Wang La, Jiang GuiTong, Wang WenJia, Ke ZunLi, Luo RuiXi, Tian WeiYi
Department of Immunology and Microbiology, School of Basic Medical Sciences, Guizhou University of Traditional Chinese Medicine, No. 4 Dongqing Road, Huaxi, Guiyang, 550025, Guizhou, PR China.
Integrated Traditional Chinese and Western Medicine Department, Cengong County People's Hospital, Kaili, Guizhou, 557801, PR China.
Heliyon. 2024 May 23;10(11):e31607. doi: 10.1016/j.heliyon.2024.e31607. eCollection 2024 Jun 15.
The aim of this study was to investigate the protective effect of HLJDD on septic rats and the underlying mechanisms.
Adult male Sprague-Dawley (SD) adult rats (150-180 g) were randomly divided into the following 5 groups (n = 7 per group): the Sham group, caecal ligation and puncture (CLP) group, HLJDD + CLP (Huang Lian Jie Du Decoction, HLJDD) group (1 g/mL/100 g), HLJDD + Rap + CLP (H. Rap) group (Rap: 3 mg/kg), and HLJDD+3-MA + CLP (H. 3-MA) group (3-MA: 30 mg/kg). Rapamycin (Rap) and 3-methyladenosine (3-MA) were used to activate and inhibit autophagy, respectively. HLJDD was purchased from Beijing Tong Ren Tang Guiyang Branch and verified by experts as a genuine product. We used CLP to establish an animal model of sepsis in the last four groups. Survival was analysed by the Kaplan‒Meier method. Then, we examined autophagy-related genes () and proteins using real-time PCR and Western blotting, respectively. The microstructure of the ileum and the number of autophagosomes were observed by transmission electron microscopy (TEM). Analyses of HE-stained pathological ileum and inflammatory factor levels were examined to assess the extent of septic injury. The effect of HLJDD on the gut microbiota was analysed by 16S rRNA gene sequencing of faeces.
In this study, we identified the protective effects of HLJDD on mortality and inflammation in septic rats. Several key proteins, including LC3-II, Beclin-1 and p62, were examined and showed that HLJDD could effectively reverse the sepsis-induced decrease in autophagy. TEM was performed and the expression of s was assessed to evaluate fluctuations in autophagy. Then, we examined the intestinal tight junction protein zona occludens (ZO-1), lipopolysaccharide (LPS) and inflammatory factors, and found that HLJDD effectively alleviated the increase in gene expression, the level of LPS and serum level of inflammatory factors caused by sepsis. These results were consistent with those obtained from pathological sectioning and TEM analysis. Moreover, autophagy activation effectively ameliorated sepsis, and autophagy inhibition exacerbated the systemic symptoms caused by infection. By examining the expression of key proteins upstream of the autophagy pathway, we found that HLJDD inhibited mTOR via the MAPK/PI3K signalling pathway to promote autophagy in septic rats. 16S rRNA sequencing revealed that HLJDD significantly affected the diversity and physiological function of the gut microbiota in septic rats.
The results of this study indicate that autophagy activation is a potential mechanism underlying the protective effect of HLJDD on the intestine in septic rats.
本研究旨在探讨黄连解毒汤（HLJDD）对脓毒症大鼠的保护作用及其潜在机制。
将成年雄性Sprague-Dawley（SD）大鼠（150 - 180 g）随机分为以下5组（每组n = 7）：假手术组、盲肠结扎穿孔（CLP）组、HLJDD + CLP（黄连解毒汤，HLJDD）组（1 g/mL/100 g）、HLJDD + Rap + CLP（H. Rap）组（Rap：3 mg/kg）和HLJDD + 3-MA + CLP（H. 3-MA）组（3-MA：30 mg/kg）。雷帕霉素（Rap）和3-甲基腺嘌呤（3-MA）分别用于激活和抑制自噬。HLJDD购自北京同仁堂贵阳分店，经专家鉴定为正品。我们用CLP法在最后四组建立脓毒症动物模型。采用Kaplan-Meier法分析生存率。然后，分别用实时PCR和蛋白质免疫印迹法检测自噬相关基因和蛋白质。通过透射电子显微镜（TEM）观察回肠的微观结构和自噬体数量。对HE染色的回肠病理切片和炎症因子水平进行分析，以评估脓毒症损伤程度。通过粪便16S rRNA基因测序分析HLJDD对肠道微生物群的影响。
在本研究中，我们确定了HLJDD对脓毒症大鼠死亡率和炎症的保护作用。检测了包括LC3-II、Beclin-1和p62在内的几种关键蛋白，结果表明HLJDD可有效逆转脓毒症诱导的自噬减少。进行TEM观察并评估自噬相关蛋白的表达以评价自噬的波动情况。然后，我们检测了肠道紧密连接蛋白闭合蛋白（ZO-1）、脂多糖（LPS）和炎症因子，发现HLJDD有效减轻了脓毒症引起的相关基因表达增加、LPS水平升高以及炎症因子血清水平升高。这些结果与病理切片和TEM分析结果一致。此外，自噬激活有效改善了脓毒症，而自噬抑制则加剧了感染引起的全身症状。通过检测自噬途径上游关键蛋白的表达，我们发现HLJDD通过MAPK/PI3K信号通路抑制mTOR以促进脓毒症大鼠的自噬。16S rRNA测序显示HLJDD显著影响脓毒症大鼠肠道微生物群的多样性和生理功能。
本研究结果表明，自噬激活是HLJDD对脓毒症大鼠肠道保护作用的潜在机制。
