Benzoylaconine Protects Skeletal Muscle Against Ischemia-Reperfusion Injury Through Activation of IF1-Dependent AMPK/Nrf2 Axis.

Background: Aconitum carmichaelii (Fuzi) has been conventionally used to cure a variety of ailments, such as pain, cold sensations, and numbness of limb muscles (Bi Zheng) in China. Our prior investigations identified Benzoylaconine (BAC) as a bioactive alkaloid derived from Aconitum carmichaelii, with other studies also demonstrating its significant pharmacological potential. Purpose: This study aimed to explore the potential of BAC as a protective agent against skeletal muscle ischemia-reperfusion (I/R) injury and to elucidate the underlying mechanisms. Methods: In vivo models involved subjecting Sprague-Dawley rats to I/R through femoral artery ligation followed by reperfusion, while in vitro models utilized C2C12 cells subjected to hypoxia/reoxygenation (H/R). CCK-8 assay was used to assess cell viability. TUNEL staining and flow cytometric analysis were used to measure cell apoptosis. Biochemical assay was used to assess skeletal muscle injury and oxidative stress. Immunofluorescence and Western blot were performed to determine protein levels. Results: BAC effectively protected muscle tissue from I/R injury, enhancing cell viability (p<0.01), elevating SOD levels (p<0.05), and reducing CK (p<0.01), LDH (p<0.01), ROS (p<0.01), MDA (p<0.01), and apoptosis-related molecules in vivo and in vitro (p<0.05, p<0.01). Mechanistically, BAC increased the expression of IF1, phosphorylated AMPK, facilitated the translocation of nuclear Nrf2, and induced the expression of HO-1 (p<0.01). Notably, AMPK inhibitor Compound C significantly hindered the ability of BAC to ameliorate H/R-induced cell injury (p<0.05), oxidative stress(p<0.01), and apoptosis (p<0.05), as well as promote Nrf2 nuclear translocation (p<0.01). Moreover, silencing of IF1 with siRNA abolished BAC-induced activation of AMPK/Nrf2 axis (p<0.01). Conclusion: Our study provides novel evidence supporting the potential of BAC as a myocyte-protective agent against I/R injury, and we establish a previously unknown mechanism involving the activation of the IF1-dependent AMPK/Nrf2 axis in mediating the protective effects of BAC.

Theoretical Prediction and Experimental Synthesis of Zr3AC2 (A = Cd, Sb) Phases.

MAX phases have great research value and application prospects, but it is challenging to synthesize the MAX phases containing Cd and Sb for the time being. In this paper, we confirmed the existence of the 312 MAX phases of Zr3CdC2 and Zr3SbC2, both from theoretical calculations and experimental synthesis. The Zr3AC2 (A = Cd, Sb) phase was predicted by the first-principles calculations, and the two MAX phases were confirmed to meet the requests of thermal, thermodynamic, and mechanical stabilities using formation energy, phonon dispersion, and the Born-Huang criteria. Their theoretical mechanical properties were also systematically investigated. It was found that the elastic moduli of Zr3CdC2 and Zr3SbC2 were 162.8 GPa and 164.3 GPa, respectively. Then, differences in the mechanical properties of Zr3AC2 (A = Cd, In, Sn, and Sb) were explained using bond layouts and charge transfers. The low theoretical Vickers hardness of the Zr3CdC2 (5.4 GPa) and Zr3SbC2 (4.3 GPa) phases exhibited excellent machinability. Subsequently, through spark plasma sintering, composites containing Zr3CdC2 and Zr3SbC2 phases were successfully synthesized at the temperatures of 850 °C and 1300 °C, respectively. The optimal molar ratio of Zr:Cd/Sb:C was determined as 3:1.5:1.5. SEM and the EDS results analysis confirmed the typical layered microstructure of Zr3CdC2 and Zr3SbC2 grains.

Benzoylaconitine: A promising ACE2-targeted agonist for enhancing cardiac function in heart failure.

Benzoylaconitine is a natural product in the treatment of cardiovascular disease. However, its pharmacological effect, direct target protein, and molecular mechanisms for the treatment of heart failure are unclear. In this study, benzoylaconitine inhibited Ang II-induced cell hypertrophy and fibrosis in rat primary cardiomyocytes and rat fibroblasts, while attenuating cardiac function and cardiac remodeling in TAC mice. Using the limited proteolysis-mass spectrometry (LiP-MS) method, the angiotensin-converting enzyme 2 (ACE2) was confirmed as a direct binding target of benzoylaconitine for the treatment of heart failure. In ACE2-knockdown cells and ACE2-/- mice, benzoylaconitine failed to ameliorate cardiomyocyte hypertrophy, fibrosis, and heart failure. Online RNA-sequence analysis indicated p38/ERK-mediated mitochondrial reactive oxygen species (ROS) and nuclear factor kappa B (NF-κB) activation are the possible downstream molecular mechanisms for the effect of BAC-ACE2 interaction. Further studies in ACE2-knockdown cells and ACE2-/- mice suggested that benzoylaconitine targeted ACE2 to suppress p38/ERK-mediated mitochondrial ROS and NF-κB pathway activation. Our findings suggest that benzoylaconitine is a promising ACE2 agonist in regulating mitochondrial ROS release and inflammation activation to improve cardiac function in the treatment of heart failure.

Gandi Capsule Improved Podocyte Lipid Metabolism of Diabetic Nephropathy Mice through SIRT1/AMPK/HNF4A Pathway.

Podocyte lipid accumulation is a potential therapeutic target for diabetic nephropathy (DN). This study was aimed at clarifying the mechanism of Gandi capsule (GDC) ameliorating DN by regulating the lipid metabolism of podocytes. Network pharmacology methods were performed to screen the key molecules and potential targets of GDC for constructing the molecular-protein interaction network of GDC and conducting signal pathway enrichment analysis. GDC was predicted to ameliorate DN through SIRT1/AMPK/HNF4A pathway. Our results showed that GDC improved renal function in db/db mice. Besides, GDC exhibited effectiveness in relieving kidney tissue damage and renal lipid accumulation in db/db mice, and same effects were present in GDC-active ingredient baicalin. We further proved the new role of HNF4A in the lipid metabolism of DN mediated by SIRT1 and AMPK signaling pathways. The results suggested decreased expression of SIRT1 and p-AMPKα in the kidney tissue and increased expression of HNF4A of db/db mice compared with the control group. GDC and baicalin could reverse these expression changes. Furthermore, similar expression changes were observed in the murine podocyte cell line (MPC-5) treated with different concentrations of GDC and baicalin. Our research suggested that GDC and its active ingredient baicalin could alleviate the abnormal lipid metabolism in the kidney of db/db mice and might exert renal protection through the SIRT1/AMPK/HNF4A pathway.

A Novel Modulator of the Renin-Angiotensin System, Benzoylaconitine, Attenuates Hypertension by Targeting ACE/ACE2 in Enhancing Vasodilation and Alleviating Vascular Inflammation.

The monoester alkaloids in Aconitum carmichaelii, including benzoylaconitine (BAC), benzoylmesaconine, and benzoylhypaconitine, were found to have anti-hypertensive effects in spontaneously hypertension rats (SHRs), of which BAC is the strongest. However, its antihypertensive target and underlying molecular mechanisms remain unclear. In this study, first, we screened the antihypertensive targets of BAC by using the CVDPlatform (www.cbligand.org/CVD) and found that ACE/ACE2 are the most possible targets. Then, we verified the effect of BAC on ACE/ACE2 by virtual docking, SPR, enzyme activity assay, and HUVECs cell experiment. We found that BAC could bind with ACE/ACE2, inhibit ACE activity and protein expression, and activate ACE2 enzyme activity. Using vascular function test in vitro, we found that BAC could target ACE/ACE2 to enhance endothelium-dependent vasorelaxation. In BAC-treated SHRs, the levels of ACE and AngII in serum were reduced while Ang (1-7) was increased significantly, and the expression of ACE was reduced, which suggested that BAC can inhibit ACE and activate ACE2 to inhibit AngI to AngII and promote AngII to Ang (1-7) to inhibit vasoconstriction and finally attenuate hypertension. Furthermore, the signaling pathways with regard to vasorelaxation and vascular inflammation were investigated. The results showed that BAC could significantly activate Akt/eNOS, increase NO production, and promote endothelial-related vasodilation; BAC could also reduce inflammatory factors TNF-α and IL6, inhibition of COX-2 expression, and IKB-α phosphorylation to reduce vascular inflammation in SHRs. In brief, BAC targets ACE/ACE2 to enhance endothelium-dependent vasorelaxation and reduce vascular inflammation to attenuate hypertension as a potential modulator of the renin-angiotensin system.

Schisandrin B targets cannabinoid 2 receptor in Kupffer cell to ameliorate CCl4-induced liver fibrosis by suppressing NF-κB and p38 MAPK pathway.

BACKGROUND: Lignans, the major bioactive components of Schisandra chinensis, displays an anti-liver fibrosis effect. However, which one is the most effective lignan and what is its molecular mechanisms are still unclear. PURPOSE: This research aimed to screen the most effective components of lignans, identify and verify its pharmacological target, and investigate its molecular mechanism against liver fibrosis. METHODS: First, the most effective lignans were screened by a comprehensive RAW264.7/CMC system and LPS-induced RAW264.7. Second, the potential targets were predicted by a liver fibrosis domain-specific chemo-genomics knowledgebase and further verified by competition binding assay. Third, the effect of anti-liver fibrosis was evaluated by employing RAW264.7, co-cultured hepatic stellate cells (HSC) and CCl4-induced liver fibrosis CB2-/- mice. The qPCR, ELISAs, western blot analyses, and immunofluorescence were used to evaluate the expression of main inflammatory factors and key proteins in NF-κB and p38 MAPK pathway. RESULTS: Schisandrin B was identified as the most effective component for attenuating liver fibrosis, and CB2 was proven to be a potential target for anti-liver fibrosis. The in vitro and in vivo assays indicated that schisandrin B ameliorated CCl4-induced liver fibrosis through suppressing NF-κB and p38 MAPK pathway in Kupffer cells by targeting CB2 receptor CONCLUSION: Schisandrin B targets CB2 receptor to inhibit Kupffer cell polarization by downregulating the NF-κB and p38 MAPK signaling pathways for ameliorating liver fibrosis.

Active-Ingredient Screening and Synergistic Action Mechanism of Shegan Mixture for Anti-Asthma Effects Based on Network Pharmacology in a Mouse Model of Asthma.

BACKGROUND: Shegan Mixture (SGM) is a traditional Chinese medicine that has anti-inflammatory and therapeutic effects on asthma. However, its active ingredients and combined action mechanism have not been fully elucidated so far. The purpose of this study was to screen the effective ingredients and targets and elucidate the synergistic action mechanism of SGM in asthma mice using the network pharmacological approach. METHODS: A mouse model of asthma model was used in this study. Mice were orally administered SGM at three doses for 4 weeks and the effect of SGM on asthma was evaluated. The active ingredients and their targets of SGM were identified by searching databases, such as Traditional Chinese Medicine Systems Pharmacology Database (TCMSP). The main active ingredients were selected with parameters OB and DL. The synergistic action mechanisms of SGM in asthma were studied through key active ingredient-target interaction network and verified using surface plasmon resonance assay (SPR). RESULTS: SGM exerts anti-asthmatic effects by reducing lung tissue damage and inflammatory factors (IFN-γ, IL-4, IL-5, and IL-13) in asthmatic mice. Twenty ingredients and 45 related proteins were selected as potential nodes using enrichment analysis and network analysis. Inflammation and smooth muscle regulation-related pathways were considered to be the main pharmacological mechanisms of SGM in the treatment of asthma. Especially, 5 molecule-target pairs (including 3 ingredients and 4 proteins) were well docked with each other and the SPR assay revealed that glabridin-PTGS2 had good binding with 44.5 µM Kd value. CONCLUSION: SGM exerts the synergistic anti-asthma effects by virtue of reducing lung-tissue damage and inflammatory factors in asthmatic mice, which explains the theoretical basis for the traditional Chinese medicine, SGM, to treat asthma. Our study thus sheds light on a variety of options including Chinese medicine that could potentially be used in the clinical treatment of asthma.

Human drug efflux transporter ABCC5 confers acquired resistance to pemetrexed in breast cancer.

AIM: Pemetrexed, a new generation antifolate drug, has been approved for the treatment of locally advanced or metastatic breast cancer. However, factors affecting its efficacy and resistance have not been fully elucidated yet. ATP-binding cassette (ABC) transporters are predictors of prognosis as well as of adverse effects of several xenobiotics. This study was designed to explore whether ABC transporters affect pemetrexed resistance and can contribute to the optimization of breast cancer treatment regimen. METHODS: First, we measured the expression levels of ABC transporter family members in cell lines. Subsequently, we assessed the potential role of ABC transporters in conferring resistance to pemetrexed in primary breast cancer cells isolated from 34 breast cancer patients and the role of ABCC5 in mediating pemetrexed transport and apoptotic pathways in MCF-7 cells. Finally, the influence of ABCC5 expression on the therapeutic effect of pemetrexed was evaluated in an in vivo xenograft mouse model of breast cancer. RESULTS: The expression levels of ABCC2, ABCC4, ABCC5, and ABCG2 significantly increased in the pan-resistant cell line, and the ABCC5 level in the MCF-7-ADR cell line was 5.21 times higher than that in the control group. ABCC5 expression was inversely correlated with pemetrexed sensitivity (IC50, r = 0.741; p < 0.001) in breast cancer cells derived from 34 patients. Furthermore, we found that the expression level of ABCC5 influenced the efflux and cytotoxicity of pemetrexed in MCF-7 cells, with IC50 values of 0.06 and 0.20 µg/mL in ABCC5 knockout and over-expression cells, respectively. In the in vivo study, we observed that ABCC5 affected the sensitivity of pemetrexed in breast tumor-bearing mice, and the tumor volume was much larger in the ABCC5-overexpressing group than in the control group when compared with their own initial volumes (2.7-fold vs. 1.3-fold). CONCLUSIONS: Our results indicated that ABCC5 expression was associated with pemetrexed resistance in vitro and in vivo, and it may serve as a target or biomarker for the optimization of pemetrexed regimen in breast cancer treatment.

ARTP/EMS-combined multiple mutagenesis efficiently improved production of raw starch-degrading enzymes in Penicillium oxalicum and characterization of the enzyme-hyperproducing mutant.

BACKGROUND: Application of raw starch-degrading enzymes (RSDEs) in starch processing for biofuel production can effectively reduce energy consumption and processing costs. RSDEs are generally produced by filamentous fungi, such as Penicillium oxalicum, but with very low yields, which seriously hampers industrialization of raw starch processing. Breeding assisted by random mutagenesis is an efficient way to improve fungal enzyme production. RESULTS: A total of 3532 P. oxalicum colonies were generated after multiple rounds of mutagenesis, by atmospheric and room-temperature plasma (ARTP) and/or ethyl methanesulfonate (EMS). Of these, one mutant A2-13 had the highest RSDE activity of 162.7 U/mL, using raw cassava flour as substrate, a yield increase of 61.1%, compared with that of the starting strain, OXPoxGA15A. RSDE activity of A2-13 further increased to 191.0 U/mL, through optimization of culture conditions. Increased expression of major amylase genes, including the raw starch-degrading glucoamylase gene, PoxGA15A, and its regulatory gene, PoxAmyR, as well as several single-nucleotide polymorphisms in the A2-13 genome, were detected by real-time reverse transcription quantitative PCR and genomic re-sequencing, respectively. In addition, crude RSDEs produced by A2-13, combined with commercial α-amylase, could efficiently digest raw corn flour and cassava flour at 40 °C. CONCLUSIONS: Overall, ARTP/EMS-combined mutagenesis effectively improved fungal RSDE yield. An RSDE-hyperproducing mutant, A2-13, was obtained, and its RSDEs could efficiently hydrolyze raw starch, in combination with commercial α-amylase at low temperature, which provides a useful RSDE resource for future starch processing.

Revealing active components, action targets and molecular mechanism of Gandi capsule for treating diabetic nephropathy based on network pharmacology strategy.

BACKGROUND: Gandi capsule is a traditional Chinese herbal formula used to promote blood circulation and removing blood stasis in clinical. Our previous study has shown that it reduces proteinuria with routine treatment in diabetic nephrophy (DN), but its pharmacological action mechanism is still unknown. METHODS: To facilitate the identification of components, a component database of Gandi capsule and target database of DN were established by ourselves. The components absorbed in blood circle were identified in rat plasma after oral administration of Gandi capsule by UHPLC-QQQ-MS/MS. The potential targets were screened by using Libdock tolls in Discovery studio 3.0. Then Pathway and Network analyses were used to enrich the screened targets. The possible targets were verified by using a surface plasmon resonance (SPR) test and the molecular mechanism focusing these targets for treating DN was clarified by western blot. RESULTS: Six components in Gandi capsule were identified detected in rat plasma after oral administration by UHPLC-QQQ-MS/MS. After molecular docking analyses in KEGG and Discovery studio, four protein targets including HNF4A, HMGCR, JAK3, and SIRT1, were screened out, and proved as effective binding with baicalin, wogonoside by SPR. And the molecular mechanism was clarified that baicalin and wogonoside inhibit the effect of high glucose (HG)-induced decreased cell viability and podocin expression, and strengthen the activation p-AKT, p-PI3K, and p-AMPK. CONCLUSION: Baicalin and wogonoside were screened out to be the active compounds in Gandi capsule and can ameliorate HG-induced podocyte damage by influencing the AMPK and PI3K-AKT signaling pathways by binding with HNF4A, HMGCR, JAK3, and SIRT1.

Total laparoscopic nephroureterectomy for upper urinary tract urothelial carcinoma under a single surgical position.

BACKGROUND: To assess the feasibility and effectiveness of total laparoscopic nephroureterectomy for upper urinary tract urothelial carcinoma (UUTUC) under a single surgical position. METHODS: The medical data of 89 UUTUC patients were collected, who were treated in our institution from Jan 2016 to Jun 2018. The 45 cases that underwent total laparoscopic nephroureterectomy with a single position were allocated in the test group, while the 44 patients who received retroperitoneal laparoscopy combined with hypogastric oblique incision were assigned in the control group. We compared the two groups in perioperative indicators and tumor recurrence rate and analyzed the clinical effect of the new surgical treatment of UUTUC. RESULTS: All 89 operations for UUTUC were successful and had no conversion to open surgery. No obvious complications occurred during the perioperative period. The test group had significantly shorter average operation time (96.58 ± 8.56 min versus 147.45 ± 9.16 min), less blood loss (39.58 ± 4.15 ml versus 46.50 ± 4.58 ml), earlier ambulation (7.47 ± 1.01 h versus 11.39 ± 1.82 h), and shorter length of stay in hospital (6.98 ± 1.14 days versus 9.89 ± 1.51 days) (P < 0.05). The visual analogue scale (VAS) scores of the test group at 1 h, 12 h, and 24 h after operation were lower compared with those of the control group (P < 0.05). No significant difference was found in the tumor stage, tumor grade, postoperative gastrointestinal function recovery time, follow-up time, and tumor recurrence rate between the two groups. CONCLUSIONS: Compared with the traditional surgical methods, the total laparoscopic treatment of UUTUC under a single surgical position had advantages of shorter operation time, less blood loss, and early postoperative ambulation. The new operative method could shorten the length of stay and accelerate recovery of patients, and it is a viable surgical procedure which deserved clinical application and promotion. TRIAL REGISTRATION: Our trial was approved and has been registered in the ethics committee of the Yantai Yuhuangding Hospital (Approval NO.[2015]171) .

Meta-analysis of efficacy and safety of custirsen in patients with metastatic castration-resistant prostate cancer.

Custirsen is the second-generation antisense oligonucleotide (ASO), which can reduce cellular levels of clusterin to increase the cytotoxic effect of chemotherapeutic drugs. Our study assessed the efficacy and safety of custirsen in patients with metastatic castration-resistant prostate cancer (mCRPC).We conducted a comprehensive search to identify all the randomized controlled trials (RCTs) of custirsen for the treatment of mCRPC. The reference lists of the retrieved studies were investigated.Three publications involving a total of 1709 patients were used in the analysis. We found that overall survival (OS) (P = .25) was not statistically significant in the comparison. Safety assessments indicated custirsen were often associated with complications resulting from neutropenia (P < .001), anaemia (P < .001), thrombocytopenia (P < .001), and diarrhea (P = .002).Our meta-analysis shows that custirsen has no obvious effect on improving the OS of patients with mCRPC. Adverse reactions were more common among those patients treated with custirsen as compared to those treated with placebo.

Revealing Synergistic Mechanism of Multiple Components in Gandi Capsule for Diabetic Nephropathy Therapeutics by Network Pharmacology.

Gandi capsule, a traditional Chinese herbal medicinal formulation that consists of eight herbs, is used as a clinical therapy for diabetic nephropathy. To clarify the potential synergistic mechanism, this study adopted a network pharmacology strategy to screen the action targets that corresponded to the active components in the Gandi capsule. We first constructed a compound database of 315 components in the Gandi capsule and a target database of diabetic nephropathy, which included 155 target proteins. Six representative compounds were selected to dock with 99 proteins found in the UniProtKB database with their PDB code, and interaction networks between the active ingredients of the Gandi capsule and their targets were mapped out. Results revealed 47 proteins with a high affinity with at least one compound molecule in the Gandi capsule. The main action pathways closely related to the development of diabetic nephropathy were the TGF-ß1, AMPK, insulin, TNF-α, and lipid metabolism pathways as per network pharmacology analysis. In the interaction network, ACC1, SOD2, COX2, PKC-B, IR, and ROCK1 proteins had the most frequent interactions with the six compounds. We performed visual molecular docking in silico and experimentally confirmed competitive component-protein binding by SPR and an enzyme activity test, which highlighted the relationships of wogonin to COX2 and SOD2, astragaloside IV to ACC1, and morroniside to ACC1. We concluded that the potential synergistic mechanism of the Gandi capsule resulted from high affinities with multiple proteins and intervention in multiple pathways in combination therapy of diabetic nephropathy.