Study On Anti-inflammatory And Immune-modulating Effects Of Baicalin And Baicalein

BackgroundBaicalein and its glycoside baicalin are major flavonoid constituents found in thetraditional Chinese medicinal herb Baikal skullcap (Scutellaria baicalensis Georgi), knownas “Huang qin” in China, which has been widely used for the treatment of various diseasessuch as pneumonia, hepatitis, and diarrhea. Previous studies have demonstrated thatbaicalin possess a wide range of pharmacological and biological activities such asanti-inflammatory, anti-tumor properties, anti-allergic, antimicrobial, antioxidant activities.Its anti-inflammatory activity has been estimated in various animal models of acute andchronic inflammation, including acute pancreatitis, asthma, ligature-induced periodontitis,embolism-induced acute lung injury, liver injury, hepatic fibrosis, and experimentalautoimmune encephalomyelitis. Baicalin administration generally could alleviateinflammatory tissue injury, reduce serum pro-inflammatory cytokine levels, and modulatecytokine production from immune cells, such as suppressing IL-4and IL-5but enhancingIL-10production in splenocytes in asthma model, suppressing IFN-γ but enhancing IL-4release from lymphocytes in experimental autoimmune encephalomyelitis model. In vitrostudy showed that baicalin inhibited lymphocyte proliferation stimulated by variousmotigens such as Con A, PMA/ionomycin, and LPS, further suggesting that lymphocytesmight be one of the important targets for baicalin. Evidence from studies using tumor celllines revealed that, in addition to anti-proliferating activity, baicalin also processpro-apoptotic activity. However, the influence of baicalin and baicalein on lymphocyteapoptosis has not been explored yet.Apoptosis of immune cells represents one of the most important mechanisms ensuresthe development of protective immunity, maintenance of self-tolerance and prevention ofautoimmunity. Defects in immune cell apoptosis are closely associated with thedevelopment of various autoimmune diseases. Evidence from transgenic mice revealed thatselective knockdown of pro-apoptotic gene in immune cells resulted in their accumulationand, in turn, chronic lymphocyte activation and development of autoimmunity. Inautoimmune animal models, as well as patients with autoimmune diseases, various defectsof apoptosis in immune cells have been detected, which result in an overabundance oflymphocytes and repeat occurrence of inflammation. This might be a major cause ofinflammatory tissue injury and dysfunction of organs. Many classical immunosuppressive agents, such as cyclosporine A, rapamycin, dexamethasone, and1,25(OH)2D3, not onlyinhibit activation and proliferation of lymphocytes, but also promote their apoptosis, whichis attributed to their immunosuppressive effects. So far, the mechanisms ofactivation-induced cell death (AICD) that related to lymphocytes death have been widelyexplored, and it appears to serve as a negative immuno-regulatory mechanism that iscritical to ensure protective immunity and avoid autoimmunity. Therefore, interferencewith regulatory mechanisms of lymphocyte apoptosis represents a promising approach forthe development of new immune-modulatory therapies for autoimmune diseases.Based on above background, the present study at first focused on the in vitro influenceof baicalin and baicalein on apoptosis of immune cells of different types and differentactivating status, and then further explored their in vivo anti-inflammatory andimmune-modulating activities, as well as their pro-apoptotic activities on lymphocytes, incollagen Ⅱ (CⅡ)-induced arthritic mice and Con A-induced immune-mediated hepatiticmice.AimThe present study is aimed to further understand the mechanisms underlying theanti-inflammatory and immune-modulating activities of baicalin and baicalein, and toexplore their therapeutic potential in treatment of autoimmune diseases, thus providingresearch basis for their clinical use.Method1. The in vitro effects of baicalin and baicalein on apoptosis of immune cells wereassayed using Con A-or LPS-stimulated mouse splenocytes, anti-CD3/CD28antibody-stimulated CD3~+T cell purified from mouse splenocytes with immunalmicrobeads, LPS-stimulated CD19~+B cells purified from mouse splenocytes with immunalmicrobeads, mouse thymocytes, bone-marrow cells, bone marrow-derived dendritic cells,PMA/ionomycin-stimulated Jurkat T cells, RAW264.7macrophages, bEnd.3endothelialcells, and L929mouse fibroblast cells. Cellular apoptosis was analyzed by flow cytometryafter stained with fluorescence-labeled antibodies against surface markers (such as CD3,CD19, and CD69) and with annexin V/propidium iodide (PI) or anti-active caspase3antibody. The effects of baicalin and baicalein on mitochondrial membrane potential insplenocyte and surface expression of Fas and FasL were assayed with flow cytometry to analyze the intracellular mechanisms of their pro-apoptotic activities.2. The in vivo anti-inflammatory, immune-modulating, and pro-apoptotic effects ofbaicalin were studied in CⅡ-induced arthritic (CIA) mice. DBA/1mice were injectedintradermally at the base of the tail with0.1ml of the emulsion containing200μg of CⅡ,and then intravenously injected with0.1ml of PBS containing200ug of CⅡ at an intervalof21days to induced arthritis. Baicalin (50mg/kg) was administrated intraperitoneallydaily from days1to14after second CⅡ immunization. Arthritis symptoms were assesseddaily after drug administration by using a scoring system (0=no signs of arthritis;1=swelling and/or redness of the paw or one digit;2=two joints involved;3=more than twojoints involved; and4=severe arthritis of the entire paw and digits). The hind limbs of themice were removed postmortem, and histochemistry study with hematoxylin and eosinstaining, Micro-CT screening were performed to evaluate the anti-arthritic effects ofbaicalin. Levels of anti-CⅡ antibody in serum samples collected on day34weredetermined by ELISA. Splenocytes were prepared and cultured in medium containing CⅡ(50μg/ml) for48h, and Th1/Th2cytokines in the culture supernatants were measured byELISA. Apoptotic cells in spleen were identified by the TUNEL assay orimmunohistochemistry assay using fluorescence-labeled antibodies against CD3, CD19,and active caspase3. Lymphocyte apoptosis in splenocytes was also analyzed with flowcytometry after stained with FITC-annexin V and fluorescence-labeled antibodies againstCD3and CD19.3. The in vivo anti-inflammatory and pro-apoptotic effects of baicalein were studied inCon A-induced immune-mediated hepatitis murine model. BALB/c mice wereintravenously injected with ConA（20mg/kg）to induced acute liver injury. Baicalein (100mg/kg) was administrated intraperitoneally2hours before ConA injection. Serum sampleswere collected8hours after ConA injection and measure for ALT levels. Liver and spleentissues were collected24hours after ConA injection, and histochemistry studies wereperformed to detect liver injury and apoptotic cells. Lymphocyte apoptosis in splenocyteswas also analyzed with flow cytometry after stained with FITC-annexin V andfluorescence-labeled antibodies against CD3and CD19.Results1. Baicalin and baicalein inhibited Con A or LPS-stimulated splenocytes proliferation dose-dependently in the dose range of5-40μM, indicating the inhibitory effects of baicalinand baicalein on lymphocytes function.2. Baicalin and baicalein induced apoptosis of ConA-stimulated splenocytesdose-dependently in the dose range of5-20μM. Further analysis of cellular composition ofapoptotic cell showed that baicalin and baicalein mainly promote apoptosis in CD3~+T cellsand CD19~+B cells, but only mildly in CD11b+macrophages.3. Comparison of pro-apoptotic activities of baicalin and baicalein (at10μM,exposure for24hours) on immune cells of different types and different activating statusrevealed that baicalin potently promote apoptosis in splenocytes, CD3~+T cells inConA-stimulated splenocytes, CD19~+B cells in LPS-stimulated splenocytes, bone-marrowcells, and PMA/ionomycin-stimulated Jurkat T cells, while only mildly in bonemarrow-derived dendritic cells, but showed almost no influence in non-stimulated Jurkat Tcells, RAW264.7macrophages, bEnd.3endothelial cells, and L929mouse fibroblast cells.These results demonstrated that baicalin does not unselectively promote apoptosis in allcells but selectively in T and B cells. It is noteworthy that Con A-activated CD3~+T cellswere more sensitive than na ve CD3~+T cells to the pro-apoptotic activity of baicalin.Similarly, PMA/ionomycin-stimulated Jurkat T cells were more sensitive thannon-stimulated Jurkat T cells, and LPS-activated CD19~+B cells were more sensitive thanna ve CD19~+B cells to the pro-apoptotic activity of baicalin. These results strongly suggestthat baicalin selectively promotes apoptosis in activated T and B cells. The action modes ofbaicalein are almost totally same as baicalin. These results suggest that baicalin andbaicalein mainly promote apoptosis in activated T and B cells.4. The pro-apoptotic activities of baicalin and baicalein on T cell were further studiedusing CD3~+T cells purified from mouse splenocytes with immunal microbeads. The resultsshowed that, in dose range of5-20μM, baicalin had no influence on apoptosis of na veCD3~+T cells but dose-dependently promoted apoptosis in anti-CD3/CD28Ab-activatedones. Baicalein promote apoptosis in both na ve and anti-CD3/CD28Ab-activated T cellsdose-dependently in dose range of5-20μM, and anti-CD3/CD28Ab-activated T cells weremuch more sensitive to baicalein than na ve CD3~+T cells.5. The pro-apoptotic activities of baicalin and baicalein on B cell were further studiedusing CD19~+B cells purified from mouse splenocytes with immunal microbeads. The results showed that, baicalin promote apoptosis in both na ve and LPS-activated CD19~+Bcells dose-dependently in dose range of5-20μM within24hours of exposure time, andLPS-activated CD19~+B cells were much more sensitive to baicalein than na ve ones.Baicalein had a more potent pro-apoptotic activity than baicalin. Within24hours ofexposure time, baicalein already significantly promote apoptosis in both na ve andLPS-activated B cells dose-dependently in dose range of5-20μM, and LPS-activated Bcells were much more sensitive to baicalein than na ve CD19~+B cells.6. Exposure of baicalin or baicalein (5-20μM) for24hours resulted indose-dependent increase in the proportion of green fluorescence-positive cells (indicatingchange in mitochondrial membrane potential, that is low△φ) in JC-1stained splenocytes,indicating that baicalin and baicalein induced the loss of mitochondrial membrane potentialin splenocytes. However, within the same dose range, baicalin and baicalein did notenhance the expressions of Fas and FasL on cell surface of splenocytes. These resultssuggest that baicalin and baicalein promote lymphocyte apoptosis through activation ofmitochondrial-dependent endogenous pathway but not Fas-dependent exogenous pathway.7. The in vivo anti-inflammatory, immune-modulating, and pro-apoptotic effects ofbaicalin were studied in CIA mice. The results showed that intraperitoneal administrationof baicalin (50mg/kg) decreased the severity of CⅡ-induced arthritis in mice. Thehistology of ankle joints from CIA mice showed severe proliferation of the synovium withsignificant infiltration of inflammatory cells, pannus formation, cartilages damage andbone erosion. Treatment with baicalin suppressed these pathologic changes, and the jointsshowed mild hyperplasia with reduced inflammatory cell infiltration, no pannus formation,and intact cartilage and bone. Baicalin treatment resulted in a significant decrease in serumlevel of anti-CⅡ IgG Abs. Production of IFN-γ, IL-2, and IL-17from collagen-stimulatedspleen cells was also reduced by baicalin treatment. These results indicated that bothhumoral and cellular immune responses against CⅡ in CIA mice were suppressed bybaicalin treatment. Immunohistochemistry and TUNEL staining of spleen sections showedthat baicalin treatment remarkably enhanced the apoptosis in lymphocytes. Flow cytometryanalysis of splenocytes revealed that baicalin treatment significantly reduced thepercentage of T and B cells in spleens, and increased their apoptosis at the same time.These results demonstrated that baicalin promotes the apoptosis of T and B lymphocytes invivo, thus exerts anti-inflammatory and immune-suppressing effects in CIA mice. 8. The in vivo anti-inflammatory and pro-apoptotic effects of baicalein were studied inCon A-induced immune-mediated hepatitis murine model. The results showed thatintraperitoneal administration of baicalein (100mg/kg) reduced serum ALT levels,alleviated Con A-induced liver injury and inflammatory infiltration, and enhancedapoptosis of T and B cells in spleens. These results demonstrated that baicalein promotesthe apoptosis of T and B lymphocytes in vivo, thus exerts anti-inflammatory inConA-induced hepatitis mice.ConclusionBaicalin and baicalein can selectively promote apoptosis in activated T and Blymphocytes possibly through modulation of mitochondrial-dependent endogenouspathway. Baicalin and baicalein exerts anti-inflammatory and immune-suppressing effectsthrough promote apoptosis of T and B lymphocytes in vivo, at least in part, in CⅡ-inducedarthritic mice and Con A-induced hepatitic mice, respectively. These findings extend ourunderstanding on the mechanism underlying the anti-inflammatory andimmune-suppressing effects of Baicalin and baicalein, and further reveal their therapeuticpotential in treatment of autoimmune diseases, thus providing research basis for theirclinical uses.