Objective  To investigate the effects of Shengjiyuhong ointment on coagulation system, endothelial progenitor cells (EPCs) differentiation and Rho kinase activity in rats with thromboangiitis obliterans by inhibiting autophagy.

Methods  The rat model of thromboangiitis obliterans was established by injecting sodium laurate solution into the left lower limb artery. 40 SD rats were randomly divided into sham operation group, model group, compound sodium aescinate gel group and Shengjiyuhong ointment group, 10 per group. The pathological morphology of vascular tissue was detected by HE staining. The expression of autophagy related proteins was detected by Western blot, coagulation system parameters were detected by coagulation detector, EPCs differentiation was detected by flow cytometry, and Rho kinase activity was detected by immunohistochemistry.

Results  The expression of autophagy marker protein LC3Ⅱ/Ⅰ, Beclin1, Rho kinase protein, the positive expression rate of CD34 in fibrinogen and EPCs of Shengjiyuhong ointment group were decreased, and the positive expression rate of serum prothrombin time, international normalized ratio, von Willebrand factor in EPCs and the expression of P62 in vascular tissue were increased (P<0.05). The in vitro experiment showed that Shengjiyuhong ointment could reduce the expression of LC3Ⅱ/Ⅰ and Beclin1, and increase the expression of P62 protein in injured endothelial cells (P<0.05).

Conclusion  Shengjiyuhong ointment can effectively improve the coagulation function, promote the differentiation of EPCs and reduce the activity of Rho kinase in rats with thromboangⅡtis obliterans. The mechanism may be related to inhibition of autophagy.

1.黄惠红, 王群花, 方芳, 等. 固肠丸治疗脾肾阳虚型溃疡性结肠炎临床观察[J]. 四川中医, 2023, 41(1): 111-114. [Huang HH, Wang QH, Fang F. Clinical observation of using guchang pill in the treatment of spleen-kidney yang-deficiency type ulcerative colitis[J]. Journal of Sichuan Traditional Chinese Medicine, 2023, 41(1): 111-114.] https://www.nstl.gov.cn/paper_detail.html?id=0fdc69f83f164d938423c3078be7f2ee.

2.Bergère G, Toquet C, Hoffmann C, et al. Effect of cannabis consumption on characteristics and evolution of thromboangitis obliterans[J]. Vasa, 2021, 50(4):301-305. DOI: 10.1024/0301-1526/a000948.

3.田野, 李新喜, 张磊, 等. 前列地尔干乳剂联合低分子肝素注射液和地塞米松注射液治疗血栓闭塞性脉管炎患者的临床研究[J].中国临床药理学杂志, 2021, 37(12): 1494-1497. [Tian Y, Li XX, Zhang L, et al. Clinical trial of alprostadil dried emulsion combined with low molecular weight heparin injection and dexamethasone injection in the treatment of patients with thromboangiitis obliterans[J]. The Chinese Journal of Clinical Pharmacology, 2021, 37(12): 1494-1497.] DOI: 10.13699/j.cnki.1001-6821.2021.12.004.

4.吴双. FoxO1对慢性血栓栓塞性肺动脉高压大鼠肺动脉内皮自噬功能的影响[D]. 福州: 福建医科大学, 2017. DOI: 10.7666/d.D01305660.

5.Rakkar K, Othman O, Sprigg N, et al. Endothelial progenitor cells, potential biomarkers for diagnosis and prognosis of ischemic stroke: protocol for an observational case-control study[J]. Neural Regen Res, 2020, 15(7): 1300-1307. DOI: 10.4103/1673-5374.269028.

6.Jiang C, Dong N, Feng J, et al. MiRNA-190 exerts neuroprotective effects against ischemic stroke through Rho/Rho-kinase pathway[J]. Pflugers Arch, 2021, 473(1): 121-130. DOI: 10.1007/s00424-020-02490-2.

7.张玉红, 虞哲波. 环状混合痔内服方联合生肌玉红膏外用促环状混合痔术后创面愈合作用研究[J].新中医, 2018, 50(9): 161-164. [Zhang YH, Yu ZB. Oral Prescription of circumferential mixed hemorrhoids combined with external application of shengji yuhong ointment has effect in promoting postoperative wound healing of circumferential mixed hemorrhoids[J]. Journal of New Chinese Medicine, 2018, 50(9):161-164.] DOI: 10.13457/j.cnki.jncm.2018.09.046.

8.杜伟鹏, 张现峰, 马立人. 生肌玉红膏联合制象皮治疗下肢静脉溃疡临床观察[J]. 实用中医药杂志, 2021, 37(1): 132-133. [Du WP, Zhang XF, Ma LR. Clinical observation on Shengji Yuhong ointment combined with processed elephant skin in the treatment of lower extremity venous ulcers[J]. Journal of Practical Traditional Chinese Medicine, 2021, 37(1): 132-133.] https://www.nstl.gov.cn/paper_detail.html?id=b72e280674c0dae630b1abe6aea15d2c.

9.刘静, 田硕, 苗明三. 三参草精油对大鼠血栓闭塞性脉管炎模型的影响[J]. 中国实验方剂学杂志, 2019, 25(4): 18-22. [Liu J, Tian S, Miao MS. Effect of Sanshencao essential oil on rat model of thromboangiitis obliterans[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2019, 25(4): 18-22.] DOI: 10.13422/j.cnki.syfjx.20190438.

10.王亚娜, 许晓琳, 马文华, 等. 芍药苷调节PI3K/AKT/NF-κB信号通路对血栓闭塞性脉管炎大鼠的治疗作用研究[J]. 中国临床药理学杂志, 2024, 40(6): 854-858. [Wang YN, Xu XL, MA WH, et al. Therapeutic effects of paeoniflorin on thromboangiitis obliterans model rats by regulating PI3K/AKT/NF-κB signaling pathway[J]. The Chinese Journal of Clinical Pharmacology, 2024, 40(6): 854-858.] DOI: 10.13699/j.cnki.1001-6821.2024.06.015.

11.王亚娜, 宋晓聪, 吴静, 等. 生肌玉红膏对血栓性脉管炎大鼠血管平滑肌细胞凋亡、炎性因子表达的影响及机制[J]. 山东医药, 2022, 62(34): 36-40. [Wang YN, Song XC, Wu J, et al.Effects of Shengji Yuhong ointment on apoptosis of vascular smooth muscle cells and expression of inflammatory factors in rats with thromboangiitis[J]. Shandong Medical Journal, 2022, 62(34): 36-40.] DOI: 10.3969/j.issn.1002-266X.2022.34.008.

12.Mungchan P, Glab-Ampai K, Chruewkamlow N, et al. Targeted nanoparticles for the binding of injured vascular endothelium after percutaneous coronary intervention[J]. Molecules, 2022, 27(23): 8144. DOI: 10.3390/molecules 27238144.

13.冯泽宇, 许岩磊, 张媞, 等. 生肌玉红膏提取液促进脂肪干细胞增殖的实验研究[J]. 中华中医药学刊, 2017, 35(2): 355-358,517. [Feng ZY, XU YL, Zhang T, et al. Experiment researches on proliferation of adipose stem cells with extracting solution of Shenji Yuhong Gao[J]. Chinese Archives of Traditional Chinese Medicine, 2017, 35(2): 355-358, 517.] DOI: 10.13193/j.issn. 1673-7717.2017.02.027.

14.Le Joncour A, Espitia O, Soudet S, et al. Women and pregnancy in thromboangiitis obliterans[J]. J Vasc Res, 2021, 58(2): 131-133. DOI: 10.1159/000512596.

15.张侠陵, 刘一, 苏杨, 等. 雷公藤多苷联合贝前列素钠治疗血栓闭塞性脉管炎[J]. 西部医学, 2022, 34(2): 284-288. [Zhang XL, Liu Y, Su Y, et al. Effects of tripterygium wilfordii polyglycosides combined with beraprost sodium in the treatment of patients withthromboangiitis obliterans on clinical efficacy, VAS and inflammatory factors[J]. Medical Journal of West China, 2022, 34(2): 284-288.] DOI: 10. 3969/j.issn.1672-3511.2022.02.026.

16.邓蕴源, 林晓光, 江湧, 等. 陈渭良教授论治血栓闭塞性脉管炎临床经验[J]. 四川中医, 2020, 38(10): 14-16. [Deng YY, Lin XG, Jiang Y, et al. Professor chen weiliang's clinical experience in treating thromboangⅡtis obliterans  [J]. Sichuan Traditional Chinese Medicine, 2020, 38(10): 14-16.] https://www.nstl.gov.cn/paper_detail.html?id=d1f6fafc6df9ebb48f6fbcbf59958cb8.

17.肖利虎, 林创辉, 欧秀华. 七叶皂苷钠联合前列地尔治疗糖尿病足的疗效及对血清IGF-1、VEGF和bFGF水平的影响[J]. 临床合理用药杂志, 2022, 15(22): 89-92. [Xiao LH, Lin CH, Ou XH. Efficacy of sodium aescinate combined with alprostadil in the treatment of diabetic foot and its effect on serum IGF-1, VEGF and bFGF levels[J]. Journal of Clinical Rational Drug Use, 2022, 15(22): 89-92.] DOI: 10.15887/j.cnki.13-1389/r.2022.22.027.

18.杨乾舸, 覃禹铭, 李武志. 左布比卡因对子宫内膜癌细胞RL-952生长和线粒体膜电位的影响[J]. 中国临床药理学杂志, 2023, 39(12): 1723-1727. [Yang GG, Qin YM, Li WZ. Effects of levobupivacaine on the growth and mitochondrial membrane potential of endometrial cancer cells RL-952[J]. The Chinese Journal of Clinical Pharmacology, 2023, 39(12): 1723-1727.] DOI: 10.13699/j.cnki.1001-6821.2023.12.011.

19.郑明慧, 白建强. 基于LKB1/AMPK信号通路探究金雀异黄酮对癫痫大鼠海马神经元凋亡和自噬的影响 [J]. 中国药师, 2022, 25(11): 1904-1910. [Zheng MH, Bai JQ. Impacts of genistein on the apoptosis and autophagy of hippocampal neurons in epileptic rats based on LKB1/AMPK signaling pathway[J]. China Pharmacist, 2022, 25(11): 1904-1910.] DOI: 10.19962/j.cnki.issn1008-049X.2022.11.006.

20.郭晓黎, 任小宁, 樊一波, 等. 沙参麦冬汤通过调节EGFR/MEK/ERK信号通路对非小细胞肺癌细胞增殖、凋亡和自噬的影响[J]. 中国药师, 2022, 25(10): 1707-1713. [Guo XL, Ren XM, Fan YB, et al. Impacts of radix ophiopogon decoction on the proliferation,apoptosis and autophagy of non-small cell lung cancer cells by regulating EGFR/MEK/ERK signaling pathway[J]. China Pharmacist, 2022, 25 (10):1707-1713.] DOI: 10.19962/j.carol carroll nki issn1008-049-x.2022.10.003.

21.史睿, 邵亚新, 田小雨, 等. PINK1/Parkin信号通路介导的线粒体自噬在重症肺炎中的作用研究[J]. 华西医学, 2024, 39(4): 567-572. [Shi R, Shao YX, Tian XY, et al. Role of mitochondrial autophagy mediated by PINK1/Parkin signaling pathway in severe pneumonia[J]. West China Medical Journal, 2024, 39(4): 567-572.] DOI: 10.7507/1002-0179.202308104.

22.Piffoux M, Eriau E, Cassier PA. Autophagy as a therapeutic target in pancreatic cancer[J]. Br J Cancer, 2021, 124(2): 333-344. DOI: 10.1038/s41416-020-01039-5.

23.周霞庆, 王灵聪. 血小板自噬在深静脉血栓形成中的研究进展[J]. 浙江临床医学, 2019, 21(12): 1734-1735,1738. [Zhou XQ, Wang LC. Research progress of platelet autophagy in deep vein thrombosis[J]. Zhejiang Clinical Medical Journal, 2019, 21(12): 1734-1735, 1738.]https://xueshu.baidu.com/usercenter/paper/show?paperid=1p7n0mf03u660ms09k4s0810p0487012&site=xueshu_se&hitarticle=1.

24.秦亚鹏, 赵玉群. 生肌玉红膏促进血栓闭塞性脉管炎(脱疽)患者伤口愈合的临床疗效[J].血管与腔内血管外科杂志, 2019, 5(6): 509-512. [Qin YP, Zhao YQ. Clinical effect of Shengjiyuhong ointment on wound healing in patients with thromboangiitis obliterans[J]. Journal of Vascular and Endovascular Surgery, 2019, 5(6): 509-512.] DOI: 10.19418/j.cnki.issn2096-0646.2019.06.009.

25.Chopra H, Han Y, Zhang C, et al. CD133(+)CD34(+) cells can give rise to EPCs: a comparative rabbit and human study[J]. Blood Cells Mol Dis, 2021, 86: 102487. DOI: 10.1016/j.bcmd.2020.102487.

26.Matsuda J, Asano-Matsuda K, Kitzler TM, et al. Rho GTPase regulatory proteins in podocytes[J]. Kidney Int, 2021, 99(2): 336-345. DOI: 10.1016/j.kint.2020.08.035.

27.张丽美, 王国强, 陈世娇, 等. RhoA/ROCK信号通路与心血管系统疾病关系的研究进展[J]. 医学研究与教育, 2021, 38(2): 8-13. [Zhang LM, Wang GQ, Chen SJ, et al. Advances on the relationship between RhoA/ ROCK signaling pathway and cardiovascular diseases[J]. Medical Research and Education, 2021, 38(2): 8-13.] DOI: 10.3969/j.issn.1674-490X.2021.02.002.