目的:探讨J型针刀通过PACAP-cAMP-PKA信号通路改善脊髓损伤后神经源性膀胱大鼠逼尿肌的作用机制。方法:36只SD雄性大鼠随机分为6组:空白组、假手术组、模型组、J型针刀治疗组、治疗+Bupivacaine抑制剂组、治疗+H-89抑制剂组,各6只。采...目的:探讨J型针刀通过PACAP-cAMP-PKA信号通路改善脊髓损伤后神经源性膀胱大鼠逼尿肌的作用机制。方法:36只SD雄性大鼠随机分为6组:空白组、假手术组、模型组、J型针刀治疗组、治疗+Bupivacaine抑制剂组、治疗+H-89抑制剂组,各6只。采用脊髓横断法制作神经源性膀胱模型。空白组为正常SD大鼠;假手术组为切开相关部位组织,无脊髓切断;其余4组予以手术脊髓切断造模。J型针刀治疗组造模后第19天予J型针刀针刺大鼠次髎穴,2 d 1次,共治疗7次。干预结束后各组行HE染色观察膀胱逼尿肌组织形态变化,Elisa检测膀胱逼尿肌组织中c AMP、PKA含量,免疫组化检测膀胱逼尿肌组织中PACAP38蛋白表达,Western blot检测膀胱逼尿肌组织中PKA、p-MLC蛋白表达。结果:HE染色结果显示,与空白组相比,模型组中膀胱逼尿肌组织严重坏死,可见大量炎症细胞浸润;与模型组相比,J型针刀治疗组、治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组均有不同程度坏死组织修复情况。免疫组化检测PACAP38蛋白表达在模型组中表达量最低,针刀治疗之后,PACAP38表达量均不同程度上调;与J型针刀治疗组相比,治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组PACAP38蛋白表达均不同程度下调(P<0.05),但高于模型组(P<0.05)。Elisa检测结果显示,cAMP、PKA表达量模型组显著下调(P<0.05),J型针刀治疗组与模型组比显著上调(P<0.05),治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组比无显著差异(P>0.05)。WB结果显示:与空白组相比,模型组PKA、p-MLC蛋白表达上调;针刀治疗组表达量下调,其中PKA蛋白表达有显著性(P<0.05),p-MLC蛋白表达无显著性(P>0.05)。与J型针刀治疗组比,PKA蛋白表达量在治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组中无显著差异(P>0.05),p-MLC蛋白表达量显著下降(P<0.05)。结论:J型针刀针刺次髎穴可改善脊髓损伤后神经源性膀胱大鼠膀胱功能,其机制与J型针刀上调膀胱逼尿肌中PACAP38、cAMP、PKA表达,激活PACAP-cAMP-PKA信号通路,从而促进逼尿肌舒张有关。展开更多
Conogethes punctiferalis is a crop and fruit pest that has caused serious economic losses to agricultural production.This pest relies heavily on its sex pheromone to ensure sexual encounters and subsequent mating succ...Conogethes punctiferalis is a crop and fruit pest that has caused serious economic losses to agricultural production.This pest relies heavily on its sex pheromone to ensure sexual encounters and subsequent mating success.However,the molecular mechanism underlying sex pheromone biosynthesis in this species remains elusive.The present study investigated the detailed mechanism underlying PBAN-regulated sex pheromone biosynthesis in C.punctiferalis by transcriptome sequencing of the C.punctiferalis pheromone glands(PGs)and subsequent functional identification of the target genes.The results showed that female mating started from the first scotophase,and peaked at the second to fifth scotophases in accordance with the release of sex pheromones.PBAN regulated sex pheromone biosynthesis by employing Ca^(2+)and cAMP as secondary messengers,as demonstrated by RNA interference(RNAi),pharmacological inhibitors,and behavioral assays.Further investigation revealed that calcineurin(CaN)and acetyl-CoA carboxylase(ACC)were activated by PBAN/Ca^(2+)signaling,and the RNAimediated knockdown of CaN and ACC transcripts significantly reduced sex pheromone production,ultimately leading to a significantly reduced ability of females to attract males.Importantly,hexokinase(HK)was found to regulate sex pheromone biosynthesis in response to the PBAN/cAMP/PKA signaling pathway,as demonstrated by RNAi,enzyme activity,and pharmacological inhibitor assays.Furthermore,Far2 and Desaturase1 were found to participate in PBAN-regulated sex pheromone biosynthesis.Altogether,our findings revealed that PBAN regulates sex pheromone biosynthesis through the PBANR/Ca^(2+)/CaN/ACC and PBANR/cAMP/PKA/HK pathways in C.punctiferalis,which enriches our comprehension of the details of sex pheromone biosynthesis in moths.展开更多
文摘目的:探讨J型针刀通过PACAP-cAMP-PKA信号通路改善脊髓损伤后神经源性膀胱大鼠逼尿肌的作用机制。方法:36只SD雄性大鼠随机分为6组:空白组、假手术组、模型组、J型针刀治疗组、治疗+Bupivacaine抑制剂组、治疗+H-89抑制剂组,各6只。采用脊髓横断法制作神经源性膀胱模型。空白组为正常SD大鼠;假手术组为切开相关部位组织,无脊髓切断;其余4组予以手术脊髓切断造模。J型针刀治疗组造模后第19天予J型针刀针刺大鼠次髎穴,2 d 1次,共治疗7次。干预结束后各组行HE染色观察膀胱逼尿肌组织形态变化,Elisa检测膀胱逼尿肌组织中c AMP、PKA含量,免疫组化检测膀胱逼尿肌组织中PACAP38蛋白表达,Western blot检测膀胱逼尿肌组织中PKA、p-MLC蛋白表达。结果:HE染色结果显示,与空白组相比,模型组中膀胱逼尿肌组织严重坏死,可见大量炎症细胞浸润;与模型组相比,J型针刀治疗组、治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组均有不同程度坏死组织修复情况。免疫组化检测PACAP38蛋白表达在模型组中表达量最低,针刀治疗之后,PACAP38表达量均不同程度上调;与J型针刀治疗组相比,治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组PACAP38蛋白表达均不同程度下调(P<0.05),但高于模型组(P<0.05)。Elisa检测结果显示,cAMP、PKA表达量模型组显著下调(P<0.05),J型针刀治疗组与模型组比显著上调(P<0.05),治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组比无显著差异(P>0.05)。WB结果显示:与空白组相比,模型组PKA、p-MLC蛋白表达上调;针刀治疗组表达量下调,其中PKA蛋白表达有显著性(P<0.05),p-MLC蛋白表达无显著性(P>0.05)。与J型针刀治疗组比,PKA蛋白表达量在治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组中无显著差异(P>0.05),p-MLC蛋白表达量显著下降(P<0.05)。结论:J型针刀针刺次髎穴可改善脊髓损伤后神经源性膀胱大鼠膀胱功能,其机制与J型针刀上调膀胱逼尿肌中PACAP38、cAMP、PKA表达,激活PACAP-cAMP-PKA信号通路,从而促进逼尿肌舒张有关。
基金supported by the National Natural Science Foundation of China(31970472,32272547)the National Science Fund of Henan Province for Distinguished Young Scholars,China(202300410191)+3 种基金the Basic Research Project of the Key Scientific Research Projects of Universities in Henan Province,China(21zx013)the Henan Agricultural Research System,China(HARS-2209-G3)the Henan Special Support for High-Level Talents Central Plains Science and Technology Innovation Leading Talents,China(224200510018)the earmarked fund for China Agricultural Research System(CARS-27)。
文摘Conogethes punctiferalis is a crop and fruit pest that has caused serious economic losses to agricultural production.This pest relies heavily on its sex pheromone to ensure sexual encounters and subsequent mating success.However,the molecular mechanism underlying sex pheromone biosynthesis in this species remains elusive.The present study investigated the detailed mechanism underlying PBAN-regulated sex pheromone biosynthesis in C.punctiferalis by transcriptome sequencing of the C.punctiferalis pheromone glands(PGs)and subsequent functional identification of the target genes.The results showed that female mating started from the first scotophase,and peaked at the second to fifth scotophases in accordance with the release of sex pheromones.PBAN regulated sex pheromone biosynthesis by employing Ca^(2+)and cAMP as secondary messengers,as demonstrated by RNA interference(RNAi),pharmacological inhibitors,and behavioral assays.Further investigation revealed that calcineurin(CaN)and acetyl-CoA carboxylase(ACC)were activated by PBAN/Ca^(2+)signaling,and the RNAimediated knockdown of CaN and ACC transcripts significantly reduced sex pheromone production,ultimately leading to a significantly reduced ability of females to attract males.Importantly,hexokinase(HK)was found to regulate sex pheromone biosynthesis in response to the PBAN/cAMP/PKA signaling pathway,as demonstrated by RNAi,enzyme activity,and pharmacological inhibitor assays.Furthermore,Far2 and Desaturase1 were found to participate in PBAN-regulated sex pheromone biosynthesis.Altogether,our findings revealed that PBAN regulates sex pheromone biosynthesis through the PBANR/Ca^(2+)/CaN/ACC and PBANR/cAMP/PKA/HK pathways in C.punctiferalis,which enriches our comprehension of the details of sex pheromone biosynthesis in moths.
