医学领域的持续探索是守护人类健康的核心动力，肝纤维化、非酒精性脂肪肝病、子宫腺肌病、应激相关血脑屏障损伤等重大疾病的发病机制解析与靶向干预，始终是临床与基础研究的重点方向。随着分子生物学与信号通路调控技术的不断突破，从关键蛋白、microRNA 到长链非编码 RNA 的靶向调控研究，正为疾病防治提供全新的科学依据与技术支撑。
本期精选 5 篇双语研究成果，聚焦核心分子与信号通路的调控作用：深入探索 Yes 相关蛋白（YAP）通过上皮间质转化调控肝纤维化的机制、miR-21 在子宫腺肌病中的致病作用及雌激素受体的调控网络、Rho/ROCK 通路介导束缚应激诱导的血脑屏障损伤机制，同时呈现 NDUFA13 蛋白过表达抑制 NLRP3 活化、lncRNA HEM2M 调控 NLRC4 炎症小体的抗肝损伤新策略。这些研究为重大疾病的病理机制研究开辟了新视角，为靶向药物研发提供了重要实验基础，邀您一同解锁医学分子调控的前沿奥秘。
01
抑制Yes相关蛋白通过抑制上皮间质转化减轻CCl4诱导的小鼠肝纤维化
Inhibiting Yes-associated protein alleviates CCl4liver fibrosis in mice by reducing epithelial mesenchymal transition
【摘要】目的 探索Yes相关蛋白（YAP）可否通过调控上皮间质转化影响肝纤维化的发生发展。方法 将8周龄C57BL/6小鼠18只随机分为对照组、肝纤维化模型组和YAP抑制剂维替泊芬干预组，6只/组。肝纤维化模型采用四氯化碳（CCl4）溶液腹腔注射8周造模；维替泊芬干预组在CCl4基础上第7～9周采用维替泊芬腹腔注射干预。HE染色、Masson染色、肝脏生化学检测观察小鼠肝脏纤维化程度；转录组、蛋白组学测序及联合生信分析探明肝纤维化过程中上皮间质转化通路是否受YAP调控；免疫组化染色和Western blotting检测YAP及上皮间质转化关键基因E-cadherin、N-cadherin、Twist等表达变化。采集健康体检、慢性乙型肝炎、乙肝肝硬化患者血清各60例，酶联免疫吸附法检测其中YAP、N-cadherin、Vimentin、Twist血清表达水平。C57BL/6小鼠24只随机分为对照组、肝纤维化模型组、Twist抑制剂干预组和Twist抑制剂与YAP激动剂共同干预组，6只/组。HE染色、Masson染色、网状纤维染色观察小鼠肝脏纤维化程度，Western blotting检测各组α-SMA、YAP和Twist表达变化。结果 小鼠肝组织病理学结果提示肝纤维化小鼠与对照组相比肝小叶结构破坏、假小叶形成，维替泊芬干预组纤维间隔变性，部分小叶结构恢复。随肝纤维化发生，血浆ALT、AST水平显著升高（P<0.01），维替泊芬干预组肝功能改善（P<0.01）。采用肝组织转录组、蛋白组测序及联合分析找到在肝纤维化形成和维替泊芬干预过程中同时在mRNA和蛋白水平差异表达的基因，发现了N-cadherin和Twist在3组间差异具有统计学意义（P<0.05），并进行PPI分析显示YAP与E-cadherin、N-cadherin存在关联。免疫组化和Western blotting结果提示N-cadherin、Twist、Vimentin随肝纤维化形成升高，E-cadherin在肝纤维化组织中表达下降（P<0.01）。抑制YAP可下调肝组织N-cadherin、Twist蛋白表达（P<0.01）。慢性乙型肝炎患者血清YAP、N-cadherin、Vimentin和Twist水平均随肝炎及肝硬化发生升高，在APRI>0.5或FIB-4>1.45患者中显著升高（P<0.01）。血清YAP在健康对照、肝炎、肝硬化患者中平均水平分别为4.09、5.69和5.36 ng/mL(P<0.01），其与N-cadherin、Vimentin、Twist水平均呈显著正相关，相关系数分别为0.626、0.435、0.526。采用Harmine抑制小鼠肝组织Twist表达，并在Harmine基础上予以YAP激动剂XMU-MP-1干预，肝组织病理学结果提示抑制Twist使肝纤维化小鼠肝组织炎症及纤维化程度减轻，同时激活YAP表达可再次加重胶原纤维沉积。Western blotting检测结果提示Harmine下调肝纤维化小鼠肝组织中α-SMA、YAP及Twist蛋白表达，同时激活YAP使肝组织α-SMA和YAP表达升高（P=0.079,P<0.05）。结论 上皮间质转化是肝纤维化发生的重要机制之一，抑制YAP可通过减少上皮间质转化发生减轻肝纤维化。
【Abstract】ObjectiveTo explore whether Yes-associated protein (YAP) affects occurrence and progression of liver fibrosis by regulating epithelial-mesenchymal transition (EMT).MethodsIn a 8-week-old C57BL/6 mouse model of CCl4-induced liver fibrosis, the effect of verteporfin (a YAP inhibitor) intervention was assessed with HE staining and by detecting liver biochemistry and expressions of YAP and EMT-related genes using immunohistochemistry and Western blotting. Transcriptome and proteomic sequencing and informatics analysis were used to investigate the main downstream pathways of YAP in liver fibrosis. Serum levels of YAP, N-cadherin, vimentin and Twist were examined in 60 healthy individuals, 60 patients with chronic hepatitis B (CHB), and 60 patients with HBV-related liver cirrhosis. In another 24 C57BL/6 mice, the effects of Twist inhibitor alone or in combination with harmine (a YAP activator) on CCl4-induced liver fibrosis were evaluated by histopathological examination and Western blotting.ResultsThe mouse models of liver fibrosis showed obvious structural damages of the liver lobes with formation of pseudolobules, and verteporfin treatment significantly improved these pathologies and lowered plasma ALT and AST levels of the mice. Transcriptome and proteomic sequencing and informatics analysis suggested that N-cadherin and Twist were differentially expressed in liver fibrosis in close correlation with YAP. Inhibition of YAP obviously downregulated hepatic N-cadherin and Twist protein expressions in the mice with liver fibrosis. In patients with CHB and liver cirrhosis, serum levels of YAP elevated obviously with the severity of liver fibrosis and were significantly correlated with N-cadherin, vimentin and Twist levels. In mice with liver fibrosis, inhibiting Twist effectively improved liver inflammation and fibrosis, while the combined treatment with YAP activator worsened hepatic collagen fiber deposition and increased hepatic YAP and α-SMA expressions.ConclusionEMT is an important pathogenic mechanism of liver fibrosis, and inhibiting YAP can alleviate liver fibrosis by reducing EMT.
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02
子宫腺肌病组织中的雌激素、雌激素受体、miR-21：致病作用和调控作用
Estrogen, estrogen receptor and miR-21 in adenomyosis: their pathogenic roles and regulatory interactions
【摘要】目的 探讨miR-21、雌激素（E2）及其受体（ER）在子宫腺肌病发病中的具体机制。方法 采用qRT-PCR检测miR-21在子宫腺肌病组织中水平，以宫颈病变为对照组。体外原代培养子宫腺肌病细胞，qRT-PCR检测E2激活组、ICI82780(ER抑制剂)+E2激活组、E2剥夺组、E2剥夺+ER抑制组及对照组各组miR-21水平。miR-21 mimic、miR-21 inhibitor转染子宫腺肌病细胞，分别过表达和干扰miR-21，从正反两方面验证miR-21对子宫腺肌病细胞增殖、凋亡、迁移及细胞自噬等超微结构的影响。结果miR-21在子宫腺肌病病灶组织中的表达水平明显高于正常子宫肌层中表达水平（P<0.05），与其余各组的差异无统计学意义（P>0.05）;E2激活状态下，E2激活组miR-21表达水平较ER抑制+E2激活组及对照组升高（P<0.05）;E2剥夺状态下，E2剥夺+ER抑制组miR-21表达水平较E2剥夺组及对照组降低（P<0.05）；干扰miR-21可以抑制子宫腺肌病细胞增殖和迁移，使线粒体内质网不同程度扩张、溶酶体增多、自噬小体出现，促进细胞的凋亡；而过表达miR-21则发挥相反的作用。结论 MiR-21在子宫腺肌病细胞中通过改变细胞超微结构而发挥促增殖、迁移及抗凋亡的作用，可能与疾病的早期形成有关；ER除与E2结合调控miR-21外还可通过其他途径调控miR-21，这可能是子宫腺肌病的发病机制之一，其中ER对miR-21的调控作用大于E2。
【Abstract】ObjectiveTo explore the pathogenic roles of miR-21, estrogen (E2), and estrogen receptor (ER) in adenomyosis.MethodsWe examined the expression levels of miR-21 in specimens of adenomyotic tissue and benign cervical lesions using qRT-PCR. In primary cultures of cells isolated from the adenomyosis lesions, the effect of ICI82780 (an ER inhibitor) on miR-21 expression levels prior to E2 activation or after E2 deprivation were examined with qRT-PCR. We further assessed the effects of a miR-21 mimic or an inhibitor on proliferation, apoptosis, migration and autophagy of the cells.ResultsThe expression level of miR-21 was significantly higher in adenomyosis tissues than in normal myometrium (P<0.05). In the cells isolated from adenomyosis lesions, miR-21 expression level was significantly higher in E2 activation group than in ER inhibition + E2 activation group and the control group (P<0.05); miR-21 expression level was significantly lower in cells in E2 deprivation+ER inhibition group than in E2 deprivation group and the control group (P<0.05). The adenomyosis cells transfected with miR-21 inhibitor showed inhibited proliferation and migration, expansion of mitochondrial endoplasmic reticulum, increased lysosomes, presence of autophagosomes, and increased cell apoptosis, while transfection of the cells with the miR-21 mimic produced the opposite effects.ConclusionMiR-21 plays an important role in promoting proliferation, migration, and antiapoptosis in adenomyosis cells by altering the cell ultrastructure, which may contribute to early pathogenesis of the disease. In addition to binding with E2, ER can also regulate miR-21 through other pathways to participate in the pathogenesis of adenomyosis, thus having a stronger regulatory effect on miR-21 than E2.
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03
束缚应激通过激活Rho/ROCK通路诱导大鼠杏仁核血脑屏障的损伤
Restraint stress induces blood-brain barrier injury in rat amygdala by activating the Rho/ROCK signaling pathway
【摘要】目的 探讨Rho/ROCK信号通路在束缚应激诱导大鼠杏仁核血脑屏障损伤过程中的作用及机制。方法 选取60只SPF级雄性SD大鼠建立束缚应激模型，将大鼠分为4组：Control组（n=15，每日禁食水6 h）;Stress组（n=15，每日束缚6 h）;Stress+Fasudil组（n=15，每日束缚6 h，束缚前0.5 h给予腹腔注射1 mg/100 g Fasudil溶液）；Fasudil组（n=15，每日给予腹腔注射1 mg/100 g Fasudil溶液）。用高架十字迷宫实验（EPM）检测各组大鼠行为学变化，ELISA检测血清CORT和S100B水平，检测脑组织伊文思蓝（EB）的渗漏情况以评估渗透性的改变，免疫荧光法和Western blotting方法检测紧密连接蛋白Claudin-5、Occludin、ZO-1的表达变化，Pull-down实验和Western blotting检测Rho/ROCK通路的激活情况，透射电镜观察脑微血管内皮细胞超微结构的形态变化。结果 与Control组相比，Stress组和Stress+Fasudil组大鼠表现出了明显的焦虑样行为；Stress组和Stress+Fasudil组血清CORT含量均显著高于Control组（P<0.001）；与Control组和Stress+Fasudil组相比，Stress组EB渗漏含量和S100B含量均显著升高（P<0.05）;Stress组紧密连接蛋白的表达显著低于Control组和Stress+Fasudil组（P<0.05）;Pull-down实验和Western blot分析证实，Stress组RhoA-GTP(P<0.001)、ROCK2(P<0.001)、p-MLC2(P<0.05)的表达显著高于Control组和Stress+Fasudil组；脑微血管内皮细胞超微结构显示，Stress组呈现显著的形态学改变。结论 束缚应激能够通过激活Rho/ROCK信号通路诱导大鼠杏仁核血脑屏障的损伤。
【Abstract】ObjectiveTo investigate the role of Rho/ROCK signaling pathway in mediating restraint stress-induced blood-brain barrier (BBB) injury in the amygdala of rats.MethodsSixty male SD rats were randomized equally into control group (with food and water deprivation for 6 h per day), restraint stress group (with restraint for 6 h per day), stress + fasudil treatment (administered by intraperitoneal injection at 1 mg/100 g 30 min before the 6-h restraint) group, and fasudil treatment alone group. The elevated plus-maze test was used to detect behavioral changes of the rats, serum corticosterone and S100B levels were determined with ELISA, and Evans Blue leakage in the brain tissue was examined to evaluate the changes in BBB permeability. The changes in expression levels of tight junction proteins in the amygdala were detected using immunofluorescence assay and Western blotting, and Rho/ROCK pathway activation was detected by Pull-down test and Western blotting. Ultrastructural changes of the cerebral microvascular endothelial cells were observed using transmission electron microscopy.ResultsCompared with those in the control group, the rats in restrain stress group and stress + fasudil group showed obvious anxiety-like behavior with significantly increased serum corticosterone level (P<0.001). Compared with those in the control group and stress+fasudil group, the rat models of restrain stress showed more obvious Evans Blue leakage and higher S100B expression (P<0.01) but lower expressions of tight junction proteins in the amygdala. Pull-down test and Western blotting confirmed that the expression levels of RhoA-GTP, ROCK2 and P-MLC 2 were significantly higher in stress group than in the control group and stress + fasudil group (P<0.05). Transmission electron microscopy revealed obvious ultrastructural changes in the cerebral microvascular endothelial cells in the rat models of restrain stress.ConclusionRestraint stress induces BBB injury in the amygdala of rats by activating the Rho/ROCK signaling pathway.
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04
NDUFA13过表达可减轻CCl4诱导的小鼠肝纤维化：基于抑制NLRP3活化
Adeno-associated virus-mediated hepatocyte-specific NDUFA13 overexpression protects against CCl4-induced liver fibrosis in mice by inhibiting hepatic NLRP3 activation
【摘要】目的 探究NDUFA13蛋白在小鼠急性肝损伤和肝纤维化中的保护作用及可能机制。方法 小鼠分为正常组（n=18）、CCl4组（n=18）、CCl4+AAV-NC组（n=18）、CCl4+AAV-NDU13组（n=18）。采用腹腔注射CCl(42次/周）建立肝纤维化小鼠模型，连续给药3、5、7周后处死取材。病毒干预组小鼠则预先分别尾静脉注射AAV8-TBG-NC对照与AAV8-TBG-NDUFA13过表达病毒，7～10 d后再经CCl4进行肝纤维化诱导。通过HE染色、Masson染色观察CCl4致小鼠肝损伤及肝纤维化情况。Western blotting检测肝组织中NDUFA13、α-SMA蛋白表达水平。免疫荧光分析NDUFA13与炎症小体NLRP3、肿瘤坏死因子TNF-α与白介素IL-1β、肝星型细胞活化标志物α-SMA与胶原纤维CollagenⅢ的表达情况。结果 HE染色、Masson染色结果显示CCl4诱导小鼠肝组织肝小叶结构紊乱，肝细胞变性坏死，炎症细胞浸润且伴大量胶原纤维沉积（P<0.001）。Western blotting结果发现CCl4模型鼠肝脏NDUFA13蛋白表达水平相比正常对照组明显降低（P<0.001）；而NDUFA13蛋白过表达后，CCl4处理小鼠的肝脏炎症细胞聚集和纤维化均明显减少（P<0.001）。免疫荧光结果显示，NDUFA13蛋白过表达的CCl4诱导鼠肝组织中炎症小体NLRP3活化明显减弱（P<0.001），并伴随炎症因子TNF-α与IL-1β分泌显著减少（P<0.001），同时肝星状细胞活化（P<0.05）及其胶原形成均受到抑制（P<0.001）。结论 肝细胞线粒体NDUFA13蛋白过表达可通过抑制NLRP3炎症信号活化发挥抗纤维化作用。
【Abstract】ObjectiveTo investigate the protective effect of NDUFA13 protein against acute liver injury and liver fibrosis in mice and explore the possible mechanisms.MethodsBALB/C mice (7 to 8 weeks old) were divided into normal group, CCl4group, CCl4+AAV-NC group and CCl4+AAV-NDU13 group (n=18). Mouse models of liver fibrosis were established by intraperitoneal injection of CCl4twice a week for 3, 5 or 7 weeks, and the recombinant virus AAV8-TBG-NC or AAV8-TBG-NDUFA13 was injected via the tail vein 7–10 days prior to CCl4injection. After the treatments, pathological changes in the liver of the mice were observed using HE and Masson staining. Hepatic expression levels of NDUFA13 and α-SMA were detected with Western blotting, and the coexpression of NDUFA13 and NLRP3, TNF-α and IL-1β, and α-SMA and collagenⅢ was analyzed with an immunofluorescence assay.ResultsHE and Masson staining showed deranged liver architecture, necrotic hepatocytes and obvious inflammatory infiltration and collagen fiber deposition in mice with CCl4injection (P<0.001). NDUFA13 expression markedly decreased in CCl4-treated mice (P<0.001), while a significant reduction in inflammatory aggregation and fibrosis was observed in mice with AAV-mediated NDUFA13 overexpression (P<0.001). In CCl4+AAV-NDU13 group, immunofluorescence assay revealed markedly weakened activation of NLRP3 inflammasomes (P<0.001), significantly decreased TNF-α and IL-1β secretion (P<0.001), and inhibited hepatic stellate cell activation (P<0.05) and collagen formation in the liver (P<0.001).ConclusionMitochondrial NDUFA13 overexpression in hepatocytes protects against CCl4-induced liver fibrosis in mice by inhibiting activation of NLRP3 signaling.
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05
过表达lncRNA HEM2M改善非酒精性脂肪肝病小鼠的肝脏损伤
Overexpression of lncRNA HEM2M alleviates liver injury in mice with non-alcoholic fatty liver disease
【摘要】目的 探讨过表达长链非编码RNA(lncRNA)HEM2M对非酒精性脂肪肝病（NAFLD）小鼠肝损伤的影响。方法 野生型C57BL/6(WT)和条件性髓系细胞lncRNA HEM2M过表达（MYKI）小鼠分别喂饲普通饮食（ND）和高脂饮食（HFD），即为WT+ND、MYKI+ND、WT+HFD和MYKI+HFD组。12周后行腹腔糖耐量及胰岛素耐量试验后处死小鼠，检测小鼠血清和肝脏组织的肝功能指标，制备肝脏组织切片后行HE染色和F4/80免疫组化染色，ELISA法检测肝脏组织中IL-6、IL-1β和TNF-α水平，qRT-PCR检测M1型（TNF-α、i NOS和IL-6）和M2型（Arg-1、YM-1和IL-10）巨噬细胞标志物mRNA表达，免疫印迹检测肝脏组织中P-AKT、T-AKT、NLRC4、caspase-1和GSDMD蛋白表达，比色法和免疫荧光测定肝脏组织caspase-1活性。结果 与HFD喂饲的WT小鼠相比，MYKI+HFD小鼠肝功能损伤减轻（P<0.01），肝脏脂肪变缓解，肝脏巨噬细胞浸润减少，糖耐量损伤及胰岛素抵抗改善（P<0.01）;MYKI+HFD小鼠肝脏组织IL-6、IL-1β和TNF-α水平降低（P<0.01）,M1型巨噬细胞标志物mRNA表达减少（P<0.01）,M2型mRNA表达增加（P<0.01）;MYKI+HFD小鼠肝脏组织NLRC4炎症小体活性降低（P<0.01），活性caspase-1减少，GSDMD-N蛋白表达降低（P<0.05）。结论 过表达lncRNA HEM2M降低NAFLD小鼠肝脏炎症因子水平，进而改善胰岛素抵抗并抑制肝脏NLRC4炎症小体激活，减少肝细胞焦亡，最终改善NAFLD小鼠肝脏损伤。
【Abstract】ObjectiveTo explore the effects of long non-coding RNA (lncRNA) HEM2M overexpression on liver injury in mice with non-alcoholic fatty liver disease (NAFLD).MethodsWild-type C57BL/6 (WT) mice and myeloid cell-specific HEM2M knock-in (MYKI) mice were fed normal (ND) or high-fat diet (HFD) for 12 weeks. After intraperitoneal glucose tolerance and insulin tolerance tests, the mice were euthanized for detection of liver function indicators in the serum and liver tissue. HE staining and F4/80 immunohistochemical staining were used to examine liver pathologies, and the levels of IL-6, IL-1β, and TNF-α in the liver tissues were determined with ELISA. The mRNA expressions of HEM2M and the markers of M1macrophages (TNF-α, iNOS, and IL-6) and M2 macrophages (Arg-1, YM-1, and IL-10) were detected using qRT-PCR, and the protein expressions of P-AKT, T-AKT, NLRC4, caspase-1 and GSDMD were assayed using immunoblotting. Caspase-1 activity in the liver tissues was determined with colorimetric measurement and immunofluorescence assay.ResultsCompared with HFD-fed WT mice, MYKI mice with HFD feeding showed milder liver function damage (P<0.01), alleviated hepatic steatosis, and reduced liver macrophage infiltration, glucose tolerance impairment and insulin resistance (P<0.01). The levels of IL-6, IL-1β, and TNF-α and mRNA expressions of M1 type macrophage markers were significantly decreased (P<0.01) and those of M2 type markers increased (P<0.01) in the liver tissues of HFD-fed MYKI mice, which also showed reduced NLRC4 inflammasome activity, caspase-1 activation, and GSDMD-N protein expression compared with their WT counterparts (P<0.05).ConclusionOverexpression of HEM2M reduces the production of hepatic inflammatory factors, improves insulin resistance and inhibits hepatic NLRC4 inflammasome activation, which leads to reduced hepatic pyroptosis and liver injury in NAFLD mice.
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