Molecular mechanism of Danshenol C in reversing peritoneal fibrosis: novel network pharmacological analysis and biological validation.

OBJECTIVE: The primary objective of this study is to elucidate the molecular mechanism underlying the reversal of peritoneal fibrosis (PF) by Danshenol C, a natural compound derived from the traditional Chinese medicine Salvia miltiorrhiza. By comprehensively investigating the intricate interactions and signaling pathways involved in Danshenol C's therapeutic effects on PF, we aim to unveil novel insights into its pharmacological actions. This investigation holds the potential to revolutionize the clinical application of Salvia miltiorrhiza in traditional Chinese medicine, offering promising new avenues for the treatment of PF and paving the way for evidence-based therapeutic interventions. METHODS: Firstly, we utilized the YaTCM database to retrieve the structural formula of Danshenol C, while the SwissTargetPrediction platform facilitated the prediction of its potential drug targets. To gain insights into the genetic basis of PF, we acquired the GSE92453 dataset and GPL6480-9577 expression profile from the GEO database, followed by obtaining disease-related genes of PF from major disease databases. R software was then employed to screen for DEG associated with PF. To explore the intricate interactions between Danshenol C's active component targets, we utilized the String database and Cytoscape3.7.2 software to construct a PPI network. Further analysis in Cytoscape3.7.2 enabled the identification of core modules within the PPI network, elucidating key targets and molecular pathways critical to Danshenol C's therapeutic actions. Subsequently, we employed R to perform GO and KEGG pathway enrichment analyses, providing valuable insights into the functional implications and potential biological mechanisms of Danshenol C in the context of PF. To investigate the binding interactions between the core active components and key targets, we conducted docking studies using Chem3D, autoDock1.5.6, SYBYL2.0, and PYMOL2.4 software. We applied in vivo and in vitro experiments to prove that Danshenol C can improve PF. In order to verify the potential gene and molecular mechanism of Danshenol C to reverse PF, we used quantitative PCR, western blot, and apoptosis, ensuring robust and reliable verification of the results. RESULTS: ① Wogonin, sitosterol, and Signal Transducer and Activator of Transcription 5 (STAT5) emerged as the most significant constituents among the small-molecule active compounds and gene targets investigated. ②38 targets intersected with the disease, among which MAPK14, CASP3, MAPK8 and STAT3 may be the key targets; The results of GO and KEGG analysis showed that there was a correlation between inflammatory pathway and Apoptosis. ④Real-time PCR showed that the mRNA expressions of MAPK8 (JNK1), MAPK14 (P38) and STAT3 were significantly decreased after Danshenol C treatment (P < 0.05), while the mRNA expression of CASP3 was significantly increased (P < 0.05)⑤Western blot showed that protein expressions of CASP3 and MAPK14 were significantly increased (P < 0.05), while the expression of STAT3 and MAPK8 was decreased after Danshenol C treatment (P < 0.05). ⑥There was no significant difference in flow analysis of apoptosis among groups. CONCLUSION: The findings suggest that Danshenol C may modulate crucial molecular pathways, including the MAPK, Apoptosis, Calcium signaling, JAK-STAT signaling, and TNF signaling pathways. This regulation is mediated through the modulation of core targets such as STAT3, MAPK14, MAPK8, CASP3, and others. By targeting these key molecular players, Danshenol C exhibits the potential to regulate cellular responses to chemical stress and inflammatory stimuli. The identification of these molecular targets and pathways represents a significant step forward in understanding the molecular basis of Danshenol C's therapeutic effects in PF. This preliminary exploration provides novel avenues for the development of anti-PF treatment strategies and the discovery of potential therapeutic agents. By targeting specific core targets and pathways, Danshenol C opens up new possibilities for the development of more effective and targeted drugs to combat PF. These findings have the potential to transform the landscape of PF treatment and offer valuable insights for future research and drug development endeavors.

Network Pharmacology for Analyzing the Key Targets and Potential Mechanism of Wogonin in Gliomas.

Objective: To analyze the key targets and potential mechanisms underlying the volatile components of Scutellaria baicalensis Georgi acting on gliomas through network pharmacology combined with biological experiments. Methods: We have extracted the volatile components of Scutellaria baicalensis by gas chromatography-mass spectrometry (GC-MS) and determined the active components related to the onset and development of gliomas by combining the results with the data from the Traditional Chinese Medicine Systems Pharmacology database. We screened the same targets for the extracted active components and gliomas through network pharmacology and then constructed a protein-protein interaction network. Using a Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, we analyzed the protein effects and regulatory pathways of the common targets. Lastly, we employed ELISA and Western blot in verifying the key targets in the regulatory pathway. Results: We ultimately determined that the active component in S. baicalensis Georgi related to the onset and development of gliomas was Wogonin. The results of the network pharmacology revealed 85 targets for glioma and Wogonin. We used gene ontology to analyze these target genes and found that they involved 30 functions, such as phosphatidylinositol phosphokinase activation, while the KEGG analysis showed that there were 10 regulatory pathways involved. Through the following analysis, we found that most of the key target genes are distributed in the PI3K-Akt and interleukin 17 signaling pathways. We then cultured U251 glioma cells for the experiments. Compared with the control group, no significant change was noted in the caspase-3 expression; however, cleaved caspase-3 expression increased significantly and was dose-dependent on Wogonin. The expression of Bad and Bcl-2 with 25 µM of Wogonin has remained unchanged, but when the Wogonin dose was increased to 100 µM, the expression of Bad and Bcl-2 was noted to change significantly (Bad was significantly upregulated, while Bcl-2 was significantly downregulated) and was dose-dependent on Wogonin. The ELISA results showed that, compared with the control group, the secretion of tumor necrosis factor alpha, IL-1ß, and IL-6 decreased as the Wogonin concentration increased. Tumor necrosis factor alpha downregulation had no significant dose-dependent effect on Wogonin, the inhibitory effect of 25 µM of Wogonin on IL-6 was not significant, and IL-1ß downregulation had a significant dose-dependent effect on Wogonin. Conclusion: Wogonin might promote the apoptosis of glioma cells by upregulating proapoptotic factors, downregulating antiapoptotic factors, and inhibiting the inflammatory response, thereby inhibiting glioma progression.

Relationship between HIF-1α and apoptosis in rats with traumatic brain injury and the influence of traditional Chinese medicine Sanqi.

Objective To explore the expression of HIF-1α, neuronal apoptosis and the influence of traditional Chinese medicine Sanqi on hematoma after brain injury in rats. Methods Ninety SD rats were divided into 3 groups randomly: blank control group, traumatic brain injury (TBI) group and Sanqi intervention group, and they were decapitated after brain injury at different time points: 6 h, 1 d, 2 d, 3 d, 5 d, 7 d. The model of cerebral hemorrhage was made by autologous non-coagulation in stereotactic locator, the expression of HIF-1α and TUNEL-positive cells (apoptotic cells) in the perihematomal area was detected by immunohistochemistry. Results In blank control group, a small amount of HIF-1α was expressed and apoptotic cells were observed. The expression of HIF-1α was up-regulated in the brain injury group from 6 h, and the apoptotic cells increased in abundance. The peak of HIF-1α was reached at 3 d, then decreased, and remained at the high level on the 7 d. Compared with blank control group, the TBI group was statistically significant (P < 0.05). The Chinese medicine Sanqi intervention group significantly up-regulated HIF-1α'expression and decreased neuronal apoptosis, which was statistically significant (P < 0.05). Conclusion HIF-1α's expression was up-regulated around the hematoma after brain injury, and the apoptosis of nerve cells was obviously increased. The traditional Chinese medicine Sanqi can significantly increase the expression of HIF-1α, reduce the apoptosis around the hematoma, and thus play a neuroprotective role.