Abstract：
BACKGROUND: Rhapontici Radix ethanol extract (RRE) is derived from the dried root of Rhaponticum uniflorum (L.) DC belonging to the Asteraceae family. RRE exhibits significant anti-inflammatory and antioxidant properties; however, the potential of RRE in mastitis treatment requires further investigation.
OBJECTIVE: This research was performed to examine the protective properties of RRE against mastitis and the mechanisms underlying the effects of RRE.
METHODS: RRE components were analyzed by HPLC-MS/MS and DPPH methods. Isochlorogenic acid B (ICAB) was obtained commercially. MTT assay was utilized to assess RRE or ICAB cytotoxicity in bovine mammary alveolar (MAC-T) cells. Immunohistochemistry were used to investigate the pathological alterations in mammary tissue. The protein levels of inflammatory cytokines and mediators were analyzed using ELISA, and the expression of MAPK and NF-κB signaling pathways, as well as p65 nuclear translocation, were analyzed through Western blotting and immunofluorescence techniques, respectively. Target proteins of RRE were screened by RNA-seq and tandem mass tag analyses. Protein interaction was revealed and confirmed using co-immunoprecipitation and CRISPR/Cas9-based knockdown and overexpression of target genes.
RESULTS: ICAB was revealed as one of the main components in RRE, and it was responsible for 84.33% of RRE radical scavenging activity. Both RRE and ICAB mitigated the infiltration of T lymphocytes in the mammary glands of mice, leading to decreased levels of inflammatory mediators (COX-2 and iNOS) and cytokines (TNF-α, IL-6, and IL-1β) in lipopolysaccharide (LPS)-induced MAC-T cells. Furthermore, RRE and ICAB suppressed the LPS-induced phosphorylation of NF-κB inhibitor and p65, thereby impeding p65 nuclear translocation in mouse mammary glands and MAC-T cells. In addition, RRE and ICAB attenuated the LPS-triggered activation of c-Jun N-terminal kinase 1/2, p38, and extracellular regulated protein kinase 1/2. Importantly, co-treated with LPS and ICAB in MAC-T cells, an upregulation of G-protein coupled receptor 161 (GPR161) and transmembrane protein 59 (TMEM59) was observed; the interact between TMEM59 and was found, leading to inhibition of NF-κB activity and inflammatory cytokine production.
CONCLUSIONS: ICAB is a prominent antioxidant in RRE. RRE and ICAB reduce mammary inflammation via MAPK and NF-κB pathways and the interaction between TMEM59 and GPR161 mediates the control of ICAB in NF-κB signaling.
OBJECTIVE: This research was performed to examine the protective properties of RRE against mastitis and the mechanisms underlying the effects of RRE.
METHODS: RRE components were analyzed by HPLC-MS/MS and DPPH methods. Isochlorogenic acid B (ICAB) was obtained commercially. MTT assay was utilized to assess RRE or ICAB cytotoxicity in bovine mammary alveolar (MAC-T) cells. Immunohistochemistry were used to investigate the pathological alterations in mammary tissue. The protein levels of inflammatory cytokines and mediators were analyzed using ELISA, and the expression of MAPK and NF-κB signaling pathways, as well as p65 nuclear translocation, were analyzed through Western blotting and immunofluorescence techniques, respectively. Target proteins of RRE were screened by RNA-seq and tandem mass tag analyses. Protein interaction was revealed and confirmed using co-immunoprecipitation and CRISPR/Cas9-based knockdown and overexpression of target genes.
RESULTS: ICAB was revealed as one of the main components in RRE, and it was responsible for 84.33% of RRE radical scavenging activity. Both RRE and ICAB mitigated the infiltration of T lymphocytes in the mammary glands of mice, leading to decreased levels of inflammatory mediators (COX-2 and iNOS) and cytokines (TNF-α, IL-6, and IL-1β) in lipopolysaccharide (LPS)-induced MAC-T cells. Furthermore, RRE and ICAB suppressed the LPS-induced phosphorylation of NF-κB inhibitor and p65, thereby impeding p65 nuclear translocation in mouse mammary glands and MAC-T cells. In addition, RRE and ICAB attenuated the LPS-triggered activation of c-Jun N-terminal kinase 1/2, p38, and extracellular regulated protein kinase 1/2. Importantly, co-treated with LPS and ICAB in MAC-T cells, an upregulation of G-protein coupled receptor 161 (GPR161) and transmembrane protein 59 (TMEM59) was observed; the interact between TMEM59 and was found, leading to inhibition of NF-κB activity and inflammatory cytokine production.
CONCLUSIONS: ICAB is a prominent antioxidant in RRE. RRE and ICAB reduce mammary inflammation via MAPK and NF-κB pathways and the interaction between TMEM59 and GPR161 mediates the control of ICAB in NF-κB signaling.
摘要：
背景：RhaponticiRadix乙醇提取物（RRE）来自单氟Rhaponticum的干燥根（L.)DC属于菊科。RRE表现出显著的抗炎和抗氧化特性;然而，RRE在乳腺炎治疗中的潜力需要进一步研究.
目的：本研究旨在研究RRE对乳腺炎的保护特性以及RRE作用的潜在机制。
方法:通过HPLC-MS/MS和DPPH方法分析RRE组分。异绿原酸B(ICAB)是商业获得的。利用MTT测定法评估RRE或ICAB在牛乳腺肺泡(MAC-T)细胞中的细胞毒性。采用免疫组织化学方法研究乳腺组织的病理改变。使用ELISA分析炎性细胞因子和介质的蛋白质水平，MAPK和NF-κB信号通路的表达,以及p65核易位，通过蛋白质印迹和免疫荧光技术进行分析，分别。通过RNA-seq和串联质量标签分析筛选RRE的靶蛋白。使用共免疫沉淀和基于CRISPR/Cas9的敲低和靶基因的过表达来揭示和确认蛋白质相互作用。
结果：显示ICAB是RRE的主要成分之一，它负责84.33%的RRE自由基清除活性。RRE和ICAB均减轻了小鼠乳腺中T淋巴细胞的浸润，导致炎症介质（COX-2和iNOS）和细胞因子（TNF-α，脂多糖（LPS）诱导的MAC-T细胞中的IL-6和IL-1β）。此外，RRE和ICAB抑制LPS诱导的NF-κB抑制剂和p65的磷酸化，从而阻碍p65在小鼠乳腺和MAC-T细胞中的核易位。此外,RRE和ICAB减弱了LPS触发的c-JunN末端激酶1/2，p38和细胞外调节蛋白激酶1/2的激活。重要的是,在MAC-T细胞中与LPS和ICAB共同处理，观察到G蛋白偶联受体161（GPR161）和跨膜蛋白59（TMEM59）的上调；发现TMEM59和TMEM59之间的相互作用，导致NF-κB活性的抑制和炎性细胞因子的产生。
结论：ICAB是RRE中一种重要的抗氧化剂。RRE和ICAB通过MAPK和NF-κB途径减轻乳腺炎症，TMEM59和GPR161之间的相互作用介导了NF-κB信号传导中ICAB的控制。
目的：本研究旨在研究RRE对乳腺炎的保护特性以及RRE作用的潜在机制。
方法:通过HPLC-MS/MS和DPPH方法分析RRE组分。异绿原酸B(ICAB)是商业获得的。利用MTT测定法评估RRE或ICAB在牛乳腺肺泡(MAC-T)细胞中的细胞毒性。采用免疫组织化学方法研究乳腺组织的病理改变。使用ELISA分析炎性细胞因子和介质的蛋白质水平，MAPK和NF-κB信号通路的表达,以及p65核易位，通过蛋白质印迹和免疫荧光技术进行分析，分别。通过RNA-seq和串联质量标签分析筛选RRE的靶蛋白。使用共免疫沉淀和基于CRISPR/Cas9的敲低和靶基因的过表达来揭示和确认蛋白质相互作用。
结果：显示ICAB是RRE的主要成分之一，它负责84.33%的RRE自由基清除活性。RRE和ICAB均减轻了小鼠乳腺中T淋巴细胞的浸润，导致炎症介质（COX-2和iNOS）和细胞因子（TNF-α，脂多糖（LPS）诱导的MAC-T细胞中的IL-6和IL-1β）。此外，RRE和ICAB抑制LPS诱导的NF-κB抑制剂和p65的磷酸化，从而阻碍p65在小鼠乳腺和MAC-T细胞中的核易位。此外,RRE和ICAB减弱了LPS触发的c-JunN末端激酶1/2，p38和细胞外调节蛋白激酶1/2的激活。重要的是,在MAC-T细胞中与LPS和ICAB共同处理，观察到G蛋白偶联受体161（GPR161）和跨膜蛋白59（TMEM59）的上调；发现TMEM59和TMEM59之间的相互作用，导致NF-κB活性的抑制和炎性细胞因子的产生。
结论：ICAB是RRE中一种重要的抗氧化剂。RRE和ICAB通过MAPK和NF-κB途径减轻乳腺炎症，TMEM59和GPR161之间的相互作用介导了NF-κB信号传导中ICAB的控制。
