Preparation,in Vitro And In Vivo Immunomodulatory Effects And Mechanism Of Deer Blood Peptides

Purpose:Deer blood peptide（DBP）is extracted from deer blood through protease hydrolysis,which has been widely developed and used in traditional Chinese medicine as a prophylactic.At the same time,the occurrence and development of many diseases are closely related to the low immunity of the host.Accordingly,the regulation of immunity is an adjuvant medical therapy for many diseases.Currently,there are few studies on the immunoregulatory activity of DBP.Therefore,this study aimed to screen the preparation methods of deer blood polypeptides with high immunoregulatory activity,and verify their immunoregulatory effects on RAW 264.7cells and immunosuppressive mice induced by cyclophosphamide（CTX）.In addition,the aim of the study was to reveal their mechanism of action.Methods:Deer blood peptide hydrolysate catalyzed by pepsin and trypsin（PTDBP）,deer blood peptide hydrolysate catalyzed by pepsin（PDBP）,and deer blood peptide hydrolysate catalyzed by trypsin（TDBP）were obtained by protease hydrolysis.The content of DBP was determined by colorimetry,and the molecular weight range of DBP was determined by the Tricine-SDS-PAGE method.Through the detection of cell viability,phagocytic activity,and nitric oxide（NO）secretion of macrophages,PTDBP with potential immunomodulatory activity was selected.The composition and content of amino acids in PTDBP were determined,which evaluated the nutritional value of PTDBP.The antioxidant capacities in PTDBP were measured by DPPH,ABTS,and OH assay.The immunoenhancement effects of PTDBP have been demonstrated in RAW264.7 cells in vitro.The pharmacological activity tests explored the immunomodulatory effect of PTDBP and observed the effect of PTDBP on cell viability,phagocytic activity,and NO secretion ability.The reactive oxygen species（ROS）generation and adhesion functions of macrophages were observed through an inverted microscope.Furthermore,NO,TNF-αand IL-6 secretion of RAW 264.7cells were detected.In addition,the cell cycle of RAW 264.7 was measured by flow cytometry.The effect of PTDBP on the expression of i NOS,TNF-α,and IL-6 m RNA were studied at the gene level.Based on the MAPK signal pathway,the mechanism of PTDBP on the immunoregulatory activity of RAW 264.7 was investigated.The in vivo immunomodulatory effects of PTDBP were investigated in CTX-induced immunosuppressed mice.The immune organ indices and the peripheral hemogram were detected.The changes in immunoglobulin and cytokines were measured.The spleen and thymus organs of CTX-induced mice were stained by hematoxylin and eosin.To explore whether the MAPK signaling pathway was involved,the expression levels of p-JNK,p-ERK,and p-p38 were detected by western blot analysis.Through an orthogonal test,the proper deodorization process ofβ-cyclodextrin and yeast was evaluated by the index.Take the stability coefficient as the index to screen a suitable stabilizer.The formula of PTDBP oral liquid was optimized by sensory attributes.The storage stability of the PTDBP oral solution was tested by observing and measuring physical and chemical indexes.Results:PTDBP,PDBP,and TDBP were successfully prepared,among which PTDBP has the highest polypeptide content.In addition,the immunomodulatory activity study showed that PTDBP had the strongest immunomodulatory activity.PTDBP could enhance proliferation,phagocytic capacity,and NO secretion of RAW264.7 cells compared with PDBP and TDBP.The amino acid analysis of PTDBP showed that PTDBP contained 18 amino acids,including 7 essential amino acids.PTDBP(IC₅₀=4.061±0.288 mg/m L,for DPPH;IC₅₀=0.629±0.015mg/m L,for ABTS;and IC₅₀=3.864±0.245 mg/m L,for OH)showed significant DPPH,ABTS,and OH radical scavenging ability.The scavenging ability of 16.0 mg/m L PTDBP to DPPH·,ABTS·,and·OH was 99.53%,72.12%,and 70.41%,respectively.The results showed that PTDBP had certain free radical scavenging activity.The results of Tricine-SDS-PAGE gel electrophoresis showed that the molecular weight of PTDBP was less than 7.8 k Da.Based on RAW 264.7 macrophages,the immunoregulatory activity of PTDBP in vitro was studied.The results showed that PTDBP had no obvious toxicity to cells and promoted the proliferation of macrophages to a certain extent.PTDBP treatment enhanced the proliferation of RAW 264.7 cells at 125.0-500.0μg/m L.It can significantly improve the phagocytic capacity,adhesion function,and secretion level of NO,TNF-α,and IL-6.PTDBP can stimulate macrophages to produce ROS and promote cell proliferation by acting on the G0/G1 phase of RAW 264.7 cells.PTDBP could up-regulate i NOS and TNF-α,and IL-6 m RNA expression levels.After exposure to PTDBP,the protein levels of p-JNK,p-ERK,and p-p38 were increased.To further explore the immunoregulatory effect of PTDBP in vivo,the immunosuppressive mouse model induced by CTX was used to study the immunomodulatory effects of PTDBP.The experimental results showed that after the intervention of PTDBP,it could alleviate the weight loss tendency,restore the blood cell indexes such as WBC,LYM,and BMNC in mice,and promote serum Ig G,TNF-α,and IL-6 in CTX-treated mice.PTDBP could increase the immune organ indices,protect the growth of the spleen and thymus,and improve pathological changes.The western blot results showed that different concentrations of PTDBP could up-regulate the level of p-p38,p-JNK,and p-ERK proteins in the spleen of CTX-induced immunosuppressive mice.PTDBP could activate the MAPK signal pathway to play an immunomodulatory role.β-cyclodextrin（4.0%）and yeast（1.5%）are recommended for PTDBP deodorization.Xanthan gum 0.07%,pectin 0.06%,and CMC-Na 0.06%are used as stabilizers.The formula of PTDBP oral liquid is PTDBP 0.5%,sucrose 4.98%,and citric acid 0.05%.Conclusion:PTDBP was prepared by two-step protease hydrolysis.The immunoregulatory activity of PTDBP was investigated in vitro and in vivo.Thus,it is possible to provide the theoretical foundation and technical support for the high-value utilization of the material and the industrialization of the immunomodulating peptides.Moreover,PTDBP could significantly enhance the immune function of immunosuppressed mice and activate RAW 264.7 cells by stimulating the MAPK signaling pathway.