| Objective:Through the observation of“nourishing kidney to produce marrow and form liver”rule of treatment in“Diwuyanggan capsule”intervention effect on CCl4-induced liver fibrosis in MSG-rats. To explore the mechanism of “Diwu Yanggan capsule”regulation of EMT/MET imbalance, reveals the EMT/MET imbalances caused by“marrow producing liver abnormally”pathogenesis and “nourishing kidney to produce marrow and form liver ” regulation of EMT / MET imbalance in the scientific connotation,providing new treatment ideas and direction for the clinical treatment of liver fibrosis.To discuss“nourishing kidney to produce marrow and form liver ”control liver regeneration improve liver and kidney essence deficiency biological basis.Methods:On the second day to tenth day(day intervals), make five times injection L-monosodium glutamate-saline solution to the subcutaneous of neonatal wistar rats, which dose for each 4mg/g. Weaning after28 days, 6-week-old division cages,select the 18 male rats which injection of MSG-saline solution were randomly divided into 3 groups :①MSG-rat blank control group( hereinafter referred to as the “control group”, ② CC14-induced liver fibrosis in MSG-rat of “Diwuyanggan capsule” treatment group(hereinafter referred to as the “treatment group”), ③ CC14-induced liver fibrosis in MSG-rat model group(hereinafter referred to as the “CCL4-MSG-model group”). Randomly select six male Wistar rats were used as model of normal control group,that④ CC14-induced liver fibrosis model group(hereinafter referred to as the “CCL4-model group”).Therefore, this experiment is divided into four groups, the control group received doses of soybean oil by subcutaneous injection for 6 weeks, 2 times per week, a dose of 1 ml/kg;the treatment group、CCL4-MSG-model group and CCL4-model group rats were given 50% CCL4/ soybean oil was established by subcutaneous injection for six weeks, 2 times per week, a dose of 1 ml/kg.In modeling at the same time, the treatment group rats were given “Diwu Yanggan capsule”solution gavage, 1 times / day, a dose of 360 mg/kg, a total of 6 weeks.And the control group、CCL4-MSG-model group and CCL4-model group rats were given the same amount of saline lavage in comparison. Modeling six weeks, the rats were sacrificed, The four groups were taken hypothalamus part of the brain do hematoxylin- eosin(HE) staining, to observe the situation MSGmodeling rats.And the four groupswere taken liver tissue do liver tissue haematin- eosin(HE) staining and Masson staining, in order to observe the degree of liver damage and the proliferation of collagen fibers extent. Real-time quantitative reverse transcription polymerase chain reaction(Q-RT-PCR) and Western blotting observed m RNA and protein expression in MSG-rats liver tissue of E-cadherin and vimentin、TGF-β1 and BMP-7,as well as hedgehog signaling pathway markers:Shh、Smo、Ptc、Gli1. Real-time quantitative RT-PCR resulting Ct value, calculated using 2- △△ Ct calculation. The image was processed by Gel-pro analyzer software, the selected analysis area, obtaining the average value of the integrated optical density IOD value. All data were processed by statistical software SPSS17.0, the result is in Mean ± SD. Group of multiple samples were compared using analysis of One-Way ANOVA, P <0.05 was considered statistically significance.Draw a diagram according to the statistical results.Results : Hypothalamic tissue biopsy(HE staining) showed hypothalamic arcuate nucleus neurons in MSG-rat development was abnormal,MSG- rat hypothalamic neurons damaged. Liver histopathology HE staining showed that “ Diwuyanggan capsule” therapy could alleviate liver cell injury, and reduce the degree of liver fibrosis, CCL4-MSG-model group is more serious than the CCL4-model group of liver fibrosis. And Masson staining showed CCL4-MSG-model group( 16.23 ± 9.76) hepatic fibrosis was significantly increased than the control group(0.04±0.05), CCL4-MSG-model group(16.23±9.76)more than CCL4-model group(10.10 ± 1.92)and more than treatment group(2.54±1.71),p<0.05.Real-time quantitative RT-PCR results showed that: ① the epithelial cell marker E-cadherin m RNA levels of the CCL4-MSGmodel group(0.75±0.42)was significantly lower than the treatment group(1.48±1.21), in contrast mesothelial cell marker Vimentin m RNA levels the CCL4-MSG- model group( 7.33 ± 5.11) was significantly higher than the treatment control group(0.95±0.64);②the TGF-β1 m RNA levels of the CCL4-MSG- model group(6.01±5.92) was significantly higher than the treatment control group(1.17±0.69), in contrast BMP-7 m RNA levels of the CCL4-MSG- model group(0.63±0.24)was significantly lower than the treatment group(1.24±0.76); the ratio of TGF-β1 /BMP-7 is CCL4-MSG- model group(9.75±4.78) was significantly higher than the treatment control group(1.04±0.76).③Liver tissue of rats in the control group Hedgehog signaling pathway in a silent state, while the Hedgehog signaling pathway in rat liver tissue CCL4-MSG- model group is active, so the Hedgehog signaling pathway marker m RNA expression CCL4-MSG- model group higher than control group. However, the Hedgehog signaling pathway by treatment group “ Diwuyanggan capsule” in the treatment of post-suppressed state, the Hedgehog signaling pathway marker m RNA expression treatment group lower than CCL4-MSG- model group,both Statistically, the significant difference, p <0.05.Western blotting results showed that: ①the epithelial cell marker E-cadherin protein expression of CCL4-MSG- model group(0.79±0.34) was significantly lower than the control group(4.51±2.27)and the treatment group(3.76±3.61); the mesothelial cells Vimentin protein expression of CCL4-MSG- model group(1.68±0.61) was significantly higher than the control group(0.20±0.13)and the treatment group(0.99±0.29);② the TGF-β1 protein expression of CCL4-MSG- model group(1.98±1.15) was significantly higher than the control group(0.13±0.07)and the treatment group(0.46±0.08),the BMP-7 protein expression of CCL4-MSG- model group(0.56±0.18) was significantly lower than the control group(2.15±0.53) and the treatment group(3.30±0.57); ③ Hedgehog signaling pathway in the control group did not activate its associated protein markers expression are very low, and in CCL4-MSG- model group, due to the activation-related marhers Hedgehog signaling pathway protein expression were significantly increased.Afte“Diwuyanggan capsule”treatment, Hedgehog signaling pathway was inhibited, so its associated protein markers expression of the treatment group was significantly lower than the CCL4-MSG-model group, both Statistically, the significant difference, p <0.05.Conclusion: CCL4-induced MSG-rat liver fibrosis model group was more serious than in normal CCL4-induced rat liver fibrosis model group, the degree of hepatic fibrosis heavier. CCl4-induced hepatic fibrosis in MSG-rats exist EMT / MET imbalance caused by etiology and pathogenesis of“marrow producing liver abnormally”, the liver tissue epithelial cell marker expression levels down, and mesenchymal cell marker expression levels increase. “ Diwuyanggan capsule ” can delay or reduce the development of liver fibrosis, regulating EMT / MET imbalance(inhibition of EMT and promote MET), to improve the“marrow producing liver abnormally”. And found Hedgehog signaling pathway is activated, the expression of TGF-β1 was significantly higher in CCL4 induced MSG-rat liver fibrosis and the expression of BMP-7 is significantly reduced, the experimental results show that the “ Diwuyanggan capsule ” may be through inhibit activation of Hedgehog pathway and reduce the ratio of TGF-β1 / BMP-7 to adjust EMT / MET imbalance, thereby reversing the development of hepatic fibrosis. |
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