Study On Pharmacological Effects And Mechanism Of Dragon’s Blood And Its Extract

Dragon’s blood (DB), a bright red resin derived from Dracaena cochinchinensis (Lour.)S.C.Chen (China), mainly found in Guangxi, Yunnan and other regions, which wasoriginally used for activating circulation to remove blood stasis. DB has been widely usedfor anti-inflammatory response, anti-oxidative stress activity, improving immunity,promoting blood circulation etc. But DB had the low bioavailability, high dose and veryhigh drug viscosity of the medicine shortcomings. Dragon’s blood extract (DBE) wereseparated from DB resin by the methods of alcohol extracting-water precipitating. The mainactive components of DB and DBE were phenolic compounds, including resveratrol,pterostilbene, loureirin A and loureirin B. It’s reported that phenolic compounds fromnaturally traditional herbs, including tea polyphenols, resveratrol and so on, reducedradiation-induced oxidative stress damage, cellular DNA injury, inflammatory responses,and myelosuppression in various mouse model studies. In our previous study, we found thatDB could alleviate radiation-induced brain and gastrointestinal injury. In this study, wehave investigated the protective effect of DB and DBE on radiation-induced hematopoieticinjury. What’s more, our research group found that DB could promote regeneration ofperipheral nerves injury, reduce focal cerebral ischemia damage and inhibit nerve cellapoptosis of hippocampus in irradiated rats. Furthermore, we have also studied the detailedmechanism of antidepressant effect of DB and DBE on mouse model experiment.Based on these results, we explored the pharmacological effects and mechanism of DBand DBE in improving bone marrow hematopoiesis and anti-depression. Our studyincluded two parts. The first part was the effect and mechanism of DB and DBE onradiation-induced bone marrow hematopoietic injury. We studied the protective effect ofDB and DBE on male adult BALB/c mouse exposed to the whole body irradiation with4Gy or5Gy60Co-γ rays. During animal experiment, we investigated the protective effect ofDB and DBE on peripheral blood cells, bone marrow histology, the colony forming units ofbone marrow derived progenitor cells, chromosome aberration and the induction ofmicronucleus of bone marrow, inflammatory response and oxidative stress injury inradiation-induced mice model. To reveal the molecular mechanism of DBE on bonemarrow hematopoiesis, we also explored the effect of DBE on cell survival, cell cycle, cellapoptosis and signaling pathways associated proteins changes in GM-CSF-depleted Mo7ecells (human megakaryocytic leukemia cell line). The second part was the effect of DB and DBE on behavior and the hippocampal neurogenesis in the rat model of depression. Westudied the protection of DB and DBE on chronic stress depression rat model exposed tochronic unpredictable mild stress for28days. In this study, we investigated the protectiveeffects and mechanism of DB and DBE on the behavioral changes such as sucrosepreference test (SP), open-field test (OF), forced swimming test (FST), novelty suppressedfeeding test (NSF), neurogenesis and migration of dentate gyrus in hippocampus,associated protein levels changes of Ngn2, DCX, and BDNF, ERK1/2, CREB inneurotrophic signaling pathway, and NMDAR, CAMKIV in long term memory signalpathway (LTP) in hippocampus. The main results were summarized as follow:Part I: The effect and mechanism of DB and DBE on radiation-induced bonemarrow hematopoietic injury.1Protective effect of DB and DBE on bone marrow hematopoiesis inmyelosuppression mice1) DB and DBE accelerated the recovery of peripheral blood cells. In4Gy60Co-γradiated mouse model, DB and DBE (0.075,0.15,0.3g/kg b.wt.) groups markedlyincreased leukocyte recovery, and DBE promoted leukocyte number to the control level onday28. DB and DBE produced a significant recovery in erythrocytes. Hemoglobin contentin DB and DBE groups showed a significantly increased recovery, and DBE inducedhemoglobin content to the control level on day28. Both DB and DBE markedly lessen thedeclined level thrombocytes and increased the recovery of them. DBE promotedthrombocyte number to the control level on day14, and DB also induced thrombocytecontent to the control level on day21. DB and DBE-treated group also markedlyaccelerated lymphocyte recovery. In5Gy60Co-γ radiated mouse model, DBE alsostimulated the recovery of peripheral blood cells, including leukocyte, erythrocyte,hemoglobin, thrombocyte and lymphocyte. Among the results in4and5Gy60Co-γ raysradiated mouse model, DBE and DB groups had a more marked action on the increase inthrombocytes.2) DB and DBE improved the pathological morphology of bone marrow and spleen. In4Gy60Co-γ radiated mouse model, DB and DBE obviously reduced the fatty tissuehyperplasia, improved abnormality in the proportions of cells, increased the proliferation ofhematopoietic cells. DB and DBE improved extramedullary hematopoiesis, increased the oflymphocyte number in the white pulp and promoted immunity. In5Gy60Co-γ radiatedmouse model, DBE also improved the morphology of bone marrow histology. 3) DB and DBE stimulated the recovery of hemopoietic progenitor cells. In4Gy60Co-γ radiated mouse model, the result of hematopoietic progenitor cell culture in vitroshowed that the numbers of CFU-GM (granulocyte/macrophage progenitors), CFU-E(erythroid), BFU-E (burst-forming unit of erythroid) and CFU-Meg (megakaryocytes) wereobviously increased in DBE group on days7,14,21after radiation. DB could significantlyincrease the colony formation of the CFU-Meg on days7,14,21after radiation. DB alsomarkedly increased the colony formation of CFU-E and BFU-E. Among the results, DBEand DB showed a more significant action on the colony formation of CFU-Meg．4) DB and DBE did not affect the colony formation of fibroblast. In4Gy60Co-γradiated mouse model, DB and DBE did not significantly affect the morphology and thecolony formation of CFU-F on days7,14,21d after irradiation.5) DB and DBE attenuated cell cycle arrest in bone marrow cells. In4Gy60Co-γradiated mouse model, DB and DBE could markedly decrease cell apoptosis and cell cyclearrest of bone marrow cells (BMCs) in myelosuppression mice. DB and DBE alsodecreased the ratio of BMCs in G0/G1phase, significantly increased in S phase and G2/Mphase, indicated that both DB and DBE could increase DNA synthesis and BMCs mitosis,promote the proliferation of bone marrow cells, accelerate the recovery of thehematopoietic function．6) DB and DBE obviously reduced DNA damage in bone marrow cells. In4Gy60Co-γradiated mouse model, DB and DBE markedly reduced different types of aberrations, suchas chromatid breaks, chromosome breaks, fragments, rings, dicentrics, polyploidy, severdamage cells (SDC), and both DB and DBE induced a significant decrease in the percentaberrant cells. DB and DBE groups also had the significant decrease in the frequencies ofmicronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromaticerythrocytes (MNCE), increase in the ratio of PCE/(PCE+NCE). Both DB and DBEreduced radiation-induced bone marrow DNA damage, minimized genomic instability ofbone marrow cells and improved hematopoietic recovery.7) DB and DBE markedly reduced the levels of inflammatory cytokines in serum. In4Gy60Co-γ radiated mouse model, DB and DBE obviously regulated the levels of IL-6,TNF-α and IFN-γ and kept them in a proper range, implied that DB and DBE could reduceinflammation response, maintain a stable hematopoietic microenvironment and acceleratehematopoiesis recovery.8) DB and DBE markedly attenuated oxidative stress in serum and spleen. In4Gy 60Co-γ radiated mouse model, DB and DBE significantly improved the activities of theantioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), andantioxidant molecule glutathione (GSH), as well as reduced lipid peroxidation productmalondialdehyde (MDA) in serum and spleen. DB and DBE could significantly reduceradiation-induced oxidative stress injury.2Effect of DBE on proliferation of GM-CSF-depleted Mo7e cells1) DBE (10,50μg/mL) showed a significant increase of cell survival inGM-CSF-depleted Mo7e cells.2) DBE significantly attenuated cell cycle arrest of Mo7e cells induced by cytokinedepletion, reduced ratio of Mo7e cells in G0/G1phase, and increased in S and G2/M,indicated that DBE advanced DNA synthesis and mitosis, enhanced cell proliferation ofMo7e cells.3) DBE markedly alleviated GM-CSF withdrawal-induced early apoptosis and totalapoptosis.4) DBE obviously reduced the level of pro-apoptotic protein Bax, improvedanti-apoptotic protein level (Bcl-2), even reduce the ratio of Bax/Bcl-2. DBE alsosignificantly inhibited the active caspase-3expression.5) DBE could induce ERK1/2phosphorylation in GM-CSF-depleted Mo7e cell, butnot Akt. In the presence of wortmannin (a PI3-K inhibitor) or imatinib mesylate (a tyrosinekinase inhibitor), DBE also did not affect the level of Akt phosphorylation. It’s shown thatDBE increased cell survival and proliferation through ERK1/2activation, but not Akt.Part II: The effect of DB and DBE on behavior and the hippocampalneurogenesis in rat model of depression1) DB and DBE markedly improved behavioral changes of chronic stress depressionmodel rats. DB (2g/kg b.wt.) and DBE (0.3g/kg b.wt.) had a marked increase in thesucrose consumption, the number of squares crossing and rearing. DB and DBE alsosignificantly decreased the immobility time of FST and the latency to eat of NSF on chronicstress depression rat model.2) DB and DBE affected neurogenesis and migration of dentate gyrus in hippocampus.Both DB and DBE increased the BrdU-cells number in the hippocampal dentate gyrus ofchronic stress depression rats. DB had a significance increase in the proliferation of neuralprogenitor cell. DB and DBE also markedly increased the number of doublecortin positivecells in hippocampus granular cell layer. 3) DB and DBE increased the levels of associated protein expression in hippocampus.DB and DBE could markedly increase the protein levels of Ngn-2, DCX, BDNF andp-CREB in neurotrophic signaling pathway, NMDAR and p-CAMKIV in long termmemory signal pathway (LTP) of hippocampus. The ERK1/2protein expression levels inthe DB group were significantly higher than those of the stress group.In conclusion, DB and DBE increased the recovery of peripheral blood cells, improvedthe morphology of bone marrow histopathology and the proliferation of bone marrowhematopoietic progenitor cells, which was likely associated with the, anti-clastogenic,anti-apoptotic anti-inflammatory and anti-oxidative properties of DB and DBE. We firstlyrevealed that DBE increased the cell survival and proliferation, attenuated cell apoptosisand cell cycle arrest of GM-CSF-depleted Mo7e cells through the decrease of Bax/Bcl-2ratio, the reduction of active caspase-3expression, the activation of ERK1/2pathway, notAkt pathway. Meanwhile, Both DB and DBE displayed the antidepressant effect throughimproving the behavioral of chronic stress depression model rats, which was likelyassociated with proliferation, differentiation and migration of granule cells in thehippocampus, the regulation of neuronal plasticity in hippocampus, and the activation ofneurotrophic signaling pathway (BDNF/CREB) and long term memory signal pathway(NMDAR/CAMKIV/CREB). DB and DBE had pharmacological effects on improving bonemarrow hematopoiesis and anti-depression.