean ± SD (n = 3). Statistical significance test for comparison with untreated group was done by t-test. ^* P < 0.05; ^** P < 0.01.

Figure 5

Effects of ORPH on macrophage-related cytokine production in macrophages. RAW 264.7 cells (3 × 10⁵ cells/well) were cultured for 24 h in the presence of media with ORPH (0–1000 μg/mL) or LPS (1 μg/mL). The amounts of TNF-α (a), IL-1β (b), and IL-6 (c) released into the culture media were measured by immunoassays. ORPH, Oviductus Ranae protein hydrolysate prepared with neutral protease. Results were expressed as means ± S.D. of four separate experiments. Statistical significance test for comparison with untreated group was done by t-test. ^* P < 0.05; ^** P < 0.01.

Figure 6

Effect of the peptide with different molecular weights on the NO and TNF-α production of RAW 264.7 cells. (a) and (b) cells were pretreated with or without test substances (500 μg/mL), respectively. The supernatant nitrite levels and TNF-α were determined using Griess reagent and ELISA kit, respectively. Control, 0 μg/mL. (c) and (d) cells were cultured in the presence of media with the <3 kDa fraction (0–1000 μg/mL) or LPS (1 μg/mL). The amounts of NO and TNF-α in the supernatant were determined using Griess reagent and ELISA kit, respectively. ORPH, Oviductus Ranae protein hydrolysate prepared with neutral protease. Results were expressed as means ± S.D. of four separate experiments. Statistical significance test for comparison with untreated group was done by t-test. ^* P < 0.05; ^** P < 0.01.

Figure 7

Effect of ORPH on nuclear NF-κB. (a) and cytoplasmic IκB-α (b) expression level by RAW 264.7 cells. Cells were cultured for 24 h in the presence of media with ORPH (0–500 μg/mL) or LPS (1 μg/mL). Western blotting was performed to detect the protein level of nuclear NF-κB and cytoplasmic IκB-α. The levels of lamin B were measured as internal loading controls. The results were analyzed by iPP 6.0 software and stated in NF-jB versus Lamin B, data are expressed as mean ± S.D. (n = 3). Statistical significance test for comparison with untreated group was done by t-test. ^* P < 0.05; ^** P < 0.01.