目的探讨外周神经元蛋白酶激活受体2-蛋白激酶A/蛋白激酶Cε(PAR2-PKA/PKCε)通路在痛转化中的作用,寻找同时干预急性痛和慢性痛的可能方案。方法 SD大鼠随机分为空白组、假诱发组、诱发组、抑制剂1组和抑制剂2组。除空白组和假诱发组,...目的探讨外周神经元蛋白酶激活受体2-蛋白激酶A/蛋白激酶Cε(PAR2-PKA/PKCε)通路在痛转化中的作用,寻找同时干预急性痛和慢性痛的可能方案。方法 SD大鼠随机分为空白组、假诱发组、诱发组、抑制剂1组和抑制剂2组。除空白组和假诱发组,所有大鼠均通过先后足部注射角叉菜胶和前列腺素E2(PGE2)建立痛觉敏化诱发模型。PGE2于角叉菜胶注射后7 d进行足部注射。抑制剂1组和抑制剂2组大鼠于PGE2注射前/后,分别给予PAR2抑制剂。观察角叉菜胶/生理盐水,注射前、注射后5 h、3 d、6 d、7 d 0.5 h、7 d 4 h和7 d 24 h大鼠机械痛阈(PWTs)的变化,检测角叉菜胶注射后7 d 24 h造模侧背根神经节(DRG)中PAR2、蛋白激酶A(PKA)和蛋白激酶(PKCε)表达。结果痛觉敏化诱发模型建立成功。角叉菜胶注射后7 d给予PGE2,显著延长了PGE2诱发疼痛的存在时间,角叉菜胶注射后7 d 24 h假诱发组大鼠PWTs与同期空白组相比差异无显著性(P>0.05),而诱发组大鼠PWTs明显低于同期空白组和假诱发组大鼠(P<0.01)。诱发组大鼠造模侧DRG中PAR2和PKCε表达在角叉菜胶注射后7 d 24 h明显提升,高于同期假诱发组和空白组(P<0.05)。给予PAR2抑制,不论时间均能显著翻转角叉菜胶注射后7 d 24 h,诱发组大鼠由PGE2诱发的疼痛(P<0.05),并抑制DRG中PKCε表达。但,给予PAR2抑制剂不能影响PGE2诱发的急性疼痛和调制DRG中PKA含量。结论抑制PAR2表达能阻断急性痛向慢性痛转化,这可能与其抑制DRG中PAR2-PKCε通路激活有关。但抑制PAR2并不能干预急性痛,这可能是因为DRG中PAR2相关通路未参与急性痛的产生。展开更多
Here,we used reverse transcription-PCR(RT-PCR) and western blot to detect protease-activated receptor(PAR) 1,PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma,and invest...Here,we used reverse transcription-PCR(RT-PCR) and western blot to detect protease-activated receptor(PAR) 1,PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma,and investigated the co-relationship between PAR expression and clinic-pathological data for esophageal cancer.The methylation of PAR4 gene promoter involved in esophageal carcinoma was also analyzed.By comparing the mRNA expressions of normal esophageal tissue and human esophageal epithelial cells(HEEpiC),we found that among the 28 cases of esophageal squamous cell carcinoma,PAR1(60%) and PAR2(71%) were elevated in 17 and 20 cases,respectively,and PAR4(68%) expression was lowered in 19 cases.Whereas,in human esophageal squamous cells(TE-1 and TE-10),PAR1 and PAR2 expression was increased but PAR4 was decreased.Combined with clinical data,the expression of PAR1 in poorly differentiated(P=0.016) and middle and lower parts of the esophagus(P=0.016) was higher; expression of PAR4 in poorly differentiated carcinoma was lower(P=0.049).Regarding TE-1 and TE-10 protein expression,we found that in randomized esophageal carcinoma,PAR1(P=0.027) and PAR2(P=0.039) expressions were increased,but lowered for PAR4(P=0.0001).In HEEpiC,TE-1,TE-10,esophageal and normal esophagus tissue samples(case No.7),the frequency of methylation at the 19 CpG loci of PAR4 was 35.4%,95.2%,83.8%,62.6% and 48.2%,respectively.Our results indicate that the expression of PAR1 and PAR2 in esophageal squamous cell carcinoma is increased but PAR4 is decreased.Hypermethylation of the promoter of the PAR4 gene may contribute to reduced expression of PAR4 in esophageal squamous cell carcinoma.展开更多
Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases,immune cell infiltration and protease-driven tissue damages....Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases,immune cell infiltration and protease-driven tissue damages.It is an urgent need to explore potential drug strategies for mitigating lung inflammation.Protease-activated receptor 2(PAR2)as a vital molecular target principally participates in various inflammatory diseases via intracellular signal transduction.However,it has been rarely reported about the role of PAR2 in lung inflammation.This study applied CRISPR-Cas9 system encoding Cas9 and sg RNA(p Cas9-PAR2)for PAR2 knockout and fabricated an anionic human serum albuminbased nanoparticles to deliver p Cas9-PAR2 with superior inflammation-targeting efficiency and stability(TAP/p Cas9-PAR2).TAP/p Cas9-PAR2 robustly facilitated p Cas9-PAR2 to enter and transfect inflammatory cells,eliciting precise gene editing of PAR2 in vitro and in vivo.Importantly,PAR2 deficiency by TAP/p Cas9-PAR2 effectively and safely promoted macrophage polarization,suppressed proinflammatory cytokine releases and alleviated acute lung inflammation,uncovering a novel value of PAR2.It also revealed that PAR2-mediated pulmonary inflammation prevented by TAP/p Cas9-PAR2was mainly dependent on ERK-mediated NLRP3/IL-1βand NO/i NOS signalling.Therefore,this work indicated PAR2 as a novel target for lung inflammation and provided a potential nanodrug strategy for PAR2 deficiency in treating inflammatory diseases.展开更多
文摘目的探讨外周神经元蛋白酶激活受体2-蛋白激酶A/蛋白激酶Cε(PAR2-PKA/PKCε)通路在痛转化中的作用,寻找同时干预急性痛和慢性痛的可能方案。方法 SD大鼠随机分为空白组、假诱发组、诱发组、抑制剂1组和抑制剂2组。除空白组和假诱发组,所有大鼠均通过先后足部注射角叉菜胶和前列腺素E2(PGE2)建立痛觉敏化诱发模型。PGE2于角叉菜胶注射后7 d进行足部注射。抑制剂1组和抑制剂2组大鼠于PGE2注射前/后,分别给予PAR2抑制剂。观察角叉菜胶/生理盐水,注射前、注射后5 h、3 d、6 d、7 d 0.5 h、7 d 4 h和7 d 24 h大鼠机械痛阈(PWTs)的变化,检测角叉菜胶注射后7 d 24 h造模侧背根神经节(DRG)中PAR2、蛋白激酶A(PKA)和蛋白激酶(PKCε)表达。结果痛觉敏化诱发模型建立成功。角叉菜胶注射后7 d给予PGE2,显著延长了PGE2诱发疼痛的存在时间,角叉菜胶注射后7 d 24 h假诱发组大鼠PWTs与同期空白组相比差异无显著性(P>0.05),而诱发组大鼠PWTs明显低于同期空白组和假诱发组大鼠(P<0.01)。诱发组大鼠造模侧DRG中PAR2和PKCε表达在角叉菜胶注射后7 d 24 h明显提升,高于同期假诱发组和空白组(P<0.05)。给予PAR2抑制,不论时间均能显著翻转角叉菜胶注射后7 d 24 h,诱发组大鼠由PGE2诱发的疼痛(P<0.05),并抑制DRG中PKCε表达。但,给予PAR2抑制剂不能影响PGE2诱发的急性疼痛和调制DRG中PKA含量。结论抑制PAR2表达能阻断急性痛向慢性痛转化,这可能与其抑制DRG中PAR2-PKCε通路激活有关。但抑制PAR2并不能干预急性痛,这可能是因为DRG中PAR2相关通路未参与急性痛的产生。
基金supported by the National Natural Foundation of China(81160302)the Major Research Project of Yunnan Province(2011FZ109)Research project of Yunnan Education Bureau(2014Y153)
文摘Here,we used reverse transcription-PCR(RT-PCR) and western blot to detect protease-activated receptor(PAR) 1,PAR 2 and PAR 4 expression in cancer tissues and cell lines of esophageal squamous cell carcinoma,and investigated the co-relationship between PAR expression and clinic-pathological data for esophageal cancer.The methylation of PAR4 gene promoter involved in esophageal carcinoma was also analyzed.By comparing the mRNA expressions of normal esophageal tissue and human esophageal epithelial cells(HEEpiC),we found that among the 28 cases of esophageal squamous cell carcinoma,PAR1(60%) and PAR2(71%) were elevated in 17 and 20 cases,respectively,and PAR4(68%) expression was lowered in 19 cases.Whereas,in human esophageal squamous cells(TE-1 and TE-10),PAR1 and PAR2 expression was increased but PAR4 was decreased.Combined with clinical data,the expression of PAR1 in poorly differentiated(P=0.016) and middle and lower parts of the esophagus(P=0.016) was higher; expression of PAR4 in poorly differentiated carcinoma was lower(P=0.049).Regarding TE-1 and TE-10 protein expression,we found that in randomized esophageal carcinoma,PAR1(P=0.027) and PAR2(P=0.039) expressions were increased,but lowered for PAR4(P=0.0001).In HEEpiC,TE-1,TE-10,esophageal and normal esophagus tissue samples(case No.7),the frequency of methylation at the 19 CpG loci of PAR4 was 35.4%,95.2%,83.8%,62.6% and 48.2%,respectively.Our results indicate that the expression of PAR1 and PAR2 in esophageal squamous cell carcinoma is increased but PAR4 is decreased.Hypermethylation of the promoter of the PAR4 gene may contribute to reduced expression of PAR4 in esophageal squamous cell carcinoma.
基金supported by the National Natural Science Foundation of China(Nos.82003784 and 81872789)the Fundamental Research Funds for the Central Universities(No.2682022ZTPY037,China)Large Instruments Open Foundation of Southwest Jiaotong University(No.2022SRII-046,China)。
文摘Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases,immune cell infiltration and protease-driven tissue damages.It is an urgent need to explore potential drug strategies for mitigating lung inflammation.Protease-activated receptor 2(PAR2)as a vital molecular target principally participates in various inflammatory diseases via intracellular signal transduction.However,it has been rarely reported about the role of PAR2 in lung inflammation.This study applied CRISPR-Cas9 system encoding Cas9 and sg RNA(p Cas9-PAR2)for PAR2 knockout and fabricated an anionic human serum albuminbased nanoparticles to deliver p Cas9-PAR2 with superior inflammation-targeting efficiency and stability(TAP/p Cas9-PAR2).TAP/p Cas9-PAR2 robustly facilitated p Cas9-PAR2 to enter and transfect inflammatory cells,eliciting precise gene editing of PAR2 in vitro and in vivo.Importantly,PAR2 deficiency by TAP/p Cas9-PAR2 effectively and safely promoted macrophage polarization,suppressed proinflammatory cytokine releases and alleviated acute lung inflammation,uncovering a novel value of PAR2.It also revealed that PAR2-mediated pulmonary inflammation prevented by TAP/p Cas9-PAR2was mainly dependent on ERK-mediated NLRP3/IL-1βand NO/i NOS signalling.Therefore,this work indicated PAR2 as a novel target for lung inflammation and provided a potential nanodrug strategy for PAR2 deficiency in treating inflammatory diseases.
