Abstract：
BACKGROUND: Buyang Huanwu Decoction (BYHWD), as a traditional Chinese medical prescription, has been used to treat intracerebral hemorrhage (ICH) for hundreds of years, but the antiapoptotic properties have not yet been studied.
OBJECTIVE: This study aims to elucidate the antiapoptotic mechanism of BYHWD in ICH.
METHODS: The therapeutic effect of BYHWD on ICH was assessed by modified neurological severity scores (mNSS), foot fault, and histopathological staining. Then, we used a modified comprehensive strategy by integrating transcriptome and network pharmacology to reveal the underlying mechanism. TUNEL assay, qRT-PCR, and western blot were further applied to evaluate the antiapoptotic effect of BYHWD on ICH. Dual-luciferase reporter assay and plasmid transfections were implemented to validate the potential competing endogenous RNAs (ceRNA) mechanism of Sh2b3.
RESULTS: Network pharmacology analysis indicated that the regulation of the apoptotic process was the highest enriched GO term, and that MAP kinase activity, ERK1, and ERK2 cascade were strongly correlated. Transcriptome analysis screened 180 differentially expressed mRNAs, which were highly enriched in the immune system process and negative regulation of programmed cell death. By checking the literature, we found that Sh2b3 was of great importance to apoptosis by modulating MAPK cascades. TUNEL assay validated the anti-apoptotic effect of BYHWD. Moreover, BYHWD was proven to regulate the Sh2b3-mediated ERK1/2 signaling pathway in ICH mice by qRT-PCR and western blot. We further explored the lncRNA-miRNA-mRNA network underlying the therapeutic effect, among which 4933404O12Rik/miR-185-5p is the upstream regulatory mechanism of Sh2b3.
CONCLUSIONS: We explored the antiapoptotic mechanism of BYHWD in treating ICH by a novel integrated strategy, which involved the 4933404O12Rik/miR-185-5p/Sh2b3 ceRNAs axis.
OBJECTIVE: This study aims to elucidate the antiapoptotic mechanism of BYHWD in ICH.
METHODS: The therapeutic effect of BYHWD on ICH was assessed by modified neurological severity scores (mNSS), foot fault, and histopathological staining. Then, we used a modified comprehensive strategy by integrating transcriptome and network pharmacology to reveal the underlying mechanism. TUNEL assay, qRT-PCR, and western blot were further applied to evaluate the antiapoptotic effect of BYHWD on ICH. Dual-luciferase reporter assay and plasmid transfections were implemented to validate the potential competing endogenous RNAs (ceRNA) mechanism of Sh2b3.
RESULTS: Network pharmacology analysis indicated that the regulation of the apoptotic process was the highest enriched GO term, and that MAP kinase activity, ERK1, and ERK2 cascade were strongly correlated. Transcriptome analysis screened 180 differentially expressed mRNAs, which were highly enriched in the immune system process and negative regulation of programmed cell death. By checking the literature, we found that Sh2b3 was of great importance to apoptosis by modulating MAPK cascades. TUNEL assay validated the anti-apoptotic effect of BYHWD. Moreover, BYHWD was proven to regulate the Sh2b3-mediated ERK1/2 signaling pathway in ICH mice by qRT-PCR and western blot. We further explored the lncRNA-miRNA-mRNA network underlying the therapeutic effect, among which 4933404O12Rik/miR-185-5p is the upstream regulatory mechanism of Sh2b3.
CONCLUSIONS: We explored the antiapoptotic mechanism of BYHWD in treating ICH by a novel integrated strategy, which involved the 4933404O12Rik/miR-185-5p/Sh2b3 ceRNAs axis.
摘要：
背景：补阳还五汤，作为传统的中医处方，数百年来一直被用于治疗脑出血(ICH)，但是抗凋亡特性尚未被研究。
目的：本研究旨在阐明BYHWD在ICH中的抗凋亡机制。
方法：BYHWD对ICH的治疗效果通过改良的神经严重度评分（mNSS）来评估，脚部故障,和组织病理学染色。然后，通过整合转录组和网络药理学，我们使用了一种改进的综合策略来揭示潜在的机制。TUNEL检测,qRT-PCR,并进一步应用westernblot评价BYHWD对ICH的抗凋亡作用。实施双荧光素酶报告基因测定和质粒转染以验证Sh2b3的潜在竞争内源RNA(ceRNA)机制。
结果：网络药理学分析表明，凋亡过程的调节是最高富集的GO术语，以及MAP激酶的活性，ERK1和ERK2级联反应密切相关。转录组分析筛选出180个差异表达的mRNA，在免疫系统过程和程序性细胞死亡的负调控中高度富集。通过查阅文献，我们发现Sh2b3通过调节MAPK级联对细胞凋亡具有重要意义。TUNEL测定验证了BYHWD的抗凋亡作用。此外，通过qRT-PCR和蛋白质印迹证明BYHWD在ICH小鼠中调节Sh2b3介导的ERK1/2信号通路。我们进一步探索了lncRNA-miRNA-mRNA网络潜在的治疗效果，其中4933404O12Rik/miR-185-5p是Sh2b3的上游调控机制。
结论：我们探索了BYHWD通过一种新的整合策略治疗ICH的抗凋亡机制，其中涉及4933404O12Rik/miR-185-5p/Sh2b3ceRNAs轴。
目的：本研究旨在阐明BYHWD在ICH中的抗凋亡机制。
方法：BYHWD对ICH的治疗效果通过改良的神经严重度评分（mNSS）来评估，脚部故障,和组织病理学染色。然后，通过整合转录组和网络药理学，我们使用了一种改进的综合策略来揭示潜在的机制。TUNEL检测,qRT-PCR,并进一步应用westernblot评价BYHWD对ICH的抗凋亡作用。实施双荧光素酶报告基因测定和质粒转染以验证Sh2b3的潜在竞争内源RNA(ceRNA)机制。
结果：网络药理学分析表明，凋亡过程的调节是最高富集的GO术语，以及MAP激酶的活性，ERK1和ERK2级联反应密切相关。转录组分析筛选出180个差异表达的mRNA，在免疫系统过程和程序性细胞死亡的负调控中高度富集。通过查阅文献，我们发现Sh2b3通过调节MAPK级联对细胞凋亡具有重要意义。TUNEL测定验证了BYHWD的抗凋亡作用。此外，通过qRT-PCR和蛋白质印迹证明BYHWD在ICH小鼠中调节Sh2b3介导的ERK1/2信号通路。我们进一步探索了lncRNA-miRNA-mRNA网络潜在的治疗效果，其中4933404O12Rik/miR-185-5p是Sh2b3的上游调控机制。
结论：我们探索了BYHWD通过一种新的整合策略治疗ICH的抗凋亡机制，其中涉及4933404O12Rik/miR-185-5p/Sh2b3ceRNAs轴。
