基于PTEN-PI3K/AKT信号通路探讨手术减压联合补阳还五汤治疗大鼠督脉瘀阻型上颈脊髓损伤模型的机制

In recent years, the incidence of spinal cord injury has been increasing year by year, and cervical spinal cord injury is the most common. Etiology and exact molecular mechanism of spinal cord injury(SCI) have not been fully explored. Recent studies have found that autophagy and pyroptosis are closely related to the occurrence of SCI. In addition, there is evidence that the PI3K/AKT signaling pathway promotes the pyroptosis of neural cells by regulating autophagy. However, PTEN can regenerate the damaged nerve by blocking the PI3K/AKT signaling pathway. It can be seen that the maladjustment of PTEN-PI3K/AKT signaling pathway is closely related to the development of SCI. Although some progress has been made in the treatment of SCI, there is still a big gap between the ideal target of treatment. Chinese medicine is extensive and profound. In recent years, it has been proved that Bu-yang Huan-wu decoction is effective in treating SCI of blockade of governor vessel, which has also been confirmed in our clinical study. However, the research on the influence of SCI on PTEN PI3K/AKT signaling pathway is still blank at home and abroad. Based on the research progress in the treatment of SCI at home and abroad, we dare to speculate that Bu-yang Huan-wu decoction can inhibit PTEN and activate PTEN-PI3K/AKT signaling pathway to regulate autophagy, decreasing the pyroptosis of nerve cells,so as to achieve nerve regeneration and the purpose of SCI treatment. Based on this, this research based on the PTEN-PI3K/AKT signal pathway as the starting point, and governor－vessel－blockade －type upper cervical spinal cord as the research object, elucidate .the mechanism of Bu-yang Huan-wu decoction to treat SCI through in vivo and in vitro experiments to provide new ideas for SCI treatment of traditional Chinese medicine.

脊髓损伤(SCI)的发病率逐年攀升,其确切分子机制尚未完全探明。新近研究发现细胞自噬和焦亡与SCI的发生密切相关。研究表明，PI3K/AKT信号通路可调控自噬促进神经细胞焦亡,而PTEN可通过阻断PI3K/AKT信号通路来实现受损神经的再生，可见该信号通路的失调与SCI的发生发展关系密切。尽管目前对SCI的治疗取得了一定进展，但疗效仍不明显。研究证明补阳还五汤治疗督脉瘀阻型SCI疗效确切，但该方对SCI的PTEN-PI3K/AKT信号通路影响的研究尚属空白。基于国内外研究进展，我们推测补阳还五汤可能通过抑制PTEN，激活PTEN-PI3K/AKT信号通路调控自噬，抑制神经细胞焦亡,从而实现神经的再生，达到治疗目的。故本研究以PTEN-PI3K/AKT信号通路为切入点，大鼠督脉瘀阻型上颈髓损伤模型为研究对象，通过体内外实验阐明补阳还五汤治疗SCI的机制，为SCI的中医药治疗提供新的思路。

脊髓损伤(SCI)的发病率逐年攀升,其确切分子机制尚未完全探明。细胞自噬和焦亡与SCI的病生理变化密切相关。相关研究表明，PTEN-PI3K/AKT信号通路可通过调控自噬改善SCI病程变化，补阳还五汤治疗督脉瘀阻型SCI疗效确切，但该方对SCI的PTEN-PI3K/AKT信号通路影响的研究尚属空白。基于国内外相关研究进展，我们推测补阳还五汤可能通过抑制PTEN，激活PI3K/AKT信号通路调控自噬，抑制神经细胞焦亡,从而达到改善SCI后神经功能的目标。本研究以PTEN-PI3K/AKT信号通路为切入点，以大鼠督脉瘀阻型上颈髓损伤模型为研究对象，通过体内实验阐明了补阳还五汤通过PTEN-PI3K/AKT信号通路调控自噬抑制焦亡从而改善了SCI后的神经功能，为发挥中医药在治疗SCI中的优势起到了很好的示范作用。本研究结果显示：1.补阳还五汤和甲强龙组大鼠的行为学（BBB评分）明显优于模型组（P<0.05），但均低于正常组，表明补阳还五汤和糖皮质激素可显著改善SCI后的神经功能；2.HE结果显示正常组结构完整，灰质和白质均较清晰；模型组大鼠的右侧脊髓结构严重受损，形成大空泡和瘢痕组织，大量中性粒细胞浸润及大量小胶质细胞增生，而补阳还五汤和甲强龙组组的病理结构明显优于模型组，这从组织学角度证实了补阳还五汤在治疗SCI中的有效性；3.WB结果显示，补阳还五汤组和甲强龙大鼠组Akt、p-Akt、MTOR、p-mTOR高于模型组（P<0.05），但均低于正常组，而p-caspase1、c-caspase1、IL-1β、PTEN均低于模型组(P<0.05)，但均高于正常组。这充分说明了脊髓损伤后自噬受损，而补阳还五汤可以通过PTEN-PI3K/AKT通路增强自噬进而调控自噬达到间接抑制焦亡的作用；4.免疫组化结果显示，补阳还五汤和甲强龙组的p-caspase1、c-caspase1、IL-1β在神经元中的棕黄色颗粒在数量和浓度上均低于模型组，但均高于正常组，这从蛋白定位的角度再次说明了补阳还五汤可以通过PTEN-PI3K/AKT通路增强自噬流进而调控自噬达到间接抑制焦亡的作用；5.Tunel结果显示补阳还五汤和甲强龙组的细胞凋亡个数低于模型组，但高于正常对照组，证明了补阳还五汤可以抑制细胞的凋亡。