| BackgroundLiver is the important metabolic, endocrine and immune organs. A variety of harmful factors can lead to liver damage, prevention and treatment of liver injury is still the subject of a global critical. Endotoxin-mediated liver injury in a variety of important factors. Liver disease can produce endotoxin, which, reciprocal causation of liver injury, and may play an important role in the development of liver disease. Viral hepatitis, alcoholic liver disease, drug and chemical induced hepatitis and so on associated with varying degrees of endotoxemia. Therefore, the study of endotoxin induced liver injury in the mechanism, to find effective means of prevention, screening hepatoprotective drugs, to explore principles of hepatoprotective effects, is of great practical significance.Hepatic portal vein and hepatic artery blood supply of both, but both are ultimately adopted by terminal arterioles and venules and liver sinusoids connected, so the completion of the liver sinusoid is the main place for the exchange of material. Acute and chronic liver disease may show different degrees of sinusoidal stenosis, congestion, circulation stasis, micro-thrombosis, can cause necrosis solubility of adjacent hepatocytes. severe Patients’hepatic sinusoidal cavity area was filled with collagen fibers, showed sinusoidal capillarization. Blocked in liver metabolism, further aggravating liver damage.Liver sinusoidal endothelial cell constitute the major component of sinusoidal wall, Can control the sinusoidal microcirculation, regulating metabolism and participate in the maintenance of liver homeostasis, plays an important "regulator" role for liver metabolism. Unlike other cells, It has a screen-like fenestrae structures and discontinuous basement membrane. Many fenestraes gathered to form endothelial sieve, play a role of Filter barrier in metabolism between-blood and hepatocytes, regulating the rate of material out of sinusoids. Pathology and aging conditions, Reduce the number and diameter of fenestrae, there may be metabolic disorders, collagen deposition, a continuous basement membrane formation occurred sinusoidal capillarization. Severe lesions, Endothelial cell loss, so that platelets and coagulation factors under the direct matrix in contact with the cells,, induced micro thrombosis, increase microcirculation, leading to liver fibrosis and even cirrhosis of the occurrence and development. Therefore, some researchers believe that LSEC may lead to liver microcirculation damage, and the trigger point of liver cell degeneration and necrosis. LSEC function can regulate the permeability of liver metabolism and hepatic sinusoidal blood flow, which coincides with the liver controlling dispersion function and many aspects and multi-target of Chinese medicine, therefore, LSEC may be one of the target of Chinese medicine in protecting liver, and associated with liver governing conveyance and dispersion.S. miltiorrhiza belongs to the plant labiatae Salvia dry root and rhizome, Of bitter flavours, slightly cold property, tropism heart and liver meridians. With blood stasis, menstrual pain, heat sedative effect, the effect that "good treatment of Blood, to Indigestion and product new, adjust the meridians and pulse" is especially good. Therefore, it is Known that"blindly Salvia, power with Siwutang". Currently, as a main component of Salvia miltiorrhiza, there is less study of polysaccharide. preliminary studies the mechanism of it against immunological liver injury were successful. Therefore, the mechanism described in depth, to clarify its target, there is great significance for reveal the theory of the Zang-fu organs.Objectives:To Investigate the mechanism of Salvia miltiorrhiza polysaccharides protect the liver and it’s possible role of target, by studying the effects of Salvia polysaccharides on acute liver injury induced LSEC morphology and function, as ICAM-1, MDA and so on. Meanwhile, try to elaborate the Correlation Between the LSEC and Liver Governing Conveyance and Dispersion, Provide a scientific basis for the study of the material basis for liver governing conveyance and dispersionan and the mechanism of the Chinese medicine protect the liverMethods:1. Preparation of Salvia miltiorrhiza polysaccharidesSalvia miltiorrhiza identified as pure by the department of Chinese medicinal herbs in the southern Medical university, polysaccharides prepared in accordance with the patent(a polysaccharide extracted from Salvia method, open No.: 200810026249.4).2. Establishment of the animal model, Grouping and treatmentMice were randomly divided into normal control group, model group, low-dose (2.6 g·kg-1) of SMPS (SMPS-L) group, middle-dose (7.8 g·kg-1)of SMPS (SMPS-M) group,and high-dose (15.6 g·kg-1) of SMPS (SMPS-H) group, DDB(0.2g·kg-1)Group, which were given daily via gavage for a period of 7 days. The control group and model group were given an equal volume of saline.7 days later, the mice were given LPS(5mg/kg) (control received saline alone) via lateral tail vein injection.3 hours after the injection of LPS, animals were anesthetized, fixed liver by portal vein infusion, Take the same parts of the liver were placed in 1.5% glutaraldehyde fixative and 10% formaldehyde fixative for electron microscopy sample preparation and pathological examination. the remaining liver tissue Ultra-low temperature refrigerator for preparation liver tissue homogenate. Blood 3000 r/min x 10 min centrifugation, serum were frozen ultra-low temperature refrigerator for next test.3. Preparation and observation of the electron microscopy samples.Gradient alcohol dehydration after repeated rinsing the liver tissues in fixative by sodium cacodylate buffer (PH7.4), embedded sections and the production of critical point drying completed by the electron microscope department.Each specimen with a low magnification to find the portal area first, located around the portal area, observed at least 5 oblique sinusoidal sieve plate-like structure, observe the number and size of sinusoidal fenestrae carefully, then look for the central vein area at least 5 fields of vision, observed sinusoidal sieve plate-like structure. Counting the number and detecting the size of sinusoidal fenestrae using ImageJ image analysis software system.4. Observed Salvia polysaccharides on the expression of ICAM-1 and integrinβ1 by immuneohistochemistry.Slice off wax, PBS rinse 3min x3, eliminate endogenous peroxidase with 3 %H2O2, PBS rinse 3min x3, Repair antigen 5min, down to room temperature naturally, PBS rinse 3min x3. add the first antibody of ICAM-1,37℃overnight, PBS rinse 3min x3. Adding the second antibody-HRP polymer 37℃for 20min, PBS rinse 3min x3, DAB (new configuration, room temperature)color 15min, hematoxylin-stained nuclei, Coverslip generally, observed. Separate negative staining on the photo, the first antibody of ICAM-1 replaced by PBS. The expression of Integrin-β1 in the same operation. 5. Observed the effect of Salvia polysaccharides on serum ALT and MDA, GSH in the liver tissue using colorimetric method.Test serum ALT according to kit instructions.preparation liver tissue homogenate. The test according to kit instructions.the contents of MDA and GSH in the liver tissue were measured by the following formula:the contents of MDA in the liver tissue= (Absorbance of test tube-Absorbance of blank tube)/(Absorbance of standard tube-Absorbance of blank tube)×Standard concentrations÷Tissue protein contentthe contents of GSH in the liver tissue= (Absorbance of test tube-Absorbance of blank tube)/(Absorbance of standard tube-Absorbance of blank tube)×Standard concentration×GSH molecular weight×Dilution÷Tissue protein content6. The statistics method.Statistical software SPSS13.0 was used to analyze the experimental data. The experimental data were indicated by mean士standard deviation(x±s). One-way ANOVA was used to analyze the data. LSD method was used in multiple comparisons between groups when variance was homogeneous. Dunnett’sT3 was used when variance is not homogeneous.Kruskal Wallis was used to analysis the difference of the stage of liver fibrosis. Difference is significant when P＜0.05.Results:1. Behavioral observationBehavior observed in fur shiny and activities responsive of mice, mice which fed Salvia miltiorrhiza polysaccharide is better than the other groups of mice. After the injection of LPS, the mental state of SMPS groups and positive control group are better than model group. There is no significant difference between SMPS groups and positive control group. 2. Ultrastructural changes of mouse liver sinusoidalElectron microscopy shows clear capillary bile duct structures on hepatocyte surface in normal group, a large number of fenestraes on LSEC surface, Microvilli of hepatocyte can through the fenestraes to reach the sinusoidal cavities. hepatocytes of model group ruptured, hepatic cord structure was destroyed, hepatocyte microvilli disappeared, Loss of fenestrae of LSEC occurred, a large number of extracellular matrix material deposition in disse cavity, some LSECs Breaking away, Within Kupffer cells and others adhesion and thrombus-like structure of matter appears in sinusoidal.In SMPS-H and SMPS-M groups, necrosis and rupture of hepatocytes reduction, LSEC fenestrae number, feret’s diameter and area were significantly increased compared with the model, there are structural integrity of the capillary bile ducts on hepatocyte surface, hepatocyte microvilli increase, cells and collagen adhesion decreased in Sinusoid. SMPS-L group compared with the model did not change significantly. There are significant differences between model and positive control groups (P＜0.05), but no significant difference between SMPS groups and positive control groups (P>0.05).3. Pathological observation of HE stainingPathology results showed that compared with the normal group, hepatic pathological changes significantly of model group(P<0.01). compared with the model,Significant improvement in hepatic pathology of SMPS-H and SMPS-M groups(P<0.01), pathological changes of SMPS-L group was not significant. Statistics showed positive control groups is significantlly different with model and SMPS-L groups (P＜0.01), while no difference with SMPS-H and SMPS-M groups(P＞0.05).4. The effect of SMPS on the expression of ICAM-1 and integrinβ1 of acute liver injury mice induced by LPS. ICAM-1, Integrin-β1 was almost no expression or weak expression of normal control group, the expression of model group was significantly higher than the normal. Compared with model group, ICAM-1, Integrin-β1 expression of SMPS-H and SMPS-M groups were significantly reduced, SMPS-L group did not change significantly. the expression of positive control groups was significantly reduced. There is no significant difference between SMPS groups and positive control group.5. The effect of SMPS on serum transaminase of acute liver injury mice induced by LPS.Compared with the normal group, model group of serum ALT levels were significantly increased(P<0.01). It showed that after intravenous injection of LPS in mice caused severe liver injury, animal model establish successfully. Compared with the model group, SMPS-H and SMPS-M groups decreased significantly(P＜0.05). SMPS-L group had no obvious effect (P>0.05). There are significant differences between model and positive control groups (P<0.05), but no significant difference between SMPS groups and positive control groups (P>0.05).6. The effect of SMPS on MDA, GSH in the liver tissue of acute liver injury mice induced by LPS.Compared with the normal group, the contend of MDA of model group were increased significantly, meanwhile, the contend of GSH were reduced significantly (P＜0.01). Compared with the model group, SMPS-H and SMPS-M groups the contend of MDA decreased significantly (P<0.05), especially more obvious effect of SMPS-H group (P＜0.01). SMPS-L group had no obvious effect (P>0.05). The contend of GSH were increased significantly in SMPS-H,SMPS-M and SMPS-L groups(P<0.01).It indicated that SMPS can reduce the lipid peroxide content in the liver tissue of acute liver injury mice induced by LPS, increased antioxidant capacity, reduce hepatocyte injury. Statistics showed positive control groups is different with model and SMPS-L groups (P＜0.05), while no difference with SMPS-H and SMPS-M groups (P>0.05).Conclusions:SMPS can reduce LSEC injury of acute liver injury mice induced by LPS, and reduce the collagen deposition, Inhibition of capillary-based situation caused by fenestrae being reduced. Improve the microcirculation, Mitigate lipid peroxidation. SMPS offers obvious effects on protecting acute liver injury induced by LPS. As well as LSEC will be it’s possible role of target, who have some correlations with liver governing conveyance and dispersion. |
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