| The Modern Medicine holds the view that inflammation, which is characterized with injury or destruction of tissues caused by various cytological and chemical reactions with typical clinical signs,such as swelling, fever, pain,dysfunction and other major syndromes, is a kind of pathology defensive reaction against harmful stimulation. From the perspective of Traditional Chinese Medicine, inflammation, no matter acute or chronic one, can be diagnosed with symptom of feverish and inflame. While being extremely heat may lead to inflammation or even loss of life, consequently, according to the different stages of the development of inflammation, eliminating heat inhibiting inflammation are of pivotal importance in the process of treating inflammation to refrain excess cytokines, thus improving microcirculation and reducing tissue damage. Countless of traditional Chinese herb are the first options of doctors, among which Paeonia lactiflora Pall. (hereinafter refer to as Pae) is the favorable one according to the related records. While most Modern Medicine studies focus research on immune anti-inflammatory activity instead of the mechanism of reaction. Therefore, the model of acute inflammation and pain response were made to determine the inflammatory and analgesic activity of Pae and histological, serological and molecular biological techniques were used to explore its molecular mechanisms of innate immune in order to provide a theoretical basis for the development of Pae and shed light on its further utilization. The results and final conclusions are as follows:1. Study on anti-inflammatory and analgesic activities of Pae in miceExperimental researchs were aimed at studying the anti-inflammatory and analgesic activities of Pae. Inflammation models, auricle swelling induced by dimethylbenzene and increased capillary permeability induced by acetic acid in mice, were used to investigate the anti-inflammatory effect, and acetic acid writhing test was made to observe its analgesic effect. To some extent, the results showed that Pae with all doses given could inhibit dimethylbenzene-induced auricle swelling and acetic acid-induced increased capillary permeability. At the dose of 10,5g crude drug/kg, Pae made different inhibitory effects on auricle swelling (P<0.05 or P<0.01) and increased capillary permeability (P<0.05). Compared with the control group, 10g crude drug/kg could significantly reduce writhing times in mice(P<0.05). Meanwhile,5g crude drug/kg showed extremely significant difference(P<0.01). With the effects of relieving inflammation and alleviating pain, conclusions can be drawn that water extracts of Pae has obvious anti-inflammatory and analgesic activities.2. EsTablelishment of LPS-induced sepsis model and intervention effects on sepsis miceImproved Kaber method was used to determine the dose of LPS to get the mice model of sepsis. Clinical symptoms, pathological and histopathological changes of mice at different times indicate the model esTablelishment is successful. Tests were set up into different groups,that is, the negative control group, Pae control group, LPS group, LPS+Pae group and LPS+dexamethasone group with observation of clinical symptoms in mice from 2h-72h..The results are as follows:at the time point of 2h after modeling, the reactions of each group of mice were normal. With the intraperitoneal injection of LPS after 6h, clinical symPtoms of mice showed:dysthymic, feather messy and short of breath. Autopsy of mice showed subcutaneous hyperemia, hemorrhage, mesenteric bleeding, lighter color of liver, partially necrosis with visible white dot. After intervention of Pae, the clinical symptoms and necropsy symptoms of LPS group were alleviated. Histology changes observed in the control group of mice liver indicated structural integrity, clear boundaries hepatic cord, liver cells arranged in neat rows. While LPS group after treatment of 6h mouse manifested hePatocytes pathological changes or disappearance, sinusoidal congestion, inflammatory cell infiltration, cell degeneration, and some diffuse necrosis; dilatation and congestion of central vein were observed and the vein wall cell losing visible sinusoid with significant congestion occurs apoptosis and necrosis. There were still inflammatory cell infiltration, liver cells dissolution, homogeneous red dye without cellular structure from microscoPic vision at 48h-72h.Liver tissue structures of LPS-induced septic mice treated with Pae and dexamethasone at 6h-24h showed different degrees of damage. While compared with the LPS group, pathological changes,such as lobular structure amount of bleeding, liver cells arrangement out of order, degeneration and necrosis were more lighter with partial visible nuclear debris. Few hepatocytes accompanied mild inflammatory infiltration and edema, cytoplasm of loose, expansion of the central vein and necrosis and inflammatory infiltration surrounded liver cell at 48h-72h. And LPS-induced septic group with intervention of Pae and dexamethasone showed no significantly differences, indicating that Pae had intervention effects on LPS-induced septic mice.3. Pae affected mRNA Expression of TLR4 and other molecules in the downstream signaling pathways in LPS-induced septic miceTLR4 can recognize pathogen-associated molecular patterns (PAMPS), thus stimulating intracellular signaling transduction and the Expression of genes through TLR4/MyD88/NF-KB signaling pathways. In this experiment, methods of fluorescence quantitative RT-PCR were esTablelished to detect mRNA Expression levels of TLR4, MyD88, NF-κB, TNF-α, IL-1β, IL-4 and IL-10 genes in mice liver. The results showed that mRNA Expression levels of TLR4, MyD88, NF-κB, TNF-α, IL-1β, IL-4 and IL-10 in LPS group were much higher than that in the negative control group. And the differences were significant or remarkably significant (P<0.05 or P<0.01). which suggests that the innate immune system may identify LPS and activate the TLR4/MyD88/NF-κB signaling pathway, aggravating inflammatory reactions. The mRNA Expression levels of the genes above in LPS-induced mice with the intervention of Pae showed significantly differences (P<0.05 or P<0.01), which were able to inhibit Expression of LPS receptor TLR4, signal transduction molecules MyD88, the transcription factor NF-κB, pro-inflammatory factor TNF-α, IL-1β and to promote Expressionof anti-inflammatory cytokines IL-4,IL-10,indicating that Pae may alleviate symptom of sepsis through TLR4/MyD88/NF-κB signaling pathway with its effective targets on TLR4, MyD88, NF-κB, TNF-a, IL-1β, IL-4 and IL-10.4. Pae affected content of serum TNF-α, IL-1p, IL-4, IL-10 and HMGB1 in LPS-stimulated septic miceSynthesis and release of critical pro-inflammatory cytokines such as TNF-α,IL-1β and high mobility group protein B1(HMGB1)cause the systemic inflammatory response syndrome. At the same time, in response to inflammatory response during LPS stimulation, host cells still produce anti-inflammatory cytokines like IL-10 and IL-4. The balance between pro-inflammatory and anti-inflammatory response plays an important role maintaining function of organism, otherwise may result in circulatory failure, the immunosuppressive state and organ dysfunction or even death. In this study, ELISA was used to detect the concentrations of serum TNF-a, IL-1β, HMGB1,IL-4and IL-10 at different time points from 2h to 72h according to the manufacturer’s protocol. Standard curves were esTablelished to assay the content of the sample, and the linear correlation coefficient R2>0.99. The results showed that the TNF-α, IL-1β, HMGB1 in LPS-challenged mice were higher than that in the control group, and the content of serum TNF-a, IL-1β, HMGB1 in LPS+Pae group were much lower than those in the LPS group (P<0.05 or P<0.01)), and the concentration of IL-4 showed significantly up-regulation while the reverse effects were observed in terms of IL-10 even though the serum content were much higher than that in the control group. All in all, the results showed that Pae inhibited release of LPS-stimulated TNF-α,IL-1β, HMGB1 promoted LPS-challenged IL-10 and IL-4 production, indicating that Pae protects mice against lethal LPS stimulation, so at least to some extent, through inhibiting TNF-α,IL-1β and HMGB1 production and accelerating IL-10,IL-4 Expression. |
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