基于KAS-seq测序的四妙勇安汤改良及其促进糖尿病足小鼠创面愈合情况的观察

Observation on the improvement of Si-Miao-Yong-An decoction based on KAS-seq sequencing and its promotion of wound healing in mice with diabetic foot

目的 基于现代科学技术对四妙勇安汤进行改良,验证其促进糖尿病足伤口愈合的效果。方法 采用乙氧二羟丁酮辅助的单链DNA测序(kethoxal-assisted single stranded DNA sequencing, KAS-seq)技术检测糖尿病足患者创面组织和正常创面组织,筛选糖尿病足相关的关键基因,并结合网络药理学,根据这些关键基因寻找有针对性的中药从而对四妙勇安汤进行改良,最后通过糖尿病足动物模型验证改良方的治疗效果。结果 通过临床样本测序分析得到CREB1、GRID2、PIK3R3、DRD2、ADORA1、TNFSF11、EDNRB、HTR1B 8个关键基因,靶向到黄柏、苍术、地黄和半夏4味中药配伍入原方。在灌胃干预第7天时改良方组(S+)小鼠伤口愈合效率显著高于对照组(Control)(P<0.05),第12天时S+组伤口愈合率显著高于原方组(S)(P<0.05)。改良方中药靶点在原方基础上更多富集到了神经系统、缺氧和血液循环调节相关通路中。结论 改良方与经方相比具有更好地促进糖尿病足伤口愈合的效果,相关机制可能与调节炎性反应、改善周围神经病变和促进伤口愈合等通路有关,同时也为中药方剂的临床加减应用提供了一种新方法和现代科学理论依据。

Objective Based on modern scientific technology,the Si-Miao-Yong-An decoction was improved to verify its effect in promoting the healing of diabetic foot wounds.Methods The kethoxal-assisted single stranded DNA sequencing(KAS-seq)technology was utilized to detect wound tissues from diabetic foot patients and normal wound tissues,to screen for key genes associated with diabetic foot.Combining network pharmacology,targeted traditional Chinese medicines were identified based on these key genes to modify the traditional formula Si-Miao-Yong-An decoction.Finally,the therapeutic effect of the modified formula was verified through an animal model of diabetic foot.Results Through clinical sample sequencing analysis,8 key genes,including CREB1,GRID2,PIK3R3,DRD2,ADORA1,TNFSF11,EDNRB,HTR1B were obtained.These genes were targeted to Huang Bai,Cang Zhu,Di Huang,and Ban Xia,four traditional Chinese medicines combined in the prescription.On the 7th day of intragastric intervention,the wound healing efficiency of the S+group mice was significantly higher than that of the control group(P<0.05),and on the 12th day,the wound healing rate of the S+group mice was significantly higher than that of the S group(P<0.05).The target points of the modified prescription were more enriched in the neural system,hypoxia,and blood circulation regulation-related pathways compared to the original prescription.Conclusions Compared to the original prescription,the modified prescription has a better effect on promoting the healing of diabetic foot wounds.The related mechanism may be associated with the regulation of inflammation,improvement of peripheral nerve damage,and promotion of wound healing pathways.This approach also provides a new perspective and a basis in modern scientific theory for the clinical application and modification of traditional Chinese medicine prescriptions.