The Mechanism Of Attenuated Hepatotoxicity Of Euphorbia Kansui After Vinegar-processing

The roots of Euphorbia kansui T.N.Liou ex T.P.Wang(called as kansui),recorded in Shennong-Bencao,has multiple medical applications for the edema,ascite,asthma in China for centuries.Many studies showed that kansui exhibited promising effects in the treatment of leukemia,cancer,pancreatitis and intestinal obstruction.However,the clinical use of kansui had some practical disadvantages because it might result in inflammation,irritation to the skin,oral,and gastrointestinal,promoting tumor.And recent studies have also shown that kansui could lead to hepatic injury.The vinegar stir-baking kansui was regularly used by Traditional Chinese Medicine(TCM)in the past period,and our studies have also shown that vinegar stir-baking could reduce the toxicity of kansui.However,its attenuated hepatotoxicity mechanism remains unknown.Thus,the main objective of this research is to carry out a systematic study on the mechanism of attenuated hepatotoxicity of Euphorbia Kansui after vinegar-processing.This research is to lay the foundation for further demonstrating attenuated hepatotoxicity mechanism of vinegar treated kansui preparations.This thesis mainly includes the following parts:Part one:Literature reviewBy means of reviewing relevant literatures,the current status of studied on the mechanism of hepatotoxicity,common TCM with hepatotoxicity and the methods to attenuate hepatotoxicity of TCM were systematic summarized,which lays the foundation for providing practical research programs and determining the study of examined indicators and the starting point on this thesis.Part two:Investigated the mechanism of toxicity attenuation on human liver cell line L-O2 cells of Euphorbia kansui after vinegar stir-baking process1.This section has compared the toxicity on normal liver cells L-O2 of the unprocessed kansui Radix,the stir-baked kansui Radix,the kansui Radix infiltrated with vinegar and Kansui Radix stir-baked with vinegar by MTT assay and the inverted phase contrast microscope.The results showed that kansui significantly inhibited the proliferation and morphological changes of L-O2 cells,and Euphorbia kansui after vinegar stir-baking process significantly attenuated the toxicity on normal liver cells L-O2 compared with the kansui Radix in concentration-dependent manner and with attenuation order as kansui Radix stir-baked with vinegar>kansui Radix infiltrated with vinegar>Stir-baked kansui Radix.And the procedures of stir with baked and infiltration with vinegar show the synergistic effect in attenuating the L-O2 cell toxicity.2.This section has studied the effects of the cell cycle and apoptosis on normal liver cells L-O2 of the unprocessed kansui Radix,the stir-baked kansui Radix,the kansui Radix infiltrated with vinegar and Kansui Radix stir-baked with vinegar by flow cytometry.The results showed that kansui significantly induced the L-O2 cells apoptosis in concentration-dependent manner.And Euphorbia kansui after vinegar stir-baking process significantly attenuated the L-O2 cells apoptosis compared with the kansui Radix in concentration-dependent manner and with attenuation order as kansui Radix stir-baked with vinegar>kansui Radix infiltrated with vinegar>Stir-baked kansui Radix.And the procedures of stir with baked and infiltration with vinegar show the synergistic effect in attenuating the L-O2 cell apoptosis.3.This section has studied the oxidative damage mechanism on normal liver cells L-O2 of Euphorbia kansui after vinegar stir-baking process.The results showed that kansui significantly induced oxidative damage on L-O2 cells and also demonstrated that the vinegar stir-baking could attenuated the hepatotoxicity of kansui by reducing oxidative damage of various index signs(P<0.01),The results indicated that kansui can lead to oxidative damage on liver cells and the vinegar stir-baking can significantly attenuate the oxidative damage on liver cells.4.This section has studied the apoptotic mechanism on normal liver cells L-O2 of Euphorbia kansui after vinegar stir-baking process.The results showed that kansui can significantly increased L-O2 cells nuclear condensation,decreased mitochondria membrane potential,promoted the Cytochrome c release(P<0.01,P<0.05,P<0.01)and increased the cell membrane permeability(P<0.01)and significantly up-regulated caspase-3 expression(P<0.01).The vinegar stir-baking can significantly decreased L-O2 cells nuclear condensation,increased mitochondria membrane potential,reduced the Cytochrome c release(P<0.05)and decreased the cell membrane permeability(P<0.05)and significantly decreased the up-regulated caspase-3 expression(P<0.01).The results indicated that kansui can significantly induced the liver cells apoptosis and the vinegar stir-baking can significantly attenuate the hepatotoxicity through reducing mitochondrial apoptotic cells.Part three:Investigated the hepatotoxicity attenuation and its mechanism of Euphorbia kansui after vinegar stir-baking process in mice1.This section has studied the normal mice by gavage administration of ethyl acetate extract of kansui and the vinegar stir-baking kansui showed that kansui can significantly increase the levels of AST,ALT,LDH and the ratio of liver weight(P<0.01,P<0.001)in the mice serum and liver tissue and significantly promote the pathological changes of liver tissue.Compared with kansui respective dose group,the vinegar stir-bakeing kansui Radix can significantly decrease the levels of ALT,AST,LDH and the ratio of liver weight(P<0.05,P<0.01,P<0.001)in the mice serum and liver tissue and significantly lighten the pathological changes of liver tissue,and showed a certain dose-effect relationship.The results indicated that kansui has significant hepatotoxicity and the vinegar stir-baking can significantly attenuate the hepatotoxicity.2.This section has studied the oxidative damage mechanism of ethyl acetate extract of Euphorbia kansui after vinegar stir-baking process in mice.The results showed that kansui can significantly decrease the level of SOD and the content of GSH(P<0.01,P<0.001),and significantly increase the content of MDA(P<0.001),and significantly decrease the level of Na+-K+-ATPase and Ca2+-Mg2+-ATPase(P<0.01)in the mice serum and liver tissue compared with control group.Compared with kansui Radix each dose group respectively,the vinegar stir-baking kansui Radix can significantly increase the level of SOD and the content of GSH(P<0.001,P<0.01),and significantly decrease the content of MDA(P<0.001),and significantly increase the level of Na+-K+-ATPase and Ca2+-Mg2+-ATPase(P<0.001)in the mice serum and liver tissue,and showed a certain dose-effect relationship.The results indicated that kansui can lead to oxidative damage hepatotoxicity of mice liver and the vinegar stir-baking can significantly attenuate the oxidative damage hepatotoxicity of mice liver.3.This section has studied the apoptotic mechanism of Euphorbia kansui after vinegar stir-baking process in mice.The results showed that kansui can significantly increase apoptosis index(P<0.01,P<0.001),up-regulate caspase-3 expression(P<0.01),down-regulate bcl-2 expression(P<0.01),and increase NF-?B and ICAM-1expression(P<0.001)in liver tissue.The vinegar stir-baking can significantly decrease apoptosis index(P<0.05,P<0.001),down-regulate caspase-3 expression(P<0.01),up-regulate bcl-2 expression(P<0.05),and decrease NF-?B and ICAM-1 expression(P<0.01,P<0.001)in liver tissue.The results indicated that kansui can significantly induce hepatotoxicity through increasing the liver cells apoptosis and inflammatory necrosis and the vinegar stir-baking can significantly attenuate hepatotoxicity through reducing the liver cells apoptosis and inflammatory necrosis.Part four:Investigated the mechanism of toxicity attenuation on human liver cell line L-O2 cells of EK-021.This section has studied the toxicity on normal liver cells L-O2 of EK-O2 by CCK-8 assay and the inverted phase contrast microscope.The results showed that EK-02 significantly inhibited the proliferation and morphological changes of L-O2 cells.The results indicated that EK-02 can significantly induce hepatotoxicity(P<0.01)and in concentration-dependent manner.2.This section has studied the effects of the cell cycle and apoptosis on normal liver cells L-O2 of EK-02 by Hoechst 33258 staining,Annexin-V-FITC/PI assay and flow cytometry.The results showed that EK-02 significantly induced the L-O2 cells apoptosis(P<0.01)and in concentration-dependent manner.3.This section has studied the apoptotic mechanism on normal liver cells L-O2 of EK-02.The results showed that EK-02 can significantly increased L-O2 cells nuclear condensation,decreased mitochondria membrane potential,promoted the Cytochrome c release(P<0.05)and increased the cell membrane permeability(P<0.01)and significantly up-regulated caspase-3,8,9,Bax,AIF,Apaf-1 expression(P<0.01)and down-regulated bcl-2 expression(P<0.01).The results indicated that EK-02 can significantly induce the liver cells apoptosis through mitochondrial apoptototic pathway.4.This section has studied that the changes of the EK-02 content evaluated by UPLC-Q-TOF-MS.The results showed that compared with kansui group,vinegar stir-baking kansui can significantly reduce the EK-02 content and the content change rate was-60.25%.The results indicated that EK-02 can be significantly reduced by vinegar stir-baking process and inferred that the possible mechanism was that reducing of Diterpenoid compounds as the representative of the toxic ingredients.