| This research is designed to study the possible material essentials and mechanism of Qizhu granule blood-stasis dissolution effect for capillarity of hepatic sinusoid.1 Theoretical investigationOn basis of several years research, the application of Traditional Chinese Medicine (TCM) in the hepatic fibrosis (HF) field and the possible mechanism of'blood-stasis in collaterals'syndrome of HF is,discussed after literature research.Western medicine has not got the effective therapeutic method for HF till now, but TCM shows reliable effect and great potential for HF. Vast of clinical practices show that collateral disease theory is suitable for HF research and treatment. According to the collateral disease theory, the main location of HF is in liver collateral. And the main mechanism is the long term effect on liver and other organs caused by damp-heat toxin and depression. The main pathological change is the abnormal circulation of qi and blood in liver collateral. And during the progress of the disease, other diseases such as jiju and flatulence are easily appeared for the chronic character of HF. A great deal of clinical practices shows that the 'blood-stasis in liver collateral' syndrome is one of the most important mechanisms of HF. So collaterals blood-stasis dissolution therapy is an effective therapeutic method for HF.Combined with researches of western medicine, capillarity of hepatic sinusoid as an important factor for the nutrition exchange between liver cell and hepatic sinusoid plays important role in the HF progress and treatment. And hepatic sinusoid capillarity may be one important contents of 'blood-stasis in liver collateral' syndrome too.2 Experiment researchAIM:To observe the effect of Qizhu granule on hepatic sinusoid capillarity mice, caused by carbon tetrachloride, and explain the collaterals blood-stasis dissolution mechanism of Qizhu granule.METHODS:Early stages of HF minces, induced by CCL4, were randomized into normal control group, model control group and Qizhu granule treatment group. And liver tissue samples were taken on weeks 4 and 8 for transmission electron microscope (TEM), immunohistochemistry (IHC) and real-time PCR examination.1mm3 samples, fixed and water deprived by glutaraldehyde (4℃2.5%), osmic acid and acetone, and embedded by ethoxyline resin, were cut in slices with colored by citric acid. The ultrastructure of liver cell and hepatic sinusoid were observed by JEOL JEM-1400 TEM.Meanwhile, other samples were fixed by 10% formaldehyde for IHC. After water deprived and wax immersion, slices were made. Then using H2O2 blocked the peroxidase and repaired by citric acid, the slices were colored by related antibodies and sealed by resin. Each section was observed and photographed by OlympusⅨ70 converted fluorescence microscope. IOD of each section was also examined by Image Pro-plus 5.0 to analyze the expression of laminin and typeⅣcollagen.And certain liver tissues were also be conserved by liquid nitrogen for RT-PCR examination. After extracting total RNA and cDNA is synthesized, RT-PCR was carried on.ΔΔCt method is adopted to analyze VEGF mRNA of each group.RESULTS:1 TEM RESULTSAbout normal control group rats, most of the liver cells are mononuclear. The nucleuses are round and in the center of the cell. Various organelles such as endoplasmic reticulum and mitochondria can be seen. Bile capillary can also be seen between some liver cells, which get microvillus in the lumen. And glycogen and lipid droplet are common in liver cell too. Liver sinusoid wall is mainly composed of endothelial cell, which is flat and has many branches. The endothelial cell gets many holes in them and many microvilli can also be seen in Disse gap.The model control groups'ones get more serious injuries. Lots of liver cells are apoptosis, and organelles are dissolved seriously. Bulb-like degeneration is appeared in the cells. The pyknosis is occurred and the cytoplasts are condensed. And with the time goes, liver sinusoid endothelial cells gradually lose their holes and continuous basilar membrane is formed. The Disse gap gets narrow and the number of microvillus is decreased sharply. Bile capillaries expanded and microvillus loss, directly resulting in bile backflow into liver cells.Qizhu granule groups rats show obvious liver cell repair. Endoplasmic reticulum increases and cell connection reappears again. The 8 weeks group ones get more serious injury than the 4 weeks', but the number of endoplasmic reticulum is more than the 4 weeks ones. Both 4 and 8 weeks groups ones also get liver sinusoid endothelial cells microvillus and holes reappeared. The width of Disse gap recovers to certain extent but the continuous basilar membrane still exists. Overall, injuries of 8 weeks group are more serious than the 4 weeks ones.2 TypeⅣcollagen and laminin observation by IHCFor the normal control groups'rats, laminin is mainly deposited on the vascular wall of liver portal area. And the typeⅣcollagen is mainly in vein and liver portal area. Both of them are brown-yellow on IHC sections. The IODs of laminin and typeⅣcollagen show significant difference between the Qizhu granule, model control groups, and normal control groups' ones (13.89±1.70vs1.61±0.65,15.92±1.96vs1.43±0.59; 12.24±1.80vs1.33±0.56,17.82±2.94vs1.48±0.33, P<0.01). And with the time goes, IODs of laminin and typeⅣcollagen also show difference significantly between 4 weeks and 8 weeks control group (13.89±1.70vs15.92±1.96; 12.24±1.80vs17.82±2.94, P<0.01). Laminin and typeⅣcollagen IODs of Qizhu granule groups are significantly lower than the control group ones (8.15±2.01vs13.89±1.70,9.64±1.97vs15.92±1.96;7.32±2.11vs12.24±1.80,11.49± 3.31vs17.82±2.94, P<0.01). With the time goes, the increase range of Laminin and type IV collagen IODs of Qizhu granule groups is smaller than the model control ones (9.64±1.97vs8.15±2.01,11.49±3.31vs7.32±2.11).3 VEGF mRNA detection by RT-PCRThe VEGF mRNA quantity of model control group shows significant difference between the normal control ones at both 4 and 8 weeks (0.33±0.02vs0.01±0.01, 0.61±0.44vs0.03±0.03, P<0.01). And the VEGF mRNA quantity of Qizhu granule groups shows significant difference between model control ones at 4 weeks (0.25±0.06vs0.33±0.02, P<0.05). The 8 weeks ones also lower than the model control ones (0.59±0.35vs0.61±0.44).CONCLUSIONS:1 Qizhu granule can protect liver cell and hepatic sinusoidal endothelial cells to certain extent, keeping the wholeness of the cell structure and promote cell repair progress, and finally intervene the progress of capillarity of hepatic sinusoid and HF.2 Qizhu granule can decrease the deposition of type IV collagen and laminin, alleviating the continuous basilar membrane formation, and intervenes the HF.3 Qizhu granule can regulate the expression of VEGF mRNA, and alleviate the progress of capillarity of hepatic sinusoid.4 The effect of Qizhu granule, that regulating VEGF mRNA expression and decreasing typeⅣcollagen and laminin deposition, may be one important mechanisms of its'collaterals blood-stasis dissolution'effect for capillarity of hepatic sinusoid and liver fibrosis.
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