Sciadonic acid attenuates high-fat diet-induced bone metabolism disorders in mice.

High-fat diet (HFD) has been associated with certain negative bone-related outcomes, such as bone metabolism disruption and bone loss. Sciadonic acid (SC), one of the main nutritional and functional components of Torreya grandis seed oil, is a unique Δ5-unsaturated-polymethylene-interrupted fatty acid (Δ5-UPIFA) that has been claimed to counteract such disorders owing to some of its physiological effects. However, the role of SC in ameliorating bone metabolism disorders due to HFD remains unclear. In the present investigation, we observed that SC modulates the OPG/RANKL/RANK signaling pathway by modifying the lipid metabolic state and decreasing inflammation in mice. In turn, it could balance bone resorption and formation as well as calcium and phosphorus levels, enhance bone strength and bone mineral density (BMD), and improve its microstructure. In addition, SC could inhibit fat vacuoles in bone, reverse the phenomenon of reduced numbers and poor continuity of bone trabeculae, and promote orderly arrangement of collagen fibers and cartilage repair. This study provides some theoretical basis for SC as a dietary intervention agent to enhance bone nutrition.

Advancements of Macrophages Involvement in Pathological Progression of Colitis-Associated Colorectal Cancer and Associated Pharmacological Interventions.

Intestinal macrophages play crucial roles in both intestinal inflammation and immune homeostasis. They can adopt two distinct phenotypes, primarily determined by environmental cues. These phenotypes encompass the classically activated pro-inflammatory M1 phenotype, as well as the alternatively activated anti-inflammatory M2 phenotype. In regular conditions, intestinal macrophages serve to shield the gut from inflammatory harm. However, when a combination of genetic and environmental elements influences the polarization of these macrophages, it can result in an M1/M2 macrophage activation imbalance, subsequently leading to a loss of control over intestinal inflammation. This shift transforms normal inflammatory responses into pathological damage within the intestines. In patients with ulcerative colitis-associated colorectal cancer (UC-CRC), disorders related to intestinal inflammation are closely correlated with an imbalance in the polarization of intestinal M1/M2 macrophages. Therefore, reinstating the equilibrium in M1/M2 macrophage polarization could potentially serve as an effective approach to the prevention and treatment of UC-CRC. This paper aims to scrutinize the clinical evidence regarding Chinese medicine (CM) in the treatment of UC-CRC, the pivotal role of macrophage polarization in UC-CRC pathogenesis, and the potential mechanisms through which CM regulates macrophage polarization to address UC-CRC. Our objective is to offer fresh perspectives for clinical application, fundamental research, and pharmaceutical advancement in UC-CRC.

Effects of topography and stand spatial structure on the diameter at breast height growth of major pioneer tree species of natural broad-leaved mixed forests in Zhejiang Province, China.

Based on the continuous inventory data of forest resources in Zhejiang Province in 2019 and 2021, we used statistical methods such as polynomial regression to analyze the impacts of topography and forest spatial structure on average annual diameter at breast height (DBH) growth of main pioneer tree species in natural broad-leaved mixed forests. The results showed that DBH of Schima superba, Quercus glauca, Quercus fabri, Lithocarpus glaber, Castanopsis eyrei, and Castanopsis sclerophylla were between 5-50.8, 5-41.5, 5-50.8, 5-43.9, 5-55.5, and 5-46.1 cm, respectively. We classified all the trees into three classes based on DBH: small (6-12 cm), medium (12-14 cm), and large (>26 cm). The average annual DBH growth of S. superba and Q. glauca was the highest on semi-shady slope and shady slope, with increases of 2.9%-15.7% and 1.1%-41.2%, respectively. The average annual DBH growth of large-diameter S. superba, L. glaber, C. eyrei and C. sclerophylla decreased with the increase of slope, with a maximum decrease of 27.0% for S. superba, with no significant difference among small- and medium-diameter trees as a whole. The slope position did not affect the annual DBH growth of small-diameter trees, while that of medium- and large-diameter S. superba, Q. glauca, and large-diameter Q. fabri, L. glaber decreased with the change of slope position from downhill, mesoslope, uphill to ridge, with a maximum decrease of 28.1% for L. glabe, and the major-diameter C. eyrei was on the contrary. Appropriate increase in the mingling was beneficial to the average annual DBH growth of medium- and large-diameter trees. Moderate mixing was suitable for S. superba, while low degree mixing and moderate mixing was suitable for Q. glauca, Q. fabri and L. glaber, and intensive mixing was suitable for C. eyrei and C. sclerophylla. No significant difference was observed for minor-diameter trees under the mingling. The neighborhood comparison only had a significant effect on the average annual DBH growth of large-diameter Q. glauca, Q. fabri, and L. glaber, which was significantly higher under subdominance-moderation than moderation-inferiority. The average annual DBH growth in the study area was mainly affected by aspect and mixing degree.

Sinomenine regulates the cholinergic anti-inflammatory pathway to inhibit TLR4/NF-κB pathway and protect the homeostasis in brain and gut in scopolamine-induced Alzheimer's disease mice.

Sinomenine (SIN), an alkaloid extracted from the Chinese herbal medicine Sinomenium acutum, has great potential in anti-inflammatory, immune regulation, analgesic and sedative, and is already a clinical drug for the treatment of rheumatoid arthritis in China. Our previous studies show SIN inhibits inflammation by regulating ɑ7nAChR, a key receptor of cholinergic anti-inflammatory pathway (CAP), which plays an important role in regulating peripheral and central nervous system inflammation. Growing evidence supports the cholinergic dysregulation and inflammatory responses play the key role in the pathogenesis of AD. The intervention effects of SIN on AD by regulating CAP and homeostasis in brain and gut were analyzed for the first time in the present study using scopolamine-induced AD model mice. Behavioral tests were used to assess the cognitive performance. The neurons loss, cholinergic function, inflammation responses, biological barrier function in the mouse brain and intestinal tissues were evaluated through a variety of techniques, and the gut microbiota was detected using 16SrRNA sequencing. The results showed that SIN significantly inhibited the cognitive decline, dysregulation of cholinergic system, peripheral and central inflammation, biological barrier damage as well as intestinal flora disturbance caused by SCOP in mice. More importantly, SIN effectively regulated CAP to suppress the activation of TLR4/NF-κB and protect the homeostasis in brain and gut to alleviate cognitive impairment.

Bushen-Yizhi formula exerts neuroprotective effect via inhibiting excessive mitophagy in rats with chronic cerebral hypoperfusion.

ETHNOPHARMACOLOGICAL RELEVANCE: Bushen-Yizhi formula (BSYZ), a traditional Chinese medicine (TCM) prescription widely used in treating mental retardation and neurodegenerative diseases with kidney deficiency, has been reported to attenuate oxidative stress-related neuronal apoptosis. Chronic cerebral hypoperfusion (CCH) is considered to be related to cognitive and emotional disorders. However, it remains to be clarified that the effect of BSYZ on CCH and its underlying mechanism. AIM OF THE STUDY: In the present study, we aimed to investigate the therapeutic effects and underlying mechanisms of BSYZ on CCH- injured rats based on the domination of oxidative stress balance and mitochondrial homeostasis through inhibiting abnormal excessive mitophagy. MATERIALS AND METHODS: The in vivo rat model of CCH was established by bilateral common carotid artery occlusion (BCCAo), while the in vitro PC12 cell model was exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) condition, and a mitophagy inhibitor (chloroquine) by decreasing autophagosome-lysosome fusion was used as reverse validation in vitro. The protective role of BSYZ on CCH-injured rats was measured by open field test, morris water maze test, analysis of amyloid fibrils and apoptosis, and oxidative stress kit. The expression of mitochondria-related and mitophagy-related proteins was evaluated by Western blot, immunofluorescence, JC-1 staining assay and Mito-Tracker Red CMXRos assay. The components of BSYZ extracts were identified by HPLC-MS. The molecular docking studies were used to investigate the potential interactions of characteristic compounds in BSYZ with lysosomal membrane protein 1 (LAMP1). RESULTS: Our result indicated that BSYZ improved the cognition and memory abilities of the BCCAo rats by diminishing the occurrence of apoptosis and abnormal amyloid deposition accumulation, suppressing oxidative stress damage for abnormal excessive mitophagy activation in the hippocampus. Moreover, in OGD/R-damaged PC12 cells, BSYZ drug serum treatment substantially enhanced the PC12 cell viability and suppressed intracellular reactive oxygen species (ROS) accumulation for protecting against oxidative stress, along with the improvement of mitochondrial membrane activity and lysosomal proteins. Our studies also showed that inhibiting of autophagosome-lysosome fusion to generate autolysosomes by using chloroquine abrogated the neuroprotective effects of BSYZ on PC12 cells regarding the modulation of antioxidant defence and mitochondrial membrane activity. Furthermore, the molecular docking studies supported the direct bindings between lysosomal associated membrane protein 1 (LAMP1) and compounds in BSYZ extract to inhibit excessive mitophagy. CONCLUSION: Our study demonstrated that BSYZ played a neuroprotective role in rats with CCH and reduced neuronal oxidative stress via promoting the formation of autolysosomes to inhibit abnormal excessive mitophagy.

Developability profiling of a panel of Fc engineered SARS-CoV-2 neutralizing antibodies.

To combat the COVID-19 pandemic, potential therapies have been developed and moved into clinical trials at an unprecedented pace. Some of the most promising therapies are neutralizing antibodies against SARS-CoV-2. In order to maximize the therapeutic effectiveness of such neutralizing antibodies, Fc engineering to modulate effector functions and to extend half-life is desirable. However, it is critical that Fc engineering does not negatively impact the developability properties of the antibodies, as these properties play a key role in ensuring rapid development, successful manufacturing, and improved overall chances of clinical success. In this study, we describe the biophysical characterization of a panel of Fc engineered ("TM-YTE") SARS-CoV-2 neutralizing antibodies, the same Fc modifications as those found in AstraZeneca's Evusheld (AZD7442; tixagevimab and cilgavimab), in which the TM modification (L234F/L235E/P331S) reduce binding to FcγR and C1q and the YTE modification (M252Y/S254T/T256E) extends serum half-life. We have previously shown that combining both the TM and YTE Fc modifications can reduce the thermal stability of the CH2 domain and possibly lead to developability challenges. Here we show, using a diverse panel of TM-YTE SARS-CoV-2 neutralizing antibodies, that despite lowering the thermal stability of the Fc CH2 domain, the TM-YTE platform does not have any inherent developability liabilities and shows an in vivo pharmacokinetic profile in human FcRn transgenic mice similar to the well-characterized YTE platform. The TM-YTE is therefore a developable, effector function reduced, half-life extended antibody platform.

Formation of transient highly-charged mAb clusters strengthens interactions with host cell proteins and results in poor clearance of host cell proteins by protein A chromatography.

Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.

Qualification of a Biolayer Interferometry Assay to Support AZD7442 Resistance Monitoring.

AZD7442, a combination of two long-acting monoclonal antibodies (tixagevimab [AZD8895] and cilgavimab [AZD1061]), has been authorized for the prevention and treatment of coronavirus disease 2019 (COVID-19). The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires methods capable of quickly characterizing resistance to AZD7442. To support AZD7442 resistance monitoring, a biolayer interferometry (BLI) assay was developed to screen the binding of tixagevimab and cilgavimab to SARS-CoV-2 spike proteins to reduce the number of viral variants for neutralization susceptibility verification. Six spike variants were chosen to assess the assay's performance: four with decreased affinity for tixagevimab (F486S:D614G and F486W:D614G proteins) or cilgavimab (S494L:D614G and K444R:D614G proteins) and two reference proteins (wild-type HexaPro and D614G protein). Equilibrium dissociation constant (KD) values from each spike protein were used to determine shifts in binding affinity. The assay's precision, range, linearity, and limits of quantitation were established. Qualification acceptance criteria determined whether the assay was fit for purpose. By bypassing protein purification, the BLI assay provided increased screening throughput. Although limited correlation between pseudotype neutralization and BLI data (50% inhibitory concentration versus KD) was observed for full immunoglobulins (IgGs), the correlations for antibody fragments (Fabs) were stronger and reflected a better comparison of antibody binding kinetics with neutralization potency. Therefore, despite strong assay performance characteristics, the use of full IgGs limited the screening utility of the assay; however, the Fab approach warrants further exploration as a rapid, high-throughput variant-screening method for future resistance-monitoring programs. IMPORTANCE SARS-CoV-2 variants harbor multiple substitutions in their spike trimers, potentially leading to breakthrough infections and clinical resistance to immune therapies. For this reason, a BLI assay was developed and qualified to evaluate the reliability of screening SARS-CoV-2 spike trimer variants against anti-SARS-CoV-2 monoclonal antibodies (MAbs) tixagevimab and cilgavimab, the components of AZD7442, prior to in vitro pseudovirus neutralization susceptibility verification testing. The assay bypasses protein purification with rapid assessment of the binding affinity of each MAb for each recombinant protein, potentially providing an efficient preliminary selection step, thus allowing a reduced testing burden in the more technically complex viral neutralization assays. Despite precise and specific measures, an avidity effect associated with MAb binding to the trimer confounded correlation with neutralization potency, negating the assay's utility as a surrogate for neutralizing antibody potency. Improved correlation with Fabs suggests that assay optimization could overcome any avidity limitation, warranting further exploration to support future resistance-monitoring programs.

Effects of precipitation seasonal distribution on net ecosystem CO2 exchange over an alpine meadow in the southeastern Tibetan Plateau.

Ecosystem carbon balance might be affected by the variability of seasonal distribution of precipitation under global climate change. Using the eddy covariance (EC) technique, long-term observations of ecosystem net CO2 exchange (NEE) were acquired over Lijiang alpine meadow in the southeastern Tibetan Plateau from January 2014 to August 2019. During the wet season (from June to October), Lijiang meadow functioned as a carbon sink (- 37.6 ± 22.5 g C m-2 month-1), while in dry season, the meadow varied between a weak carbon source and sink with an average monthly NEE of - 3.9 ± 11.9 g C m-2 month-1. Monthly CO2 fluxes were mainly controlled by air temperature and soil water content. A large annual variation of CO2 uptake was observed. The annual NEE was - 140.3 g C m-2 year-1 in 2014 while - 247.0 g C m-2 year-1 in 2016. Correspondingly, the precipitation in wet season accounted 90% of annual precipitation in 2014 and 74% of that in 2016 despite the annual precipitation was larger than 1200 mm in both years. More precipitation in dry season can lead to longer period of net CO2 uptake, while more precipitation concentrated in wet season depressed the meadow's light response through the decrease of the magnitude of light-saturated net CO2 exchange (NEEsat) at the onset and the end of growing season.

Sinomenine inhibits macrophage M1 polarization by downregulating α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1.

BACKGROUND: Sinomenine (SIN) is an anti-inflammatory drug that has been used for decades in China to treat arthritis. In a previous study, SIN acted on α7 nicotinic acetylcholine receptor (α7nAChR) to inhibit inflammatory responses in macrophages, which indicates a new anti-inflammatory mechanism of SIN. However, the level of α7nAChR was increased in the inflammatory responses and was downregulated by SIN in vitro, so the underlying mechanisms of SIN acting on α7nAChR remain unclear. PURPOSE: To analyze the role of α7nAChR in inflammation and the effect and mechanism of SIN regulation of α7nAChR. METHODS: The effects of SIN on α7nAChR in endotoxemic mice and LPS-stimulated macrophages were observed. Nicotine (Nic) was used as a positive control, and berberine (Ber) was used as a negative control targeting α7nAChR. The antagonists of α7nAChR, α-bungarotoxin (BTX) and mecamylamine (Me), were used to block α7nAChR. In RAW264.7 macrophage cells in vitro, α7nAChR short hairpin RNA (shRNA) was used to knock down α7nAChR. Macrophage polarization was analyzed by the detection of TNF-α, IL-6, iNOS, IL-10, Arg-1, and Fizz1. U0126 was used to block ERK phosphorylation. The cytokines α7nAChR, ERK1/2, p-ERK1/2 and Egr-1 were detected. RESULTS: SIN decreased the levels of TNF-α, IL-6 and the expression of α7nAChR increased by LPS in endotoxemic mice. The above effects of SIN were attenuated by BTX. In the α7nAChR shRNA transfected RAW264.7 cells, compared with the control, α7nAChR was knocked down, and M1 phenotype markers (including TNF-α, IL-6, and iNOS) were significantly downregulated, whereas M2 phenotype markers (including IL-10, Arg-1, and Fizz1) were significantly upregulated when stimulated by LPS. SIN inhibited the expression of p-ERK1/2 and the transcription factor Egr-1 induced by LPS in RAW264.7 cells, and the above effects of SIN were attenuated by BTX. The expression of α7nAChR was suppressed by U0126, which lessened the expression of p-ERK1/2 and Egr-1. CONCLUSIONS: SIN acts on α7nAChR to inhibit inflammatory responses and downregulates high expression of α7nAChR in vivo and in vitro. The increase of α7nAChR expression is correlated with inflammatory responses and participates in macrophage M1 polarization. SIN downregulates α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1, which contributes to inhibiting macrophage M1 polarization and inflammatory responses.

Explore the Anti-Acne Mechanism of Licorice Flavonoids Based on Metabonomics and Microbiome.

Acne vulgaris is one of the most common inflammatory dermatoses in dermatological practice and can affect any gender or ethnic group. Although in previous studies, we had found that licorice flavonoids (LCF) play an anti-acne role by inhibiting PI3K-Akt signaling pathways and mitochondrial activity, the mechanism of LCF regulating skin metabolism, serum metabolism and skin microbes is still unclear. Here, we performed a full spectrum analysis of metabolites in the skin and serum using UHPLC-Triple TOF-MS. The results showed that LCF could treat acne by regulating the metabolic balance of amino acids, lipids and fatty acids in serum and skin. Similarly, we performed Illumina Hiseq sequencing of DNA from the skin microbes using 16S ribosomal DNA identification techniques. The results showed that LCF could treat acne by regulating the skin microbes to interfere with acne and make the microecology close to the normal skin state of rats. In summary, this study confirmed the anti-acne mechanism of LCF, namely by regulating metabolic balance and microbial balance. Therefore, this discovery will provide theoretical guidance for the preparation development and clinical application of the drug.

Electrostatically Driven Protein-Protein Interactions: Quantitative Prediction of Second Osmotic Virial Coefficients to Aid Antibody Design.

Electrostatically driven attractions between proteins can result in issues for therapeutic protein formulations such as solubility limits, aggregation, and high solution viscosity. Previous work showed that a model monoclonal antibody displayed large and potentially problematic electrostatically driven attractions at typical pH (5-8) and ionic strength conditions (∼10-100 mM). Molecular simulations of a hybrid coarse-grained model (1bC/D, one bead per charged site and per domain) were used to predict potential point mutations to identify key charge changes (charge-to-neutral or charge-swap) that could greatly reduce the net attractive protein-protein self-interactions. A series of variants were tested experimentally with static and dynamic light scattering to quantify interactions and compared to model predictions at low and intermediate ionic strength. Differential scanning calorimetry and circular dichroism confirmed minimal impact on structural or thermal stability of the variants. The model provided quantitative/semiquantitative predictions of protein self-interactions compared to experimental results as well as showed which amino acid pairings or groups had the most impact.

The value of positron emission tomography computed tomography in predicting invasiveness of ground glass nodules: A protocol for systematic review and meta-analysis.

BACKGROUND: The study was conducted to investigate the value of Positron emission tomography computed tomography (PET/CT) in predicting invasiveness of ground glass nodule (GGN) by the method of meta-analysis. METHODS: Two researchers independently searched for published literature on PET/CT diagnosis of GGN as of November 30, 2020. After extracting the data, RevMan5.3 was used to evaluate the quality of the included literature. The Stata14 software was used to test the heterogeneity of the original study that met the inclusion criteria, to calculate the combined sensitivity, specificity, positive likelihood ratio and negative likelihood ratio, the prior probability and posttest probability. The summary receiver operator characteristic curve was drawn and the area under the curve was calculated. Using Deeks funnel plot to evaluate publication bias. RESULTS: Five studies were finally included, including 298 GGN cases. The included studies had no obvious heterogeneity and publication bias. The combined sensitivity and specificity of PET/CT for predicting invasive adenocarcinoma presenting as GGN were 0.74 (95% confidence interval [CI]: 0.68-0.79), 0.82 (95% CI: 0.71-0.90), positive likelihood ratio and negative likelihood ratio were 4.1 (95% CI: 2.5-6.9), 0.32 (95% CI: 0.25-0.40), and the diagnostic odds ratio was 13 (95% CI: 7-26). The prior probability is 20%, the probability of GGN being invasive adenocarcinoma when PET/CT was negative was reduced to 7%, and the probability of GGN being invasive adenocarcinoma when PET/CT was positive was increased to 51%. The area under the curve of the summary receiver operator characteristic curve was 0.85. CONCLUSION: PET/CT has high diagnostic accuracy for invasive adenocarcinoma presenting as GGN.

Sinomenine increases adenosine A2A receptor and inhibits NF-κB to inhibit arthritis in adjuvant-induced-arthritis rats and fibroblast-like synoviocytes through α7nAChR.

Sinomenine (SIN) is a clinical drug for treating rheumatoid arthritis (RA) in China. Our previous study found SIN inhibited inflammation via alpha7 nicotinic acetylcholine receptor (α7nAChR) in macrophages in vitro. Adenosine receptor A2A has anti-inflammatory and immunosuppressive function. However, the mechanisms of SIN acting on α7nAChR and the effect on adenosine A2A receptor (A2A R) in RA are not clear. In the present study, the effects of SIN on adjuvant-induced-arthritis (AIA) rats in vivo and on fibroblast-like synoviocytes (FLSs) in vitro were investigated. Indomethacin (Indo) and methotrexate (MTX), the clinical anti-arthritis drugs, were used as controls. Nicotine (Nic), a specific agonist of α7nAChR, was used as a control for targeting α7nAChR. Alpha-bungarotoxin (α-BTX), the antagonist of α7nAChR or small interference RNA (siRNA) was used to block or knock down α7nAChR. Results showed that SIN decreased arthritis index, hind paw volume, erythrocyte sedimentation (ESR) and serum TNF-α in AIA rats, and α-BTX attenuated the earlier-mentioned effects of SIN and Nic, but not Indo and MTX. The expressions of A2A R in synovium declined in AIA rats, but remarkably increased after the intervention of SIN. The expression of A2A R decreased by LPS or TNF-α, but increased by SIN; cAMP also increased by SIN in FLSs in vitro. SIN inhibited the expression of MCP-1, IL-6, and vascular endothelial growth factor in LPS-induced FLSs. SIN inhibited the activation of NF-κB. Meanwhile, α-BTX or α7nAChR siRNA blocked the earlier-mentioned effects of SIN in FLSs. Results suggested the expressions of A2A R in synovium and FLSs are negatively correlated with the arthritis progression of AIA rats and the activation of FLSs. SIN increases A2A R and inhibits the activation of NF-κB pathway via α7nAChR in AIA rats and FLSs.

The effects of Danggui-Shaoyao-San on neuronal degeneration and amyloidosis in mouse and its molecular mechanism for the treatment of Alzheimer's disease.

The abnormal deposition of the extracellular amyloid-ß peptide is the typical pathological hallmark of Alzheimer's disease. Strategies to reduce the amyloid-ß deposition effectively alleviate the neuronal degeneration and cognitive deficits of Alzheimer's disease. Danggui-Shaoyao-San has been considered a useful therapeutic agent known for the treatment of Alzheimer's disease. However, the mechanism of Danggui-Shaoyao-San for the treatment of Alzheimer's disease remains unclear. We investigated Danggui-Shaoyao-San's effect on amyloidosis and neuronal degeneration in an APP/PS1 mouse model. We found Danggui-Shaoyao-San alleviated the cognitive deficits in APP/PS1 mice. Additionally, Danggui-Shaoyao-San ameliorated the neuronal degeneration in these mice. Danggui-Shaoyao-San reduced the amyloidosis and amyloid-ß1-42 deposition in APP/PS1 mouse brain and down-regulated the receptor for advanced glycation end products, and up-regulated the level of low-density lipoprotein receptor-related protein-1. However, the protein expression of the ß-amyloid precursor protein, ß-Secretase and presenilin-1 (PS1) in the amyloid-ß production pathway, and the expression of neprilysin and insulin-degrading enzyme in the amyloid-ß degradation pathway were not altered. Our findings collectively suggest that Danggui-Shaoyao-San could ameliorate the amyloidosis and neuronal degeneration of Alzheimer's disease, which may be associated with its up-regulation lipoprotein receptor-related protein-1 and down-regulation of the receptor for advanced glycation end products.

Qiangji Jianli Decoction Alleviates Hydrogen Peroxide-Induced Mitochondrial Dysfunction via Regulating Mitochondrial Dynamics and Biogenesis in L6 Myoblasts.

Oxidative stress can cause the excessive generation of reactive oxygen species (ROS) and has various adverse effects on muscular mitochondria. Qiangji Jianli decoction (QJJLD) is an effective traditional Chinese medicine (TCM) that is widely applied to improve muscle weakness, and it has active constituents that prevent mitochondrial dysfunction. To investigate the protective mechanism of QJJLD against hydrogen peroxide- (H2O2-) mediated mitochondrial dysfunction in L6 myoblasts. Cell viability was determined with MTT assay. Mitochondrial ultrastructure was detected by transmission electron microscope (TEM). ROS and mitochondrial membrane potential (MMP) were analyzed by fluorescence microscope and flow cytometry. The superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) level were determined by WST-1, TBA, and DTNB methods, respectively. The mRNA and protein levels were measured by quantitative real-time PCR (qRT-PCR) and Western blot. The cell viability was decreased, and the cellular ROS level was increased when L6 myoblasts were exposed to H2O2. After treatment with QJJLD-containing serum, the SOD and GSH-Px activities were increased. MDA level was decreased concurrently. ROS level was decreased while respiratory chain complex activity and ATP content were increased in L6 myoblasts. MMP loss was attenuated. Mitochondrial ultrastructure was also improved. Simultaneously, the protein expressions of p-AMPK, PGC-1α, NRF1, and TFAM were upregulated. The mRNA and protein expressions of Mfn1/2 and Opa1 were also upregulated while Drp1 and Fis1 were downregulated. These results suggest that QJJLD may alleviate mitochondrial dysfunction through the regulation of mitochondrial dynamics and biogenesis, the inhibition of ROS generation, and the promotion of mitochondrial energy metabolism.

Blocking endothelial lipase with monoclonal antibody MEDI5884 durably increases high density lipoprotein in nonhuman primates and in a phase 1 trial.

Cardiovascular disease (CVD) is the leading global cause of death, and treatments that further reduce CV risk remain an unmet medical need. Epidemiological studies have consistently identified low high-density lipoprotein cholesterol (HDL-C) as an independent risk factor for CVD, making HDL elevation a potential clinical target for improved CVD resolution. Endothelial lipase (EL) is a circulating enzyme that regulates HDL turnover by hydrolyzing HDL phospholipids and driving HDL particle clearance. Using MEDI5884, a first-in-class, EL-neutralizing, monoclonal antibody, we tested the hypothesis that pharmacological inhibition of EL would increase HDL-C by enhancing HDL stability. In nonhuman primates, MEDI5884 treatment resulted in lasting, dose-dependent elevations in HDL-C and circulating phospholipids, confirming the mechanism of EL action. We then showed that a favorable lipoprotein profile of elevated HDL-C and reduced low-density lipoprotein cholesterol (LDL-C) could be achieved by combining MEDI5884 with a PCSK9 inhibitor. Last, when tested in healthy human volunteers, MEDI5884 not only raised HDL-C but also increased HDL particle numbers and average HDL size while enhancing HDL functionality, reinforcing EL neutralization as a viable clinical approach aimed at reducing CV risk.

Kuijieling decoction suppresses NLRP3-Mediated pyroptosis to alleviate inflammation and experimental colitis in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) is an autoimmune disease. Although the mortality rate of UC is not very high, it has a considerable morbidity rate and an unsatisfactory cure rate. Without effective treatment, UC is likely to develop into colon cancer. Kuijieling (KJL) is an effective empirical formula to treat UC in the clinical setting, and it has been proven to have curative effects against UC. AIM OF THE STUDY: In a previous study, we demonstrated that KJL could suppress NOD-like receptor protein 3 (NLRP3) to reduce inflammatory cytokines and alleviate UC. In this study, we investigated the mechanism of KJL in more detail, from the perspective of pyroptosis. MATERIALS AND METHODS: We established a dextran sulfate sodium-induced UC mouse model and RAW264.7 cells to measure different indicators with different experimental methods. The efficiency of KJL was evaluated by measuring the length and unit weight of mouse colons, and assessment of pathological injury was performed using HE staining. We detected different expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, gasdermin-D C-terminal domain (GSDMD-C), gasdermin-D N-terminal domain (GSDMD-N), IL-1ß, and IL-18 in colon tissues and cells using RT-qPCR and western blotting. Immunohistochemistry was used for tissues and immunofluorescence for cells to confirm protein expression. IL-1ß and IL-18 were measured with enzyme-linked immunosorbent assay in serum, tissue, and cell culture supernatant. MiR-223 was detected using RT-qPCR. RESULTS: After administration of KJL suspension, colon damage in KJL groups was milder than in model groups. ASC, caspase-1, IL-1ß, and IL-18 mRNA levels in colon tissue were decreased to different degrees in the KJL groups. Protein expression of NLRP3, caspase-1, GSDMD-N, IL-1ß, and IL-18 in vivo decreased significantly in the KJL groups. In addition, Mir-223 level decreased in colon tissue of the KJL groups. In vitro, NLRP3, ASC, caspase-1, GSDMD-N, IL-1ß, and IL-18 levels decreased to varying degrees, at both mRNA and protein levels. Mir-223 was lower in the KJL groups than in the model group. Furthermore, KJL was shown to regulate the level of miR-223, which returned to normal after its expression was inhibited or promoted, and the levels of associated indicators also returned to normal after transfection. CONCLUSIONS: KJL is able to inhibit pyroptosis to alleviate UC, but these suppression effects were not mediated through miR-223 regulation.

Isoorientin Inhibits Inflammation in Macrophages and Endotoxemia Mice by Regulating Glycogen Synthase Kinase 3ß.

Isoorientin has anti-inflammatory effects; however, the mechanism remains unclear. We previously found isoorientin is an inhibitor of glycogen synthase kinase 3ß (GSK3ß) in vitro. Overactivation of GSK3ß is associated with inflammatory responses. GSK3ß is inactivated by phosphorylation at Ser9 (i.e., p-GSK3ß). Lithium chloride (LiCl) inhibits GSK3ß and also increases p-GSK3ß (Ser9). The present study investigated the anti-inflammatory effect and mechanism of isoorientin via GSK3ß regulation in lipopolysaccharide- (LPS-) induced RAW264.7 murine macrophage-like cells and endotoxemia mice. LiCl was used as a control. While AKT phosphorylates GSK3ß, MK-2206, a selective AKT inhibitor, was used to activate GSK3ß via AKT inhibition (i.e., not phosphorylate GSK3ß at Ser9). The proinflammatory cytokines TNF-α, IL-6, and IL-1ß were detected by ELISA or quantitative real-time PCR, while COX-2 by Western blotting. The p-GSK3ß and GSK3ß downstream signal molecules, including NF-κB, ERK, Nrf2, and HO-1, as well as the tight junction proteins ZO-1 and occludin were measured by Western blotting. The results showed that isoorientin decreased the production of TNF-α, IL-6, and IL-1ß and increased the expression of p-GSK3ß in vitro and in vivo, similar to LiCl. Coadministration of isoorientin and LiCl showed antagonistic effects. Isoorientin decreased the expression of COX-2, inhibited the activation of ERK and NF-κB, and increased the activation of Nrf2/HO-1 in LPS-induced RAW264.7 cells. Isoorientin increased the expressions of occludin and ZO-1 in the brain of endotoxemia mice. In summary, isoorientin can inhibit GSK3ß by increasing p-GSK3ß and regulate the downstream signal molecules to inhibit inflammation and protect the integrity of the blood-brain barrier and the homeostasis in the brain.