Integrated Network Pharmacology and in vivo Experimental Validation Approach to Explore the Potential Antioxidant Effects of Annao Pingchong Decoction in Intracerebral Hemorrhage Rats.

Annao Pingchong decoction (ANPCD) is a traditional Chinese decoction which has definite effects on treating intracerebral hemorrhage (ICH) validated through clinical and experimental studies. However, the impact of ANPCD on oxidative stress (OS) after ICH remains unclear and is worth further investigating. To investigate whether the therapeutic effects of ANPCD on ICH are related to alleviating OS damage and seek potential targets for its antioxidant effects. The therapeutic candidate genes of ANPCD on ICH were identified through a comparison of the target genes of ANPCD, target genes of ICH and differentially expressed genes (DEGs). Protein-protein interaction (PPI) network analysis and functional enrichment analysis were combined with targets-related literature to select suitable antioxidant targets. The affinity between ANPCD and the selected target was verified using macromolecular docking. Subsequently, the effects of ANPCD on OS and the selected target were further investigated through in vivo experiments. Forty-eight candidate genes were screened, in which silent information regulator sirtuin 1 (SIRT1) is one of the core genes that has antioxidant effects and ICH significantly affected its expression. The good affinity between 6 compounds of ANPCD and SIRT1 was also demonstrated by macromolecular docking. The results of in vivo experiments demonstrated that ANPCD significantly decreased modified neurological severity scoring (mNSS) scores and serum MDA and 8-OHdG content in ICH rats, while significantly increasing serum SOD and CAT activity, complicated with the up-regulation of ANPCD on SIRT1, FOXO1, PGC-1α and Nrf2. Furthermore, ANPCD significantly decreased the apoptosis rate and the expression of apoptosis-related proteins (P53, cytochrome c and caspase-3). ANPCD alleviates OS damage and apoptosis after ICH in rats. As a potential therapeutic target, SIRT1 can be effectively regulated by ANPCD, as are its downstream proteins.

Zhou X ，Wang X ，Li J ，Zhang M ，Yang Y ，Lei S ，He Y ，Yang H ，Zhou D ，Guo C ... -  《Drug Design Development and Therapy》

Annao Pingchong decoction alleviate the neurological impairment by attenuating neuroinflammation and apoptosis in intracerebral hemorrhage rats.

Intracerebral hemorrhage (ICH) is a central nervous system disease that causes severe disability or death. Even though Annao Pingchong decoction (ANPCD), a traditional Chinese decoction, has been used clinically to treat ICH in China, its molecular mechanism remains unclear. To study whether the neuroprotective effect of ANPCD on ICH rats is achieved by alleviating neuroinflammation. This paper mainly explored whether inflammation-related signaling pathways (HMGB1/TLR4/NF-κB P65) plays a role in ANPCD treatment of ICH rats. Liquid chromatography-tandem mass spectrometry was used to analyze the chemical composition of ANPCD. ICH models were established by injecting autologous whole blood into the left caudate nucleus of Sprague-Dawley (SD) rats. Modified neurological severity scoring (mNSS) was used to assess the neurological deficits. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were analyzed using enzyme-linked immunosorbent assay (ELISA). Pathological changes in the rat brains were observed using hematoxylin-eosin, Nissl, and TUNEL staining. The protein levels of HMGB1, TLR4, NF-κB p65, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) were measured by western blotting and immunofluorescence analysis. Ninety-three ANPCD compounds were identified, including 48 active plasma components. Treatment with ANPCD effectively improved the outcome, as observed by the neurological function scores analysis and brain histopathology. Our results showed that ANPCD exerts its anti-inflammatory effects by significantly downregulating the expression of HMGB1, TLR4, NF-κB p65, TNF-α, IL-1β, and IL-6. ANPCD also exerted anti-apoptotic effects by significantly decreasing the apoptosis rate and Bax/Bcl-2 ratio. We found that ANPCD had neuroprotective effect in clinical work. Here, we also found that the action mechanism of ANPCD might be related to attenuate neuroinflammation and apoptosis. These effects were achieved by inhibiting the expression of HMGB1, TLR4 and NF-κB p65.

Guo C ，Zhou X ，Wang X ，Wang H ，Liu J ，Wang J ，Lin X ，Lei S ，Yang Y ，Liu K ，Long H ，Zhou D ... -  《-》

Decoding the underlying mechanisms of Di-Tan-Decoction in treating intracerebral hemorrhage based on network pharmacology.

Chinese medicine usually acts as "multi-ingredients, multi-targets and multi-pathways" on complex diseases, and these action modes reflect the coordination and integrity of the treatment process with traditional Chinese medicine (TCM). System pharmacology is developed based on the cross-disciplines of directional pharmacology, system biology, and mathematics, has the characteristics of integrity and synergy in the treatment process of TCM. Therefore, it is suitable for analyzing the key ingredients and mechanisms of TCM in treating complex diseases. Intracerebral Hemorrhage (ICH) is one of the leading causes of death in China, with the characteristics of high mortality and disability rate. Bring a significant burden on people and society. An increasing number of studies have shown that Chinese medicine prescriptions have good advantages in the treatment of ICH, and Ditan Decoction (DTT) is one of the commonly used prescriptions in the treatment of ICH. Modern pharmacological studies have shown that DTT may play a therapeutic role in treating ICH by inhibiting brain inflammation, abnormal oxidative stress reaction and reducing neurological damage, but the specific key ingredients and mechanism are still unclear. To solve this problem, we established PPI network based on the latest pathogenic gene data of ICH, and CT network based on ingredient and target data of DTT. Subsequently, we established optimization space based on PPI network and CT network, and constructed a new model for node importance calculation, and proposed a calculation method for PES score, thus calculating the functional core ingredients group (FCIG). These core functional groups may represent DTT therapy for ICH. Based on the strategy, 44 ingredients were predicted as FCIG, results showed that 80.44% of the FCIG targets enriched pathways were coincided with the enriched pathways of pathogenic genes. Both the literature and molecular docking results confirm the therapeutic effect of FCIG on ICH via targeting MAPK signaling pathway and PI3K-Akt signaling pathway. The FCIG obtained by our network pharmacology method can represent the effect of DTT in treating ICH. These results confirmed that our strategy of active ingredient group optimization and the mechanism inference could provide methodological reference for optimization and secondary development of TCM.

Zhen Z ，Xue DJ ，Chen YP ，Li JH ，Gao Y ，Shen YB ，Peng ZZ ，Zhang N ，Wang KX ，Guan DG ，Huang T ... -  《-》

A novel strategy of integrating network pharmacology and transcriptome reveals antiapoptotic mechanisms of Buyang Huanwu Decoction in treating intracerebral hemorrhage.

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. This study aims to elucidate the antiapoptotic mechanism of BYHWD in ICH. 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. 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. 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.

Cheng M ，Li T ，Hu E ，Yan Q ，Li H ，Wang Y ，Luo J ，Tang T ... -  《-》

The therapeutic mechanism of Yuye decoction on type 2 diabetes mellitus based on network pharmacology and experimental verification.

Yuye decoction (YYD) has been widely used as a folk Chinese herbal formula in clinical treatment of type 2 diabetes mellitus(T2DM) for many years. However, its mechanism is still unclear. The aim of this study was to explore the potential mechanism of YYD against T2DM initially by UHPLC-MS/MS combining with network pharmacology, molecular docking techniques and experimental validation. The main ingredients in the water extract of YYD were initially identified using UHPLC-MS/MS analysis. Combined with network pharmacology and molecular docking techniques, the YYD key compounds-core targets-key signaling pathways network was constructed and the binding activity of key components to core targets was validated. The T2DM rat model was induced by Streptozotocin combined with high glucose and high fat diets. The apoptosis cell model of mouse islet β-cell of Min6 was induced by high-glucose and palmitic acid. Histopathological and immunofluorescence satining were used to evaluate pancreatic islet β-cell function and apoptosis in rats. Min6 cell viability and apoptosis ratio were evaluated by CCK-8 and TUNEL staining. The predicted targets and pathways were validated by experiments in vitro and in vivo. The 56 compounds from YYD were identified by UHPLC-MS/MS. The potential targets of the above compounds were predicted by online compound target database, among of which 362 targets were associated with T2DM. Protein-protein interaction analysis identified the main targets such as SRC, MAPK1, PIK3R1, AKT1, HRAS and HSP90AA1, which were considered as the therapeutic targets of YYD on against T2DM. Functional enrichment analysis revealed that PI3K/AKT, FoxO and apoptosis signaling pathways were significantly enriched. Molecular docking results showed that compounds of monolinolein, neomangiferin, mangiferin, pelargonidin-3-O-glucoside and acacetin from YYD had high binding activities to PIK3R1, AKT1, Sirt1 and FoxO1. Therefore, PI3K/AKT1, Sirt1/FoxO1 and apoptotic signaling pathways were considered as predicted targets for experimental validation study. Animal experiments showed that YYD reduced blood glucose levels, improved pancreatic dysfunction and pancreatic islet β-cells apoptosis in T2DM rats which contributed to the activation of AKT1 and FoxO1 and their related signaling molecules. These results were confirmed in Min6 cell model induced by high-glucose and palmitic acid. In summary, this study systematically visualized the possible therapeutic effects and mechanisms of YYD on T2DM through the network pharmacology approach and experimental study. The results indicated that YYD could prevent pancreatic islet dysfunction and reverse islet of β-cells apoptosis possibly via PI3K/AKT1, Sirt1/FoxO1 signaling pathways.

Guo F ，Yao L ，Zhang W ，Chen P ，Hao R ，Huang X ，Jiang J ，Wu S ... -  《-》