| 王喆,周昱彤,周梦璇,王顺富,郭冬雅,张娃,金文璋.胡椒碱通过AMPK/mTOR通路调控自噬促进炎性创面愈合的机制[J].浙江中西医结合杂志,2026,36(7): |
| 胡椒碱通过AMPK/mTOR通路调控自噬促进炎性创面愈合的机制 |
| Mechanism of piperine in promoting the healing of inflammatory wounds by regulating autophagy through the AMPK/mTOR pathway |
| 投稿时间：2025-10-25 修订日期：2026-06-09 |
| DOI： |
| 中文关键词: 胡椒碱 AMPK/mTOR信号通路 自噬 炎症创面愈合 |
| 英文关键词:Piperine AMPK/mTOR pathway autophagy healing of inflammatory wounds |
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| 中文摘要: |
| 目的 探究胡椒碱对炎性创面的愈合作用并探究其可能的作用机制。方法 通过胡椒碱浓度梯度的细胞毒性与抗炎试验确定其实验浓度。然后，将24只C57BL/6小鼠随机分为对照组、模型组、实验组和抑制组。模型组、实验组和抑制组利用脂多糖（LPS）构建炎症创面模型，对照组构建普通创面模型作为空白对照。实验组和抑制组小鼠创面滴加50 μmol/L的胡椒碱混悬液，对照组和模型组创面滴加等量的生理盐水，此外抑制组小鼠通过尾静脉注射10 mg/kg单磷酸腺苷活化蛋白激酶（AMPK）抑制剂化合物C（Compound C）。通过记录创面愈合率，苏木精-伊红（HE）和马松（Masson）染色来评价创面的愈合情况。利用酶联免疫吸附测定（ELISA）法检测肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和超氧化物歧化酶（SOD）、丙二醛（MDA）。采用免疫印迹法（Western blot, WB）检测创面组织的自噬调控通路相关蛋白AMPK、磷酸化腺苷酸活化激酶（pAMPK）、雷帕霉素靶蛋白（mTOR）、磷酸化雷帕霉素靶蛋白（pmTOR）、UNC-51样激酶1（ULK1）、磷酸化UNC-51样激酶1（pULK1）及自噬相关蛋白微管相关蛋白轻链3（LC3）、选择性自噬接头蛋白P62（p62）在创面组织中的表达水平。利用免疫荧光染色实验检测创面组织中LC3蛋白的表达水平。结果 对照组作为普通创面模型，其愈合情况最佳。相比之下，模型组由于构建了炎症创面模型，创面愈合明显延迟，炎症浸润明显，胶原沉积少，炎症因子（TNF-α、IL-1β、IL-6）与氧化应激相关因子（SOD、MDA）表达明显升高，AMPK/mTOR通路被抑制，自噬水平降低。与模型组相比，实验组小鼠创面愈合率[（67.86±2.48）％比（49.89±6.47）％，P＜0.05]增大，炎症浸润面积[（14.53±1.57）％比（28.50±3.34）％，P＜0.01]减小，胶原沉积面积[（72.98±2.66）％比（57.79±3.16）％，P＜0.01）]增大，TNF-α[（113.51±12.55）pg/mL比（202.85±9.04）pg/mL，P＜0.01]、IL-1β[（79.96±4.61）pg/mL比（121.13±13.40）pg/mL，P＜0.01]、IL-6[（58.94±2.71）pg/mL比（85.17±7.62）pg/mL，P＜0.01]下降，SOD[（4.33±0.42）U/mg比（1.90±0.22）U/mg，P＜0.01]升高，MDA[（5.89±0.57）nmol/mg比（9.16±0.64）nmol/mg，P＜0.01]下降，pAMPK/AMPK[（1.073±0.078）比（0.671±0.057），P＜0.01]、pULK1/ULK1[（0.874±0.078）比（0.592±0.010），P＜0.01]升高，pmTOR/mTOR[（0.562±0.015）比（0.869±0.043），P＜0.01]、p62[（0.597±0.019）比（0.913±0.029），P＜0.01]降低，LC3-Ⅱ/LC3-Ⅰ[（1.674±0.078）比（1.471±0.037），P＜0.01]、LC3荧光强度[（17.47±0.59）比（11.38±1.24），P＜0.01]升高。与实验组相比，抑制组小鼠创面愈合率[（54.08±2.96）％比（67.86±2.48）％，P＜0.05]减小，炎症浸润面积[（20.82±1.52）％比（14.53±1.57）％，P＜0.05]增大，胶原沉积面积[（65.87±1.57）％比（72.98±2.66）％，P＜0.05）]减小，TNF-α[（157.21±6.72）pg/mL比（113.51±12.55）pg/mL，P＜0.05]、IL-1β[（110.96±9.48）pg/mL比（79.96±4.61）pg/mL，P＜0.05]、IL-6[（72.27±3.20）pg/mL比（58.94±2.71）pg/mL，P＜0.05]升高，SOD[（2.30±0.52）U/mg比（4.33±0.42）U/mg，P＜0.01]降低，MDA[（7.62±0.44）nmol/mg比（5.89±0.57）nmol/mg，P＜0.05]上升，pAMPK/AMPK[（0.593±0.018）比（1.073±0.078），P＜0.05]、pULK1/ULK1[（0.719±0.038）比（0.874±0.078），P＜0.05]下降，pmTOR/mTOR[（0.747±0.031）比（0.562±0.015），P＜0.05]、p62[（0.801±0.016）比（0.597±0.019），P＜0.01]升高，LC3-Ⅱ/LC3-Ⅰ[（1.017±0.099）比（1.674±0.078），P＜0.01]、LC3荧光强度[（10.45±0.66）比（17.47±0.59），P＜0.01]下降。结论 胡椒碱可能通过激活AMPK/mTOR通路，增强自噬，从而减轻炎症进展，加速炎性创面愈合。 |
| 英文摘要: |
| Objective To investigate the healing effect of piperine on inflammatory wounds and explore its potential mechanism of action. Methods The experimental concentration of piperine was determined through cytotoxicity and anti-inflammatory tests using a concentration gradient of piperine. Subsequently, 24 C57BL/6 mice were randomly divided into a control group, a model group, an experimental group, and an inhibition group. The model group, the experimental group and the inhibition group were used to construct inflammatory wound models using LPS, while the control group was used to construct ordinary wound models as a blank control. Wounds in the experimental and inhibition groups were treated with 50 μmol/L piperine suspension, while wounds in the control and model groups were treated with an equal amount of normal saline. Additionally, mice in the inhibition group were administered 10 mg/kg of the AMPK inhibitor Compound C via tail vein injection. Wound healing was evaluated by recording the wound healing rate and performing Hematoxylin-Eosin (HE) and Masson staining. The levels of inflammation-related factors (TNF-α, IL-1β, IL-6) and oxidative stress-related factors (SOD, MDA) in the wound tissue were detected using Enzyme-Linked Immunosorbent Assay (ELISA). The expression levels of autophagy-related pathway proteins AMPK, pAMPK, mTOR, pmTOR, ULK1, pULK1, and autophagy-related proteins LC3 and p62 in the wound tissue were measured using Western blot (WB). The expression of LC3 was further assessed by immunofluorescence staining. Results The control group, serving as the ordinary wound model, exhibited the best healing outcome. In contrast, wound healing in the model group—established as an inflammatory wound model via LPS stimulation—was significantly delayed. This group exhibited pronounced inflammatory infiltration, reduced collagen deposition, and markedly elevated expression levels of inflammatory cytokines (TNF-α, IL-1β, IL-6) and oxidative stress-related factors (SOD, MDA). The AMPK/mTOR pathway is inhibited, and the level of autophagy is decreased.Compared with the model group, the experimental group exhibited a significantly increased wound healing rate [(67.86±2.48)% vs. (49.89±6.47)%, P < 0.05], a reduced inflammatory infiltration area [(14.53 ±1.57)% vs. (28.50±3.34)%, P < 0.01], and an enhanced collagen deposition area [(72.98± 2.66)% vs. (57.79±3.16)%, P < 0.01]. Levels of TNF-α [(113.51±12.55) pg/mL vs. (202.85± 9.04) pg/mL, P < 0.01], IL-1β[(79.96±4.61) pg/mL vs. (121.13±13.40) pg/mL, P < 0.01], and IL-6 [(58.94±2.71) pg/mL vs. (85.17±7.62) pg/mL, P < 0.01] were decreased. SOD activity was elevated [(4.33±0.42) U/mg vs. (1.90±0.22) U/mg, P < 0.01], while MDA content was reduced [(5.89±0.57) nmol/mg vs. (9.16±0.64) nmol/mg, P < 0.01]. The ratios of pAMPK/AMPK [(1.073±0.078) vs. (0.671±0.057), P < 0.01] and pULK1/ULK1 [(0.874± 0.078) vs. (0.592±0.010), P < 0.01] were increased, whereas the ratios of pmTOR/mTOR [(0.562±0.015) vs. (0.869±0.043), P < 0.01] and p62 expression [(0.597±0.019) vs. (0.913± 0.029), P < 0.01] were decreased. Additionally, the LC3-Ⅱ/LC3-Ⅰ ratio [(1.674±0.078) vs. (1.471±0.037), P < 0.01] and LC3 fluorescence intensity [(17.47±0.59) vs. (11.38±1.24), P < 0.01] were elevated.In comparison with the experimental group, the inhibition group showed a decreased wound healing rate [(54.08±2.96)% vs. (67.86±2.48)%, P < 0.05], an increased inflammatory infiltration area [(20.82±1.52)% vs. (14.53±1.57)%, P < 0.05], and a reduced collagen deposition area [(65.87±1.57)% vs. (72.98±2.66)%, P < 0.05]. Levels of TNF-α [(157.21±6.72) pg/mL vs. (113.51±12.55) pg/mL, P < 0.05], IL-1β [(110.96±9.48) pg/mL vs. (79.96±4.61) pg/mL, P < 0.05], and IL-6 [(72.27±3.20) pg/mL vs. (58.94±2.71) pg/mL, P < 0.05] were elevated. SOD activity was lower [(2.30±0.52) U/mg vs. (4.33±0.42) U/mg, P < 0.01], while MDA content was higher [(7.62±0.44) nmol/mg vs. (5.89±0.57) nmol/mg, P < 0.05]. The ratios of pAMPK/AMPK [(0.593±0.018) vs. (1.073±0.078), P < 0.05] and pULK1/ULK1 [(0.719±0.038) vs. (0.874±0.078), P < 0.05] were decreased, whereas the ratios of pmTOR/mTOR [(0.747±0.031) vs. (0.562±0.015), P < 0.05] and p62 expression [(0.801± 0.016) vs. (0.597±0.019), P < 0.01] were increased. Moreover, the LC3-Ⅱ/LC3-Ⅰ ratio [(1.017 ±0.099) vs. (1.674±0.078), P < 0.01] and LC3 fluorescence intensity [(10.45±0.66) vs. (17.47 ±0.59), P < 0.01] were reduced.. Conclusion Piperine may promote the healing of inflammatory wounds by activating the AMPK/mTOR pathway, enhancing autophagy, and thereby mitigating inflammatory progression. |
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