Immunoregulatory Effects Of Pyrroloquinoline-quinone On RAW 264.7 Cells And Mice

Pyrroloquinoline quinone(PQQ)is a new type of water-soluble anionic redox compound,within anti-oxidative,anti-inflammatory,hepatoprotective,neuroprotective,antiosteoporosis,and some other biological activities.Immune system,as an important protective system against foreign substances and dangerous signals in the body,plays a vital role in maintaining homeostasis.Immune modulators have been widely used in the prevention and treatment of various immune diseases,while the regulation of PQQ on the immune system and its mechanism are less studied.The aim of this study was to investigate the immunoregulatory effect of PQQ on RAW 264.7 cells and mice and its potential immunomodulatory mechanism.Our study will provide a theoretical basis for its use as an immunomodulatory adjuvant or functional food in the future.(1)Effects of PQQ on immunoregulatory activity of RAW 264.7 cellsMethods: LPS was used as positive drug to investigate the immune activity of PQQ on RAW 264.7 cells.The effects of PQQ on the cell proliferation,cell morphology,NO content,phagocytosis ability and inflammatory cytokines of RAW 264.7 cells were mainly investigated.In addition,the effects of PQQ on the expression of related proteins in the signaling pathways of mitogen-activated protein kinases(MAPKs)and nuclear factor-κB(NF-κB)in RAW 264.7 cells were also investigated by western blot.Results: Different concentrations of PQQ significantly promoted the relative proliferation rate of RAW 264.7cells.After PQQ treatment,macrophages became larger and grew more pseudopods,and the phagocytosis capacity of macrophages,the secretion capacity of NO and inflammatory cytokines(IL-1β,IL-6,TNF-α)were also significantly increased.Meanwhile,western blot results showed that PQQ could activate RAW 264.7 cells through MAPKS-NF-κB signaling pathway.(2)Effect of PQQ on immune function of immunosuppressive mice that induced by cyclophosphamide(CTX)Methods: The immunosuppressive mice model was established by intraperitoneal injection of CTX.After the modeling was established,different concentrations of PQQ(5,10 and 20 mg/kg)was given to the immunosuppressive mice for 14 days.The following relevant immunological indexes were then determined,such as body weight,viscera index,spleen histological morphology,serum cytokines(IL-6,IL-1β,TNF-α)content,serum immunoglobulin(Ig M,Ig A,Ig G)content,serum NO content,peritoneal macrophage NO secretion,spleen lymphocyte proliferation ability,and so on.Moreover,the expression of related proteins in the MAPKs-NF-κB pathway of mice spleen was also analyzed by western blot.Results: After intraperitoneal injection of CTX,serious body weight loss was observed and the immune indexes of mice were disorder.After treatment with PQQ,the mice began to gain weight and their organ indexes returned to normal levels.The morphology of spleen in mice was observed to be improved by HE staining,and the marginal areas of white and red pulp were clear,indicating that PQQ had a repair effect on the damaged spleen.Moreover,the serum levels of IL-1β,IL-6,TNF-α,and NO content in the supernatant of peritoneal macrophages were also increased in dose dependence manner.The proliferation ability of splenic lymphocytes was also significantly enhanced by treating with PQQ.Furthermore,western blot results indicated that PQQ could enhance the expression of related proteins in the MAPKs-NF-κB signaling pathway.(3)The effect of PQQ on intestinal microflora of immunosuppressive mice that induced by CTXMethods: The feces of mice were collected under aseptic conditions,and the intestinal flora of the feces was detected and analyzed by 16 S r RNA sequencing technology and bioinformatics analysis software.Results: The correlation analysis of environmental factors showed that PQQ and related environmental factors(CTX,TNF-α,IL-6,Ig G,Ig M,Ig A)were strongly and significantly correlated with microbial community structure,indicating that the use of PQQ and CTX was closely related to intestinal microflora.The α-diversity and β-diversity analysis showed that PQQ improved the disturbance of intestinal microbiota that caused by CTX,increased the diversity and richness of intestinal microbiota,and regulated the overall structure of intestinal microbiota.The community taxonomic composition and LEf Se analysis showed that PQQ increased the level of Bacteroidetes and Lactobacillaceae and decreased the level of pathogenic bacteria(Proteobacteria,Ruminococcaceae,Lachnospiraceae).In conclusion,PQQ can effectively alleviate the disturbance of intestinal flora caused by CTX.In conclusion,this study showed that PQQ had good immunomodulatory activity both in vitro and in vivo,which laid a good experimental foundation for its application as an immunomodulatory adjuvant or functional food in the future.