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扶正化瘀方对肝硬化小鼠模型肝细胞消亡与再生的影响
DOI: 10.12449/JCH240417
Effect of Fuzheng Huayu prescription on hepatocyte extinction and regeneration in a mouse model of liver cirrhosis
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摘要:
目的 探讨扶正化瘀方对纤维化肝脏肝细胞消亡与再生的影响，及其药物促进肝细胞再生的作用机制。 方法 以CCl4腹腔注射6周诱导建立肝硬化小鼠模型。正常对照组9只，模型组10只，索拉非尼组10只，扶正化瘀方组10只。自造模第4周起，扶正化瘀方组、索拉非尼组分别给予4.8 g/kg、4 mg/kg小鼠体质量相应药物灌胃，连续3周；正常组和模型组给予等体积羧甲基纤维素钠。检测血清肝功能；METAVIR评分系统评价肝组织炎症及纤维化分期；天狼星红染色和肝组织羟脯氨酸含量评价胶原沉积量；免疫组化检测Ⅳ型胶原、CD31与CD32b、Ki67、CyclinD1、谷氨酰胺合成酶（GS）、Wnt2以及HGF蛋白的表达；Western Blot检测肝组织Wnt2、LRP6、β-catenin、p-β-catenin、CyclinD1表达。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。 结果 与模型组比较，扶正化瘀方组和索拉非尼组血清ALT、AST水平和肝组织羟脯氨酸含量均降低（P值均<0.01），METAVIR评分减低（P值均<0.05）；Ⅳ型胶原、CD31表达减少（P值均<0.05），CD32b表达增加（P<0.01）；肝组织实质病变消亡数量减少，Ki67、CyclinD1表达上升（P值均<0.01）；Wnt2、LRP6、β-catenin、CyclinD1蛋白表达水平上调、p-β-catenin表达显著下调（P值均<0.05）；肝组织CD32b与Wnt2共染阳性细胞显著增多。 结论 扶正化瘀方可通过抑制肝窦毛细血管化，改善肝窦内皮细胞Wnt2外分泌功能，激活肝细胞再生相关Wnt/β-catenin信号通路，最终逆转肝硬化。 Abstract:Objective To investigate the effect of Fuzheng Huayu prescription on hepatocyte extinction and regeneration in fibrotic liver and its mechanism of action in promoting hepatocyte regeneration. Methods Mice were given intraperitoneal injection of CCl4 for 6 weeks to establish a model of liver cirrhosis， and there were 10 mice in the model group， 10 in the sorafenib group， 10 in the Fuzheng Huayu prescription group， and 9 in the normal control group. Since week 4 of modeling， the mice in the Fuzheng Huayu prescription group and the sorafenib group were given the corresponding drug by gavage at a dose of 4.8 g/kg and 4 mg/kg， respectively， for three consecutive weeks， and those in the normal group and the model group were given an equal volume of sodium carboxymethyl cellulose. Serum liver function parameters were measured； the METAVIR scoring system was used to evaluate liver inflammation and fibrosis stage； Sirius Red staining and hydroxyproline （Hyp） content in liver tissue were used to evaluate collagen deposition； immunohistochemistry was used to measure the protein expression levels of type IV collagen， CD31， CD32b， Ki67， CyclinD1， glutamine synthetase， Wnt2， and HGF， and Western blot was used to measure the expression levels of Wnt2， LRP6， β-catenin， p-β-catenin， and CyclinD1 in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results Compared with the model group， the Fuzheng Huayu prescription group and the sorafenib group showed the following changes： significant reductions in the serum levels of alanine aminotransferase and aspartate aminotransferase and the content of Hyp in liver tissue （all P<0.01）； a significant reduction in METAVIR score； significant reductions in the expression levels of type Ⅳ collagen and CD31 （all P<0.05） and a significant increase in the expression level of CD32b （P<0.01）； significant reductions in the number of parenchymal extinction lesions and significant increases in the expression levels of Ki67 and CyclinD1 in liver tissue （all P<0.01）； significant increases in the protein expression levels of Wnt2， LRP6， β-catenin， and CyclinD1 and a significant reduction in the protein expression level of p-β-catenin （all P<0.05）； significant increases in the number of cells stained positive for both CD32b and Wnt2. Conclusion Fuzheng Huayu prescription can inhibit hepatic sinusoidal capillarization， improve the Wnt2 exocrine function of liver sinusoidal endothelial cells， activate the Wnt/β-catenin signaling pathway associated with hepatocyte regeneration， and finally reverse liver cirrhosis. -
Key words:
- Liver Cirrhosis /
- Liver Regeneration /
- Fuzheng Huayu Recipe
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图 1 扶正化瘀方对CCl4肝硬化小鼠模型肝组织炎症、胶原沉积的影响（×100）
Figure 1. Effect of FZHY on liver inflammation， collagen deposition in CCl4 mice （×100）
图 2 扶正化瘀方对肝组织羟脯氨酸含量的影响
Figure 2. Effect of FZHY on hydroxyproline content in liver tissue
图 3 扶正化瘀方对CCl4肝硬化小鼠模型肝组织Ⅳ型胶原、CD31、CD32b表达的影响
Figure 3. Effect of FZHY on the expression of collagen type Ⅳ， CD31 and CD32b in CCl4 mice
图 4 扶正化瘀方对CCl4肝硬化小鼠模型肝组织Ⅳ型胶原、CD31、CD32b的影响（×200）
Figure 4. Effect of FZHY on the collagen type Ⅳ， CD31 and CD32b in CCl4 mice （×200）
图 5 扶正化瘀方对CCl4肝硬化小鼠模型肝组织PEL程度的影响（GS免疫组化染色）
Figure 5. Effect of FZHY on PEL in CCl4 mice （GS immunohistochemical staining）
图 6 扶正化瘀方对肝组织PEL数量和对肝组织中央静脉与汇管区距离的影响
Figure 6. Effect of FZHY on PEL number and distance between central vein and portal area of liver tissue
图 7 扶正化瘀方对肝组织Ki67和HGF表达的影响
Figure 7. Effect of FZHY on the expression of Ki67 and HGF in CCl4 mice
图 8 扶正化瘀方对CCl4肝硬化小鼠模型肝组织Ki67和HGF的影响（×200）
Figure 8. Effect of FZHY on Ki67 and HGF in CCl4 mice （×200）
图 9 扶正化瘀方对模型小鼠LSEC中CD32b与Wnt2表达的影响（×200）
注： CD32b，黄色；Wnt2，红色；DAPI：蓝色。
Figure 9. Effect of FZHY on the expression of CD32b and Wnt2 in mice（×200）
图 10 扶正化瘀方对CCl4肝硬化小鼠模型Wnt/β-catenin信号通路相关蛋白的影响（×200）
Figure 10. Effect of FZHY on Wnt/β-catenin signaling pathway-related protein in CCl4 mice（×200）
图 11 扶正化瘀方对Wnt2和CyclinD1表达的影响
Figure 11. Effect of FZHY on the expression of Wnt2 and CyclinD1 in CCl4 mice
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