Safe Concentration Range Research Articles

Kaempferol alleviates LD-mitochondrial damage by promoting autophagy: Implications in Parkinson's disease

Emerging evidence indicates that unexpected lipid droplet (LD) deposition and peroxidation can accelerate organelle stress and plays a crucial role in the pathogenesis of neurodegenerative diseases (NDDs). In our previous study, we confirmed that kaempferol (Ka), a natural flavonoid small molecule, exhibited neuroprotective effects on mice with LPS-induced Parkinson's disease (PD). In addition, previous studies have shown that autophagy plays an important role in the regulation of cellular LD deposition. In the current study, we showed that Ka protected against TH+ neuronal loss and behavioral deficits in MPTP/p-induced PD mice, accompanied by reduced lipid oxidative stress in the substantia nigra pars compacta (SNpc). In cultured neuronal cells, Ka exhibited a relatively safe concentration range and significantly suppressed LD accumulation and cellular apoptosis induced by MPP+. Further study indicated that the protective effect of Ka was dependent on autophagy, specifically lipophagy. Critically, Ka promoted autophagy to mediate LD degradation in lysosomes, which then alleviated lipid deposition and peroxidation and the resulting mitochondrial damage, consequently reducing neuronal death. Furthermore, AAV-shAtg5-mediated Atg5 knockdown abolished the neuroprotective effects of Ka against lipid oxidation in PD mice. This work demonstrates that Ka prevents dopaminergic neuronal degeneration in PD via the inhibition of lipid peroxidation-mediated mitochondrial damage by promoting lipophagy and provides a potential novel therapeutic strategy for PD and related NDDs.

Drd2 biased agonist prevents neurodegeneration against NLRP3 inflammasome in Parkinson’s disease model via a β-arrestin2-biased mechanism

Activated astrocytes secrete inflammatory cytokines such as interleukin-1β (IL-1β) into the extracellular milieu, damaging surrounding neurons and involving in the pathogenesis of neurodegenerative diseases, such as Parkinson’s disease (PD). Dopamine receptor D2 (Drd2) expresses both in neurons and astrocytes, and neuronal Drd2 is a significant target in therapy of PD. Our previous study reveals that astrocytic Drd2 exerts anti-inflammatory effect via non-classical β-arrestin2 signaling in PD model. Therefore, seeking new biased ligands of Drd2 with better efficacy and fewer side effects to treat PD is desirable and meaningful. In the present study, we evaluated the effects of UNC9995, a novel biased Drd2 agonist on astrocyte-derived neuroinflammation and dopaminergic (DA) neuron degenerationin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. We showed that UNC9995 rescued the TH+ neurons loss and inhibited glial cells activation in mouse substantia nigra in a Drd2 dependent manner. Focusing on astrocytes, we found UNC9995 shows a relatively safe concentration range and significantly suppresses astrocytic NLRP3 inflammasome activation induced by lipopolysaccharide plus ATP. Further study revealed that the anti-inflammatory effect of UNC9995 is independent of Drd2 / Gαi protein pathway. It activates β-arrestin2 by recruiting it to cell membrane. Critically, UNC9995 enhances β-arrestin2 interacting with NLRP3 to interfere inflammasome assembly, which consequently reduces IL-1β production. On the other hand, UNC9995 inhibits IL-1β-induced inflammatory pathway activation in DA neurons and rescues subsequent apoptosis via β-arrestin2 interacting with protein kinases, such as JNK and suppressing their phosphorylation. Furthermore, β-arrestin2 knockout abolishes the anti-inflammatory and neuroprotective effects of UNC9995 in PD mouse model, supporting that UNC9995 is a β-arrestin2-biased Drd2 agonist and revealing its novel function in PD treatment. Collectively, this work illustrates that Drd2 agonist UNC9995 prevents DA neuron degeneration in PD and provides a new strategy for developing the β-arrestin2-biased ligands in the therapy of NDDs.

Construction of Inflammatory Directed Polymer Micelles and Its Application in Acute Lung Injury.

Currently, there is no specific treatment for acute lung injury (ALI) in clinical practice. In order to efficiently and accurately treat ALI, the advantages of cationic carriers were combined to accelerate the cell uptake. Polycaprolactone-polyethylene glycol carrier (PCL-PEG-COOH, PPC) with good biocompatibility, polycaprolactone-polyethylmethacrylate cationic carrier (PCL-PDMAEMA, PCD), and polycaprolactone-polyethylene glycol carrier connected with high-affinity targeting peptide (Esbp) targeting inflammatory endothelial cells (PCL-PEG-Esbp, PPE) were used to construct the high-molecular polymer micelles (PCD/PPC/PPE). The particle size of the prepared DEX-loaded micelles was 130 ± 4.41nm, and the Zeta potential was 28.3 ± 0.76mV. The CMC value of the prepared polymer micelles was 0.643μg/mL, and it was not easy to depolymerize in the blood circulation. Only about 40% DXM was released from the drug-loaded polymer micelles after 12h compared with free DXM, indicating that the micelle material had a certain sustained-release performance in vitro release experiments. The safe concentration range of polymer was determined by biocompatibility test. It was recommended that the concentration of polymer micelles should not exceed 0.40mg/mL to obtain a good compatibility in organisms. The results of cytotoxicity measurement showed that when the content of PCD increased to 50%, the concentration of blank micelles should not exceed 500μg/mL and the concentration of DXM-loaded micelles should not be higher than 100μg/mL. It was proved in the cell uptake experiment that the cation carrier of the micelles accelerated the cell uptake. The targeting ability of the targeted micelle group was higher compared with the non-targeted micelle group (P< 0.01, **). Meanwhile, the targeting ability of the non-targeted micelle group was higher compared with the free group (P< 0.001, ***). The targeting ability of the non-targeted micelle group was about 2.30 times and the targeted micelle group was about 3.16 times larger than that of the free group. It was also proved in the in vivo targeting experiments that the targeted micelles had a good targeting ability. The results of in vivo imaging of mice showed that the DXM of the micelle group gathered more in the lungs, and the micelle group had a better targeting ability compared with the free DID group. The order of lung targeting intensity was targeted micelles > non-targeted micelles >> free DID group. The targeting ability of polypeptide Esbp to ALI was confirmed. In conclusion, the prepared PCD/PPC/PPE polymer micelles had obvious in vitro and in vivo targeting ability and good biocompatibility. They could be used as a new targeted delivery system for the treatment of ALI in the future.

Oxidation and Characterization of Low-Concentration Gas in a High-Temperature Reactor

To achieve the efficient utilization of low-concentration mine gas, reduce resource waste and alleviate environmental pollution, the high-temperature oxidation of low-concentration gas at a concentration range of 1.00% to 1.50%, which is directly discharged into the atmosphere during coal mine production, was carried out to recover heat for reuse. The gas oxidation equipment was improved for the heating process and the safety of low-concentration gas oxidation under a high-temperature environment was evaluated. The experimental results showed that the reactor could provide a 1000 °C high-temperature oxidation environment for gas oxidation after installing high-temperature resistant ceramics. The pressure variation curves of the reactor with air and different concentrations of gas were similar. Due to the thermal expansion, the air pressure slightly increased and then returned to normal pressure. In contrast, the low-concentration gas exhibited a stable pressure response in the high-temperature environment of 1000 °C. The outlet pressure was significantly greater than the inlet pressure, and the pressure difference between the inlet and outlet exhibited a trend to increase with the gas concentration. The minimum pressure difference was 4 kPa (air) and the maximum was 11 kPa (1.50% gas). The explosion limit varied with the temperature and the blend of oxidation products. The ratio of measured gas pressure to air pressure after oxidation was below the explosion criterion, indicating that the measured concentration of gas is still safe after the shift of the explosion limit, which provides a safe concentration range for the efficient use of low-concentration gas in the future.

Porcupine Inhibitor LGK974 Downregulates the Wnt Signaling Pathway and Inhibits Clear Cell Renal Cell Carcinoma.

Targeted therapy for kidney cancer has achieved significant clinical results. However, because most patients who use targeted therapy will develop drug resistance, we still need to constantly explore new therapeutic targets. Although porcupine (PORCN) as a palmitoyltransferase plays a crucial role in the activation and secretion of Wnt proteins and affects the activity of the Wnt signaling pathway, little is known about the role of PORCN in clear cell renal cell carcinoma (ccRCC). We found that PORCN is highly expressed in renal cancer cell lines and patients with renal cell carcinoma with high expression of PORCN have a poor prognosis. Pathway analysis of PORCN and its related proteins showed that PORCN played a role through the Wnt signaling pathway, and there was a strong coexpression relationship between PORCN and Wnt proteins. Therefore, PORCN may be a potential and effective target for ccRCC. In the present study, we found that LGK974 could inhibit proliferation and colony formation and induce apoptosis in ccRCC cells. We also found that LGK974 could inhibit the migration and invasion of renal cell carcinoma and reduce the expression of mesenchymal markers. After treatment with LGK974, the expression level of β-catenin, a key protein in the classical Wnt pathway, was significantly decreased, and the expression levels of the target genes cyclin D1, c-Myc, MMP9, and MMP2 in the Wnt signaling pathway were also significantly decreased, which represented a significant decrease in the activity of the Wnt signaling pathway. At the same time, the cycle of renal cancer cells was significantly blocked. In conclusion, our results indicate that LGK974 could significantly inhibit the progression of renal cancer cells in a safe concentration range, so PORCN may be a safe and effective target for patients with renal cancer.

Experimental study on the inhibition of Formononetin on the differentiation of osteoclasts induced by RANKL

To establish the in vitro study model of osteoclasts induced by RANKL, to elaborate the effect of formononetin (FO) , an effective component of Caulis Spatholobi, on the differentiation and function of bone marrow mononuclear macrophages (BMMs) into osteoclasts, and to explore the molecular mechanism of its inhibition. The BMMs in femur and tibia were isolated from 20 clean C57/BL6 mice of 4 to 6 weeks old, 10 males and 10 females, each weighing (20± 2) g. The BMMs in femur and tibia were cultured and proliferated in vitro with α-MEM medium. BMMs were cultured with MCSF and different concentrations of anthocyanin (5 to 50 μm) respectively for 4 days, and CCK8 of cell proliferation and toxicity was detected. BMMs in good growth condition were added to M-CSF and RANKL to induce osteoclast differentiation in turn. There was no special treatment in the control group. DMSO was added to the control group with DMSO solvent. Each observation group was added with different concentrations of awnasin (1 to 20 μm) . After 6 days of culture, the osteoclasts were counted and statistically analyzed. The expression of NFATc1, c-Fos and ERK, the key transcription factors in osteoclast differentiation, were detected by Western blot, RNA was extracted at 4 days, and the activity of ctsk, trap, MMP9 and Car2 were detected by real time PCR. CCK8 test results showed that awnstein could inhibit the activity of BMMs in a dose-dependent manner, and had no significant toxic effect on the growth of bmms within the safe concentration range of ≤20 μM (P= 0.278>0.05) . The results of trap staining showed that awnstein could inhibit osteoclast production in a dose-dependent manner in the concentration range of (1 to 20 μM) , especially in 10 μM (P=0.000<0.05) . Western blot showed that 10 μ m could significantly inhibit the expression of NFATc1 and c-Fos, but not the expression of ERK. In terms of osteoclast function, the expression of ctsk (P=0.000<0.05) , trap (P=0.000<0.05) , MMP9 (P=0.000<0.05) and Car2 (P=0.000<0.05) related to osteoclast function were detected by real time PCR. The effective component of Caulis Spatholobi can inhibit the proliferation and differentiation of primary mononuclear macrophages into osteoclasts, and down regulate the expression of osteoclast bone resorption related proteins and genes, which may be one of the mechanisms of its prevention and treatment of bone destruction and collapse in osteonecrosis of femoral head.

Hyaluronic acid/ carboxylated Zeolitic Imidazolate Framework film with improved mechanical and antibacterial properties

Hyaluronic acid (HA), a naturally sourced polysaccharide, has shown remarkable effectiveness on wound healing, but its low mechanical strength and instability limits its frequent application in this field. In order to minimize this shortcoming, hyaluronic acid based wound dressings were blended with functionalized ZIF-8, which not only provides high mechanical strength, but also introduces antibacterial properties and promotes fibroblast migration and proliferation. To analyze physiochemical and biological characteristics of prepared wound dressings, tests including DLS, XRD, FTIR as well as antibacterial and cell adhesion assays were carried out. Results indicated that HA film modification boosted the Young’s modulus from 138 to 176 K Pa, and reduced the water contact angle from 37.4 to 27.7 proving enhancement in hydrophilicity. Ameliorated antibacterial properties and better cell adhesion were also observed. Suitable cell viability was observed in samples with FZIF-8, since released Zn ions maintained within a safe concentration range.

Transport characteristics of Shuxiong prescription across Caco-2 cell monolayer model

Shuxiong prescription (Notoginseng Radix et Rhizoma, Chuanxiong Rhizome and Carthami Flos) has the function of activating blood circulation to dissipate blood stasis, activating meridians to stop pain. This paper was mainly aimed to discuss the transport characteristics of Shuxiong prescription across Caco-2 cell monolayer. Safe concentration range of Shuxiong prescription against Caco-2 cell monolayer model was determined by MTT assay. The mechanism of Shuxiong prescription bidirectional transport was investigated by Caco-2 cell monolayer model. The apparent permeability coefficient Papp of digoxin was determined by high performance liquid chromatography (HPLC). The test results showed that the Papp of extract from Notoginseng Radix et Rhizoma, Chuanxiong Rhizome, Carthami Flos, Chuanxiong Rhizome+Carthami Flos and Shuxiong prescription transport from apical (AP) side to basolateral (BL) side was (3.12±0.73)×10⁻⁶, (2.58±0.41)×10⁻⁶, (4.97±0.64)×10⁻⁶, (4.63±0.57)×10⁻⁶, (5.79±0.68)×10⁻⁶ cm·s⁻¹, respectively, indicating that the transport of digoxin across Caco-2 cell monolayer model was active absorption, and the P-gp protein took part in the process. Chuanxiong Rhizome could significantly decrease the transport of digoxin from BL→AP(P<0.01) and increase its transport from AP→BL(P<0.05) significantiy. After the addition of Shuxiong prescription, the transport of digoxin from BL→AP was significantly inhibited(P<0.01). The results suggested that the extract of safflower had no effect on P-gp transport, nor on the independence diffusion of digoxin. The transport of digoxin could be degraded by the extract of Chuanxiong Rhizome and the extract of Shuxiong prescription from BL→AP(P<0.01), significantly; pseudo-ginseng had no effect on the independence diffusion of digoxin; the extract of safflower+Chuanxiong Rhizome had the same experimental result as Chuanxiong Rhizome extract.

Synthesis and evaluation of cationic polymeric micelles as carriers of lumbrokinase for targeted thrombolysis

To achieve targeted thrombolysis, a targeted delivery system of lumbrokinase (LK) was constructed using RGDfk-conjugated hybrid micelles. Based on the specific affinity of RGDfk to glycoprotein complex of GPⅡb/Ⅲa expressed on the surface of membrane of activated platelet, LK loaded targeted micelles (LKTM) can be delivered to thrombus. The hybrid micelles were composed of polycaprolactone-block-poly (2-(dimethylamino) ethyl methacrylate) (PCL-PDMAEMA), methoxy polyethylene glycol-block- polycaprolactone (mPEG-PCL) and RGDfk conjugated polycaprolactone-block- polyethylene glycol (PCL-PEG-RGDfk). PCL-PDMAEMA was synthesized via ring open polymerization (ROP) and atom transfer radical polymerization (ATRP). PCL-PEG-RGDfk was synthesized via ROP and carbodiimide chemistry. The prepared LKTM was characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). Colloidal stability assay showed the prepared LKTM was stable. Biocompatibility assay was performed to determine the safe concentration range of polymer. The assay of fluorescent distribution in vivo demonstrated that LKTM can be efficiently delivered to thrombi in vivo. Thrombolysis in vivo indicated the thrombolytic potency of LKTM was optimal in all groups. Notably, the laboratory mice treated with LKTM exhibited a significantly shorter tail bleeding time compared to those treated with LK or LK-loaded micelles without RGDfk, which suggested that the targeted delivery of LK using RGDfk-conjugated hybrid micelles effectively reduced the bleeding risk.

Roselle Polyphenols Exert Potent Negative Inotropic Effects via Modulation of Intracellular Calcium Regulatory Channels in Isolated Rat Heart.

Roselle (Hibiscus sabdariffa Linn.) calyces have demonstrated propitious cardioprotective effects in animal and clinical studies; however, little is known about its action on cardiac mechanical function. This study was undertaken to investigate direct action of roselle polyphenols (RP) on cardiac function in Langendorff-perfused rat hearts. We utilized RP extract which consists of 12 flavonoids and seven phenolic acids (as shown by HPLC profiling) and has a safe concentration range between 125 and 500μg/ml in this study. Direct perfusion of RP in concentration-dependent manner lowered systolic function of the heart as shown by lowered LVDP and dP/dt max, suggesting a negative inotropic effect. RP also reduced heart rate (negative chronotropic action) while simultaneously increasing maximal velocity of relaxation (positive lusitropic action). Conversely, RP perfusion increased coronary pressure, an indicator for improvement in coronary blood flow. Inotropic responses elicited by pharmacological agonists for L-type Ca2+ channel [(±)-Bay K 8644], ryanodine receptor (4-chloro-m-cresol), β-adrenergic receptor (isoproterenol) and SERCA blocker (thapsigargin) were all abolished by RP. In conclusion, RP elicits negative inotropic, negative chronotropic and positive lusitropic responses by possibly modulating calcium entry, release and reuptake in the heart. Our findings have shown the potential use of RP as a therapeutic agent to treat conditions like arrhythmia.

Docosahexenoic acid treatment ameliorates cartilage degeneration via a p38 MAPK-dependent mechanism.

Osteoarthritis (OA) is a common chronic inflammatory disease, characterized by cartilage degradation. The aberrant expression of matrix metalloproteinase-13 (MMP-13) plays a vital role in the pathogenesis of OA. The anti-inflammatory property of docosahexenoic acid (DHA) was previously revealed and showed that DHA retards the progress of many types of inflammatory disease. To evaluate the prophylactic function of DHA in OA, the effect of DHA on cartilage degeneration was assessed in interleukin-1β (IL-1β) stimulated human chondrosarcoma SW1353 cells or a rat model of adjuvant-induced arthritis (AIA). The safe concentration range (0–50 µg/ml in vitro) of DHA was determined by flow cytometry and MTT assay. The inhibitory effects of DHA on MMP-13 mRNA and protein expression were confirmed by RT-qPCR, ELISA and western blotting. Furthermore, findings of an in vivo study showed that DHA can increase the thickness of articular cartilage and decrease MMP-13 expression in cartilage matrix in a rat AIA model. We also revealed the mechanism by which DHA ameliorates cartilage degeneration from OA. The DHA-mediated inhibition of MMP-13 expression was partially attributed to the inactivation of the p38 mitogen-activated protein kinases pathway by suppressing p-p38 in IL-1β-stimulated SW1353 cells and a rat AIA model. Our findings suggested that DHA is a promising therapeutic agent that may be used for the prevention and treatment of OA.

Potential risk of mitomycin C at high concentrations on peripheral nerve structure.

Although the local application of mitomycin C may prevent epidural adhesion after laminectomy, mitomycin C can induce neurotoxicity in optic and acoustic nerves at high concentrations. To determine the safe concentration range for mitomycin C, cotton pads soaked with mitomycin C at different concentrations (0.1, 0.3, 0.5, and 0.7 mg/mL) were immediately applied for 5 minutes to the operation area of rats that had undergone laminectomy at L1. Rat sciatic nerves, instead of dorsal nerves, were used in this study. The results showed that mitomycin C at 0.1-0.5 mg/mL did not damage the structure and function of the sciatic nerve, while at 0.7 mg/mL, mitomycin C significantly reduced the thickness of the sciatic nerve myelin sheath compared with lower concentrations, though no functional change was found. These experimental findings indicate that the local application of mitomycin C at low concentrations is safe to prevent scar adhesion following laminectomy, but that mitomycin C at high concentrations (> 0.7 mg/mL) has potential safety risks to peripheral nerve structures.

Tolerance of Physocypria kraepelini (Crustacean, Ostracoda) to water-borne ammonia, phosphate and pH value

This study evaluated the median lethal concentration (LC50) and safe concentration of water-borne ammonia, phosphate and pH value on Physocypria kraepelini, a freshwater Ostracoda with a static renewal test system. The results indicated that the LC50 values of ammonia for P kraepelini were 1026.71, 859.98, 771.79 and 583.82 mg/L at 24, 48, 72 and 96 h exposure, respectively, and the safe concentration range of ammonia for the long-term survival of P. kraepelini was less than 58.38 mg/L. The safe range of pH value for the survival of P. kraepelini was from 6.59 to 7.61. P. kraepelini has a high tolerance to ammonia, phosphate and pH value which are the main environmental factors in the serious eutrophication water.