An herbal formulation "Shenshuaifu Granule" alleviates cisplatin-induced nephrotoxicity by suppressing inflammation and apoptosis through inhibition of the TLR4/MyD88/NF-κB pathway.

Shenshuaifu Granule (SSF) is an in-hospital preparation approved by the Guangdong Food and Drug Administration of China. It has been clinically used against kidney diseases for more than 20 years with a definite curative effect. Cisplatin (CDDP) is a first-line chemotherapeutic drug in clinical practice, primarily excreted by the kidney with nephrotoxicity as a common side effect. Approximately 5-20% of cancer patients develop acute kidney injury (AKI) after chemotherapy; however, prevention and control strategies are currently unavailable. Therefore, it is important to identify safe and effective drugs that can prevent the nephrotoxicity of CDDP. SSF is an herbal formulation with 8 herbs, and has been used to protect the kidney in China. Nonetheless, its mechanism in relieving CDDP nephrotoxicity remains unclear. Therefore, this work attempt to prove that SSF can alleviate CDDP nephrotoxicity. We also explore its mechanism. First, Thin Layer Chromatography (TLC) of a few herbs in SSF were performed for quality control. Several open-access databases were used to identify the active ingredients of SSF, their corresponding targets, and CDDP-induced nephrotoxicity targets. We performed Protein-Protein Interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Next, the results of network pharmacology were validated using CDDP-induced nephrotoxicity mouse models. Renal function in the mice was assessed by analyzing the levels of serum creatinine (Scr) and blood urea nitrogen (BUN). On the other hand, renal damage was assessed by determining the level of tubular injury and apoptotic cells using Periodic acid-Schiff (PAS) staining and Terminal Dutp Nick End-Labeling (TUNEL) staining, respectively. The expression of inflammatory and apoptotic-related targets including IL-1β, IL-6, TNF-α, Cox-2, Bax, Bcl-2, Cleaved-caspase 3, and Cleaved-caspase 9 was determined using Western Blot (WB) and Immunohistochemistry (IHC). Furthermore, WB was used to analyze the expression of proteins associated with the TLR4/MyD88/NF-κB pathway in the kidneys of mice with CDDP-induced nephrotoxicity. Finally, molecular docking simulations were performed to evaluate the binding abilities between major active ingredients of SSF and core targets. Through network pharmacology, we identified 127 active ingredients of SSF and their corresponding 134 targets. Additional screening identified 14 active ingredients and 17 targets for further analysis. In biological process (BP), the targets were enriched in inflammation and apoptosis, among others. In KEGG terms, they were enriched in apoptosis and NF-κB pathways. Animal experiments revealed that SSF significantly reduced the levels of Scr and BUN and prevented renal tubular damage in mice treated with CDDP. In addition, SSF inhibited inflammation and apoptosis by targeting the TLR4/MyD88/NF-κB pathway. Molecular docking revealed good binding capacities of active ingredients and core targets. In summary, the experimental findings were consistent with the network pharmacological predictions. SSF can inhibit inflammation and apoptosis by targeting the TLR4/MyD88/NF-κB pathway. Taken together, our data suggest that SSF is an alternative agent for the treatment of CDDP-induced nephrotoxicity.

Jin X ，He R ，Liu J ，Wang Y ，Li Z ，Jiang B ，Lu J ，Yang S ... -  《-》

Shenshuaifu Granule Attenuates Acute Kidney Injury by Inhibiting Ferroptosis Mediated by p53/SLC7A11/GPX4 Pathway.

Acute kidney injury (AKI) is a common clinical condition resulting in a rapid decline in renal function, and requires improvement in effective preventive measures. Ferroptosis, a novel form of cell death, is closely related to AKI. Shenshuaifu granule (SSF) has been demonstrated to prevent AKI through suppressing inflammation and apoptosis. This study aimed to explore whether SSF can inhibit ferroptosis in AKI. Active ingredients in SSF were detected through HPLC-MS/MS, and their binding abilities with ferroptosis were evaluated by molecular docking. Then, male C57/BL/6J mice were randomly divided into control, cisplatin, and cisplatin+SSF groups. In the latter two groups, mice were intraperitoneally injected with 20 mg/kg of cisplatin. For five consecutive days prior to cisplatin injection, mice in the cisplatin+SSF group were gavaged with 5.2 g/kg of SSF per day.72 h after cisplatin injection, the mice were sacrificed. Serum creatinine (SCr) and blood urea nitrogen (BUN) were measured to evaluate renal function. H&E and PAS staining were used to observe pathological damage of kidney. Cell death was observed by TUNEL staining, and iron accumulation in kidneys of mice was detected by Prussian blue staining. Western blotting, immunohistochemistry, and immunofluorescence were used to investigate the presence of inflammation, oxidative stress, mitochondrial dysfunction, iron deposition, and lipid peroxidation in mouse kidneys. Active ingredients in SSF had strong affinities with ferroptosis. SSF reduced SCr (p<0.01) and BUN (p<0.0001) levels, pathological damage (p<0.0001), dead cells in the tubular epithelium (p<0.0001) and iron deposition (p<0.01) in mice with cisplatin induced AKI. And SSF downregulated macrophage infiltration (p<0.01), the expressions of high mobility group box 1 (HMGB1, p<0.05) and interleukin (IL)-17 (p<0.05), upregulated superoxide dismutase (SOD) 1 and 2 (p<0.01), and catalase (CAT, p<0.05), and alleviated mitochondrial dysfunction (p<0.05). More importantly, SSF regulated iron transport and intracellular iron overload and reduced the expression of ferritin (p<0.05). Moreover, it downregulated the expressions of cyclo-oxygenase-2 (Cox-2, p<0.001), acid CoA ligase 4 (ACSL4, p<0.05), and solute carrier family 7, member 11 (SLC7A11, p<001), upregulated glutathione peroxidase 4 (GPX4, p<0.01) and p53 (p<0.01), and decreased 4-hydroxynonenal (4-HNE) level (p<0.001). SSF attenuates AKI by inhibiting ferroptosis mediated by p53/SLC7A11/GPX4 pathway.

Jin X ，He R ，Lin Y ，Liu J ，Wang Y ，Li Z ，Liao Y ，Yang S ... -  《Drug Design Development and Therapy》

[Mechanism of Fushen Granules treat peritoneal dialysis-related peritonitis by regulating TLR4/NF-κB pathway:based on network pharmacology and animal experiments].

Xing HT ，Ding FM ，Li JQ ，Zhou QY ，Yang HT ... -  《-》

An herbal formulation "Shugan Xiaozhi decoction" ameliorates methionine/choline deficiency-induced nonalcoholic steatohepatitis through regulating inflammation and apoptosis-related pathways.

Shugan Xiaozhi (SGXZ) decoction is a traditional Chinese medicine used for treating nonalcoholic steatohepatitis (NASH). It has been used clinically for over 20 years and proved to be effective; however, the molecular mechanism underlying the effects of SGXZ decoction remains unclear. We analyzed the chemical components, core targets, and molecular mechanisms of SGXZ decoction to improve NASH through network pharmacology and in vivo experiments. The chemical components, core targets, and related signaling pathways of SGXZ decoction intervention in NASH were predicted using network pharmacology. Molecular docking was performed to verify chemical components and their core targets. The results were validated in the NASH model treated with SGXZ decoction. Mouse liver function was assessed by measuring ALT and AST levels. TC and TG levels were determined to evaluate lipid metabolism, and lipid deposition was assessed via oil red O staining. Mouse liver damage was determined via microscopy following hematoxylin and eosin staining. Liver fibrosis was assessed via Masson staining. Western blot (WB) and immunohistochemical (IHC) analyses were performed to detect inflammation and the expression of apoptosis-related proteins, including IL-1β, IL-6, IL-18, TNF-α, MCP1, p53, FAS, Caspase-8, Caspase-3, Caspase-9, Bax, Bid, Cytochrome c, Bcl-2, and Bcl-XL. In addition, WB and IHC were used to assess protein expression associated with the TLR4/MyD88/NF-κB pathway. Quercetin, luteolin, kaempferol, naringenin, and nobiletin in SGXZ decoction were effective chemical components in improving NASH, and TNF-α, IL-6, and IL-1β were the major core targets. Molecular docking indicated that these chemical components and major core targets might interact. KEGG pathway analysis showed that the pathways affected by SGXZ decoction, primarily including apoptosis and TLR4/NF-κB signaling pathways, interfere with NASH. In vivo experiments indicated that SGXZ decoction considerably ameliorated liver damage, fibrosis, and lipid metabolism disorder in MCD-induced NASH mouse models. In addition, WB and IHC verified the underlying molecular mechanisms of SGXZ decoction as predicted via network pharmacology. SGXZ decoction inhibited the activation of apoptosis-related pathways in MCD-induced NASH mice. Moreover, SGXZ decoction suppressed the activation of TLR4/MyD88/NF-κB pathway in MCD-induced NASH mice. SGXZ decoction can treat NASH through multiple targets and pathways. These findings provide new insights into the effective treatment of NASH using SGXZ decoction.

Wang S ，Chen B ，Du R ，Zhong M ，Zhang C ，Jin X ，Cui X ，Zhou Y ，Kang Q ，Xu H ，Li Y ，Wu Q ，Tong G ，Luo L ... -  《-》

Hua-Feng-Dan Alleviates LPS-induced Neuroinflammation by Inhibiting the TLR4/Myd88/NF-κB Pathway: Integrating Network Pharmacology and Experimental Validation.

Neuroinflammation is the pathological basis of many neurological diseases, including neurodegenerative diseases and stroke. Hua-Feng-Dan (HFD) is a well-established traditional Chinese medicine that has been used for centuries to treat stroke and various other brain-related ailments. Our study aims to elucidate the molecular mechanism by which HFD mitigates neuroinflammation by combining network pharmacology and in vitro experiments. TCMSP and SymMap databases were used to extract active compounds and their related targets. The neuroinflammation-related targets were obtained from the GeneCards database. The common targets of HFD and neuroinflammation were used to construct a protein-protein interaction (PPI) network. MCODE plug-in was used to find the hub module genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to dissect the hub module genes. The lipopolysaccharide (LPS)-induced BV2 microglial neuroinflammation model was utilized to assess the therapeutic effects of HFD on neuroinflammation. Western blotting analysis was performed to examine the core target proteins in the TLR4/My- D88/NF-κB signaling pathway, potentially implicated in HFD's therapeutic effects on neuroinflammation. Hoechst 33342 staining and JC-1 staining were employed to evaluate neuronal apoptosis. Through network pharmacology, 73 active compounds were identified, with quercetin, beta-sitosterol, luteolin, and (-)-Epigallocatechin-3-Gallate recognized as important compounds. Meanwhile, 115 common targets of HFD and neuroinflammation were identified, and 61 targets were selected as the hub targets utilizing the MCODE algorithm. The results of in vitro experiments demonstrated that HFD significantly inhibited microglial-mediated neuronal inflammation induced by LPS. Integrating the predictions from network pharmacology with the in vitro experiment results, it was determined that the mechanism of HFD in mitigating neuroinflammation is closely related to the TLR4/MyD88/NF-κB pathway. Furthermore, HFD demonstrated the capacity to shield neurons from apoptosis by curbing the secretion of pro-inflammatory factors subsequent to microglial activation. The findings demonstrated that HFD had an inhibitory effect on LPS-induced neuroinflammation in microglia and elucidated its underlying mechanism. These findings will offer a theoretical foundation for the clinical utilization of HFD in treating neurodegenerative diseases associated with neuroinflammation.

Zhang Z ，Pei Y ，Zheng Y ，Liu Y ，Guo Y ，He Y ，Cheng F ，Wang X ... -  《-》