Immunomodulatory Effects Of Caffeoyl Glycoside Monomer From Picrorhiza Scrophulariiflora In Vitro

The genus Picrorhiza contains two species Picrorhiza kurrooa Royle(P.kurrooa) and Picrorhiza scrophulariiflora Pennel(P.scrophulariiflora).The two species of Picrorhiza have revealed many medical benefits including hepatoprotection, immunomodulation,antiinflammation,antiasthma,nerve growth factor(NGF)-potentiating, and antioxidative activities.Recently,our lab successfully isolated a monomer,1,6-di-O- caffeoyl-β-D-glucopyranoside, named Caffeoyl Glycoside(CG)from the roots of P. scrophulariiflora.In the present studies,the immunomodulatory effects of CG were investigated in vitro.We found that CG stimulated cell proliferation of plenocytes and peritoneal macrophages peritoneal macrophages energy metabolism and phagocytic function.CG enhanced activation of Natural killer cells(NK cells).CG increased CD4 and CD8 cell population.CG increased S period cell population.Furthermore,to further understand the mechanism,several cytokines were examined.We found that the level of Th1(IL-2,IL-12,IFN-γ,TNF-a)significantly increased,and Th2(IL-4,IL-10)decreased after CG treatment.Token together,these results indicated that CG might have potential effects on regulating immune system.Cell proliferation and the assessment of substances that either promote or inhibit cell proliferation are crucial to the study of cell biology and to drug-discovery research. In the present studies,CG significantly increased proliferation of splenocytes and peritoneal macrophages at middle range of dosages(5-125μg/mL)without any mitogen stimulation.These effects were markedly enhanced by presence of either LPS or Con A.Both LPS and Con A are lymphocytes motigen that encourages a cell to commence cell division,triggering mitosis.Mitogens are often used to stimulate lymphocytes and therefore assess immune function.Con A directly acts upon T cells, while LPS acts upon B cells.T cells and B-cells are the major cellular components of the adaptive immune response.T cells are involved in cell-mediated immunity whereas B cells are primarily responsible for humoral immunity.CG could facilitate either LPS or Con A to stimulate proliferation of lymphocytes,therefore,CG could enhance both cellular immunity and humoral immunity.These conclusions were further supported by CG enhanced CD4~+ and CD8~+ cell population experiment.CD4 recognize antigens present by a MHCⅡand mediate both cellular immune response through Th1 cells and humoral immune response through Th2 cell.CD8 recognize antigens present by MHCⅠand mediate cellular immune response through cytotoxic T cells.Both adaptive immunity and innate immunity play an essential role in fighting diseases.NK cells are a part of innate immune system and play a major role in defending the host from both tumours and virally infected cells.NK cells distinguish infected cells and tumours from normal and uninfected cells by recognizing alterations in levels of a surface molecule called MHCⅠ.In the present paper,we demonstrated that CG significantly enhanced NK function to kill target K562 cell.The result together with CG promoted proliferation of peritoneal macrophages,another innate immune component,indicated that CG not only enhanced adaptive immunity but also activated innate immunity.As NK cells are activated in response to a family of cytokines,interferons and proliferation of activated T-cells is mediated by the release of IL-2,we speculated that CG enhanced immunity through it directly stimulating cytokines secretion from target cells.Cytokines are proteins and peptides that are used for cell signaling.They are produced by many types of cells to communicate with other cells.Due to their central role in the immune system,cytokines are involved in a variety of immunological, inflammatory,and infectious diseases.When the immune system is fighting pathogens, cytokines signal immune cells such as T-cells and macrophages to travel to the site of infection.In addition,cytokines activate those cells,stimulating them to produce more cytokines.Quality and quantity of cytokines are critical in initiation and execution of immunity.A variety of experiments have shown that excessive or insufficient production of cytokines may significantly contribute to the pathophysiology of a range of disease responses and are thought to be decisive for pathological or physiological consequences.After activation,CD4 T helper cells differentiate into either Th1-type cells,secreting IL-2,IL-12,IFN-γand TNF-a,or Th2- type cells secreting IL-4,IL-5, IL-10,and IL-13.Indeed,the balance between Th1 and Th2 cytokines is critical for the orientation of the inflammatory response toward cell-mediated or humoralmediated responses.In this study,we found that CG had a significant stimulatory effect on CD4~+ and CD8~+ T cells,thereby confirming its general effect on the cell-mediated immune response.We also found that CG could significantly increase the proliferation of Con A and LPS stimulated spleenic lymphocytes,increase Th1/Th2 ratio.This suggested that CG might enhance non-specific,humoral and cellular immunity in vitro.