| Angelica sinesis, originated in Minxian county of Gansu province, was the root of Angelica sinensis (Oliv.) Diels. Angelica sinesis was used to treat anemia and dysmenorrheal in Chinese herbal medicine for thousands of years. Angelica sinesis polysaccharide (ASP), the main active ingredient of Angelica sinesis, was extracted from the roots of Angelica sinensis (Oliv.) Diels. It is well documented that ASP has various important bioactivities, not only hematopoietic activity and immunological enhancement but also hypoglycemic activity. However, the mechanism of hepatoprotective activity is still undefined.The raw Angelica sinensis polysaccharide was achieved from the roots of Angelica sinensis (Oliv.) Diels by boiling water extraction and alcohol precipitation method. The refined polysaccharide, named ASP, was received by freeze-drying after purification. The purity and primary structure were analyzed by spectrum. The distribution in rat liver, particle size and binding in TLR and MR were analyzed to study the targeting effects of ASP after labeled with fluorescent tags. The study of mechanism in ConA-induced liver injury and ethonal-induced hepatic damage in vivo and vitro would lay a solid foundation for the application of ASP.Part ? The study of ASP in the liver targeting effectThe raw Angelica sinensis polysaccharide was achieved from the roots of Angelica sinensis (Oliv.) Diels by boiling water extraction and alcohol precipitation method. After treated by multigelation and millipore filter, the raw polysaccharide was purified by using Sephadex G-50 column. Total sugar contents of the purified polysaccharide (ASP) were about 90.25% with the molecular weight of 94,000 Da. The UV spectra of ASP had no absorption bands at 260 or 280 nm, indicating the absence of nucleic acids and proteins. The FT-IR spectra showed that ASP was a?-D-pyranoid polysaccharide.Studies showed that the yellow-orange product labeled with FITC (FITC-ASP, FA) was stable and retained the original biological activity in PBS and serum, which could be used in the study of the targeting effects. FA(3,6,12 and 24 mg/kg) was intravenously administered in Sprague Dawley rats. The concentration of FA in liver was detected at 0.25,0.5,1,1.5,2,3 and 4 h with the method of high-performance gel permeation. According to the concentration time curve, there was a peak at about 1.5 h, and the FA was hardly detected after 4 h.As showed by atomic force microscope and Brookhaven granulometer, ASP (1 ?g/mL-20 mg/mL) was uniformly dispersed in solution in size between 200 nm and 3 ?m. Therefore, ASP could be engulfed by Kupffer cells, resulting in the passive targeting effect in liver.Antagonistic experiment in vitro indicated that ASP could combine with MR rather than TLR4. It could be suggested that MR played a part of action on the active targeting effect of ASP, which laid a solid foundation for the study of hepatoprotective effect produced by ASP.Part II The effect of ASP on the ConA-induced liver injuryAnimals were randomly divided into five groups (n= 10 each):the normal control group, the ASP (6 mg/kg) group, the ConA group, the ConA+ASP (1.5 mg/kg) group, and the ConA+ASP (6 mg/kg) group. The ConA+ASP (1.5 mg/kg) group and the ConA+ ASP (6 mg/kg) group were intravenously administered single doses of 1.5 and 6 mg/kg body weight of ASP per day for two weeks, respectively. A single intravenous (i.v.) administration of a dose (6 mg/kg body weight) of ASP was given to the ASP (6 mg/kg) group per day for two weeks. The normal control group and the ConA group were intravenously administered with the same volume of PBS per day for two weeks. The ConA group, the ConA+ASP (1.5 mg/kg) group and the ConA+ASP (6 mg/kg) group were challenged intravenously with ConA (15 mg/kg body weight) on the 15th day, while the normal control group and the ASP (6 mg/kg) group were challenged intravenously with the same volume of PBS on the 15th day. Blood samples were collected 8 h after ConA or PBS injection. After 20 h of ConA challenge, all mice were sacrificed, and the liver tissues were collected.Morphologic examination of external surface of entire liver showed that pretreatment with ASP relieved congestion of liver after challenged intravenously with ConA. Hematoxylin and eosin staining of the liver was performed in order to evaluate the pathological changes in liver. In the ConA group, massive hepatocyte necrosis in the ConA group was widely presented. However, after ConA challenge, the extent and area of necrosis were almost absent in animals pretreated with ASP. Besides, ConA treatment increased the inflammatory cell infiltration observed in liver, and these effects were reduced by the prior administration of ASP.The liver index decreased in mice pretreated with ASP. The results of serum aminotransferase levels were in good agreement with histopathological changes. The results showed that serum ALT and AST levels increased 8 h after intravenous ConA administration compared with the normal control group. However, compared with the ConA group, ASP markedly decreased the elevation of serum ALT and AST levels.-The results above indicated that ASP could reduce the liver damage in ConA-induced liver injury.Serum TNF-a, IFN-?, IL-2, IL-6 and MDA contents and liver ROS and SOD levels were analyzed using colorimetric test and ELISA kits. It was found that after challenged intravenously with ConA, the serum levels of TNF-a, IFN-y, IL-2 and IL-6 in the ConA group were higher than those in the normal control group. However, the increased serum levels of TNF-a, IFN-?, IL-2 and IL-6 were prevented by pretreatment with ASP in the ConA+ASP (1.5 mg/kg) group and the ConA+ASP (6 mg/kg) group, respectively. Administration of ConA decreased the activities of SOD and increased the levels of ROS and MDA, respectively. Compared with the ConA group, treatment with ASP to ConA-intoxicated mice significantly resulted in an increase in the activities of SOD and decrease in the levels of ROS and MDA. It was suggested that ASP might inhibit the process of hepatic injury by alleviating inflammatory response and oxidative stress induced by ConA.The results tested by Western blot analysis showed that the expressions of NF-?B, IKKa, p-I?Ba, Caspase-3, Caspase-8, cleaved Caspase-8, p-JNK, Bax, STAT3, p-STAT3, TLR4 and TNFR1 were significantly enhanced after ConA challenge, and ASP pretreatment inhibited the expression of those proteins markedly compared with that in the ConA administration group. What's more, ASP inhibited the decrease of Bcl-2, but did not enhance Akt phosphorylation after challenged intravenously with ConA. The underlying mechanism of ASP pretreatment in ConA-induced liver damage was listed as followed.(1) ASP might attenuate the JNK-mediated mitochondrial apoptosis pathway and Caspase-8-dependent apoptosis in ConA-induced hepatitis.(2) ASP was able to block NF-?B activation and subsequently attenuate the transcription of target genes including various proinflammatory cytokines and chemokines, thus protecting mice from ConA-induced hepatitis.(3) Treatment with ASP significantly reduced the release of total STAT3 and inhibited liver STAT3 phosphorylation in ConA-induced liver damage in mice. Thus, it could be suggested that ASP had a potential role in inhibition of IL-6/STAT3 signaling pathway in acute liver injury induced by ConA.The% divided and the Prol. Index of CD19+B cells were similar between the control group and the ConA group. However, the% divided in the ConA+ASP group increased from 56.6% to 92.2% and the Prol. Index in the ConA+ASP group increased from 1.53 to 2.59 compared with the ConA group. For CD4* T cells, the% divided in the ConA+ASP group fall to 70.5% from 90.9% in the ConA group. Moreover, the Prol. Index declined to 1.78 from 2.31 compared with the ConA group. Both the% divided and the Prol. Index in the ConA+ASP group were decreased, indicating the significant effect of ASP on the proliferation of CD4-T cells stimulated with ConA. The MTT analysis showed that ASP (10-1000?g/mL) had no effect on HepG and HT 29 cells, indicating that ASP did not combine directly with the damage liver cells to play a protective role.Part ? The effect of ASP on the ethonal-induced liver injuryAnimals were randomly divided into four groups (n= 10 each):the normal control group, the ethonal group, the ethonal+ASP (1.5 mg/kg) group, and the ethonal+ASP (6 mg/kg) group. The ethonal+ASP (1.5 mg/kg) group and the ethonal+ASP (6 mg/kg) group were intravenously administered single doses of 1.5 and 6 mg/kg body weight of ASP per day for two weeks, respectively. The normal control group and the ethonal group were intravenously administered with the same volume of PBS per day for two weeks. The ethonal group, ethonal+ASP (1.5 mg/kg) group and ethonal+ASP (6 mg/kg) group were challenged with ethonal (15 mg/kg body weight) by gavage on the 15th day, while the normal control group was challenged with the same volume of PBS on the 15th day. Blood samples were collected 6 h after ethonal or PBS injection. After 12 h of ethonal challenge, all mice were sacrificed, and the liver tissues were collected.Hematoxylin and eosin staining of the liver was performed in order to evaluate the pathological changes in liver. In the ethonal group, massive inflammatory cell infiltration and cavitation were widely presented. However, these phenomena were almost absent in animals pretreated with ASP. The liver index decreased in mice pretreated with ASP. The results of serum aminotransferase levels showed that serum ALT and AST levels increased 6 h after intragastric administration compared with the normal control group. However, compared with the ethonal group, ASP markedly decreased the elevation of serum ALT and AST levels. The results above indicated that ASP could reduce the liver damage in ethonal-induced liver injury.TNF-a, IFN-?, IL-6, SOD, CAT, GR, GSH-Px, GSH, ROS, MDA and LPS in serum and liver were analyzed using colorimetric test and ELISA kits. It was found that after challenged with ethonal, the serum and liver levels of TNF-a, IFN-y, ROS, MDA and LPS in the ethonal group were higher than those in the normal control group, and SOD, CAT, GR, GSH-Px and GSH levels decreased after challenged with ethonal. Compared with the ethonal group, the increased levels of TNF-a, IFN-y, ROS, MDA and LPS were prevented by pretreatment with ASP in the ethonal+ASP (1.5 mg/kg) group and the ethonal+ASP (6 mg/kg) group, respectively. What's more, administration of ASP increased the activities of SOD, CAT, GR, GSH-Px and GSH, respectively. However, there was no significant difference in the levels of IL-6 among each group. It was suggested that ASP might inhibit the process of hepatic injury by alleviating inflammatory response and oxidative stress induced by ethonal.To investigate the effect of ASP on oxidative stress and lipid peroxidation stimulated by ethonal, ROS production and DPPH removal ability were monitored by flow cytometry and fluorescence imaging technique, and TG in serum and liver was analyzed using colorimetric test. Moreover, the microscope images of liver and HepG2 cells were observed by Oil Red O staining. As shown in the results, the increased level of ROS was prevented by treatment with ASP. And ASP (1-20 mg/mL) could remove DPPH free radical. Administration of ASP reduced the levels of TG in liver and HepG2 cells, and decreased the lipid droplets in liver and HepG2 cells by Oil Red O staining.The MTT analysis showed that the ethonal might induce HepG2 cells apoptosis, but ASP (more than 50?g/mL) could inhibit the cytotoxicity of ethonal. It could be suggested that ASP had an effect on the inhibition of cell apoptosis stimulated by ethonal.The results tested by Western blot analysis showed that the expressions of Caspase-3, Bax, TLR4 and CYP2E1 in liver and HepG2 cells and NF-?B, p-I?Ba in liver were significantly enhanced after ethonal challenge, and ASP pretreatment inhibited the expression of those proteins markedly compared with that in the ethonal administration group. What's more, ASP inhibited the decrease of Bcl-2 in liver and HepG2 cells, but did not decrease the expressions of NF-?B and p-IicBa in HepG2 cells after challenged with ethonal. The underlying mechanism of ASP pretreatment in ethonal-induced liver damage was listed as followed.(1) ASP might reduce the ratio of Bax and Bcl-2 so that attenuate the Caspase-mediated apoptosis pathway in ethonal-induced hepatitis.(2) ASP was able to block NF-?B activation and subsequently attenuate the transcription of target genes including various proinflammatory cytokines and chemokines.(3) Treatment with ASP significantly inhibited the expression of CYP2E1 in ethonal-induced liver damage in mice. Thus, ASP had a potential role in inhibition the release of ROS and attenuated inflammatory response and oxidative stress induced by ethonal. |
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