| Tibetan medicine is one of the “Great four” ethnic medicine in China,and is also the source of excellent ethnic culture,vital healthcare and high value economy in Tibet autonomous region.Now it has been developing into one of the pillar industries in Tibet.Tibetan medicinal materials are important material basis for healthy development and precious source for continuous development to Tibetan medicine industry.Tibetan medicine Bazhu is the roots of Oxybaphus himalaicus Edgew.(Nyctaginaceae).Bazhu,as the superior of “Five Roots” in Tibetan medicine,can warm the lower part of body,by which to utilized to treat nephritis edema,etc.,recorded by Tibetan medicine classics,Tu Jian.However,the modern pharmacological study of the traditional medication of Bazhu is still deficient to date.Acute kidney injury(AKI)is the combination of a sort of clinical syndromes,which refer to abrupt decline of renal function in a short term.The main similarity of AKI under different inducers is the inflammation response in kidney.Therefore,to evaluate the pharmacological activity of Bazhu on “nephritis edema” from the site of modern pharmacology,the roots of Bazhu were extracted by ethanol and separated for the further study.Then,the anti-inflammatory fraction of Bazhu was screened out to explore whose anti-inflammation mechanism.Meanwhile,the AKI mice model was established by lipopolysaccharide to evaluate the effect of the antiinflammatory fraction of Bazhu on improving AKI in mice.Concretely,this study contained the following three respects:1.Extraction,partition,and activity screening of Bazhu(1)The roots of Bazhu were extracted by ethanol reflux to obtain the total ethanolic extract(TE).(2)Bazhu ethanolic extract was separated with D101 macroporous resin to obtain the water Fraction A,Fraction B,Fraction C,Fraction D and Fraction E,totally 5fractions.(3)The anti-inflammatory activity of each fraction was screened via in vitro inflammatory model set up by adding 1 μg/m L lipopolysaccharide(LPS)into RAW264.7macrophages.Fraction B from Bazhu ethanolic extract(Labelled as OE)was found to maximally reduce Nitric oxide(NO)release in macrophages with low cytotoxicity,indicating that OE was the main anti-inflammatory fraction.And OE was taken into the further study(4)Ultrahigh performance liquid chromatography tandem mass spectrometry was used to identify the components in OE.41 compounds were matched in mz Cloud database with mz Cloud Best Match scores higher than 85.2.Evaluation of in vitro anti-inflammatory activity of Bazhu(1)OE reduced NO release in a concentration-dependent manner in LPS-induced RAW264.7 macrophage.Western blotting assay revealed OE significantly reversed LPSinduced protein expression of inflammatory-related protein,such as i NOS,COX-2,TNF-α and IL-6.q PCR assay indicated OE significantly reduced the m RNA level of i NOS,COX-2,TNF-α,IL-6 and MCP-1.3.Study on anti-inflammatory mechanism of OE(1)NF-κB refers to be vital transcription factor in modulation inflammatory signaling transduction.Immunofluorescence and western blotting assay manifested OE reversed LPS-induced nuclear translocation and protein phosphorylation of p65,which is a subunit of transcription factor,NF-κB.And OE also supressed degradation of IκB.Above mentioned results indicated OE inhibited the activation of NF-κΒ.(2)NF-κB cascade activation can be induced by complex formation between TLR4 and is coreceptor MD2.Co-immunoprecipitation assay was applied to investigated the effect of OE on TLR4 and MD2.Data revealed OE supressed TLR4-mediated My D88 and TRIF dependent pathway by inhibiting TLR4/MD2 complex formation in RAW264.7macrophages.(3)The activation of TLR4 will further lead to the activation of MAPK and encoding of type Ⅰ interferon,such as IFN-β.Western blotting assay uncovered OE significantly downregulated TLR4-mediated phosphorylation of MAPK,including JNK,ERK and p38.q PCR assay revealed m RNA level of TRIF dependent pathway-mediated IFN-β was significantly downregulated by OE.(4)si NRA transfection was applied to validate the anti-inflammatory mechanism of OE through modulating TLR4 signaling.Western blotting assay revealed si TLR4 interference significantly reversed LPS-induced p65 nuclear translocation and protein phosphorylation,IκB degradation and the protein phosphorylation of MAPK signaling while combined OE treatment do not showed further reduction.Those manifested the effect of OE was neutralized by si TLR4.(5)Reactive oxygen species(ROS)are important modulators in inflammation response.DCFH-DA assay was used to measure the ROS level in RAW264.7 macrophages.Data revealed OE,like endocytosis inhibitor Dynasore,significantly declined ROS production induced by LPS in macrophages.(6)The underlying mechanism of OE on regulating ROS production was investigated in further.Immunofluorescent staining manifested that OE,similar to Dynasore,reversed the cytoplasmic co-localization between ROS production-regulating protein,NOX2,and endosome marker EEA1 induced by LPS in macrophages.In addition,western blotting assay revealed OE significantly reduced cytoplasmic protein expression level of NOX2,while this effect of OE was counteracted by si TLR4 interference.Taken together,OE modulated My D88 and TRIF dependent signaling and NOX2 endocytosis activation by inhibiting TLR4/MD2 complex formation,whereby the inflammatory cytokines and ROS production were declined to ameliorate LPS-induced inflammation.4.Study on OE alleviating AKI in miceMice were continuously administered with 100,200 and 400 mg/kg OE by gavage for 7 days,followed by intraperitoneal injection of LPS(10 mg/kg)to induced AKI.And the in vitro pharmacological activity and underlying mechanism of OE were explored.(1)Within 7 days of intragastric administration,there was no significant difference in body weight among all groups.After sampling,histomorphology analysis showed that the kidneys in control mice was black-purple,while in AKI mice was reddish-brown.And OE treatment improved it near to the appearance as the control mice.(2)OE significantly decreased Creatinine(CRE)and blood urea nitrogen(BUN)level in AKI mice serum.(3)HE staining showed that OE alleviated epithelial cell shedding and inflammatory infiltration in kidney tissue of AKI mice.(4)Immunohistochemistry assay revealed OE treatment obviously reduced the expression and distribution of macrophage marker CD68 and F4/80,and chemokine MCP-1 in the kidneys of AKI mice.(5)Western blotting and q PCR assay showed OE downregulated the protein expression level of i NOS,COX-2,TNF-α and IL-6,and the m RNA level of i NOS,COX-2,TNF-α,IL-6 and MCP-1.5.In vivo validation of mechanism of OE(1)Western blotting analysis revealed that OE inhibited the degradation of IκB and phosphorylation of p65,leading to the inactivation of NF-κB.(2)Coimmunoprecipitation assay showed OE inactivated both My D88 and TRIF dependent signaling by inhibiting TLR4/MD2 complex formation.(3)DHE staining found OE declined DHE fluorescent intensity on mice kidney section,which indicated OE reduced ROS production in mice kidney.(4)Western blotting assay showed OE downregulated protein expression level of NOX2 in mice kidneys.Collectively,OE mitigated inflammation through reducing inflammatory cytokine and ROS production via inhibiting TLR4/MD2 complex formation,whereby alleviating LPS-induced AKI in mice.In this thesis,the total extract of Bazhu was partitioned by D101 macroporous resin to screen the anti-inflammatory fraction.OE was found to be the main anti-inflammatory fraction and the components in OE were identified.Further pharmacological studies have shown that OE exerts anti-inflammatory activity in vitro and in vivo,by inhibiting the formation of TLR4/MD2 complexes,thereby protecting LPS-induced AKI in mice.This study initially explained the mechanism of Bazhu used in the treatment of nephritis edema,and also provided basis for the modern development of Bazhu. |
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