recording-inward-rectifying-k-currents-freshly-isolated-basilar

Recording of Inward Rectifying K<sup>+</sup> Currents in Freshly Isolated Basilar Artery Smooth Muscle Cells by Patch Clamp Technique

Cerebrovascular disease is a prevalent condition among the elderly, with its incidence steadily rising. The basilar artery is a critical cerebral vessel ...

Shanxi Medical University

Therapeutic hypothermia is the most promising non-pharmacological neuroprotective strategy against ischemic injury. However, shivering is the most common adverse reaction. Many studies have shown that dantrolene is neuroprotective in in vitro and in vivo ischemic injury models. In addition to its neuroprotective effect, dantrolene neutralizes the adverse reaction of hypothermia. Dantrolene may be an effective adjunctive therapy to enhance the neuroprotection of hypothermia in treating ischemic stroke. Cortical neurons isolated from rat fetuses were exposed to 90 minutes of oxygen-glucose deprivation followed by reoxygenation. Neurons were treated with 40 μM dantrolene, hypothermia (at 33°C), or the combination of both for 12 hours. Results revealed that the combination of dantrolene and hypothermia increased neuronal survival and the mitochondrial membrane potential, and reduced intracellular active oxygen cytoplasmic histone-associated DNA fragmentation, and apoptosis. Furthermore, improvements in cell morphology were observed. The combined treatment enhanced these responses compared with either treatment alone. These findings indicate that dantrolene may be used as an effective adjunctive therapy to enhance the neuroprotective effects of hypothermia in ischemic stroke.

Dantrolene enhances the protective effect of hypothermia on cerebral cortex neurons.

Highly up-regulated in liver cancer (HULC) is a long non-coding RNA (lncRNA). We found that HULC up-regulated sphingosine kinase 1 (SPHK1), which is involved in tumor angiogenesis. Levels of HULC were positively correlated with levels of SPHK1 and its product, sphingosine-1-phosphate (S1P), in patients HCC samples. HULC increased SPHK1 in hepatoma cells. Chicken chorioallantoic membrane (CAM) assays revealed that si-SPHK1 remarkably blocked the HULC-enhanced angiogenesis. Mechanistically, HULC activated the promoter of SPHK1 in hepatoma cells through the transcription factor E2F1. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) further showed that E2F1 was capable of binding to the E2F1 element in the SPHK1 promoter. HULC increased the expression of E2F1 in hepatoma cells and levels of HULC were positively correlated with those of E2F1 in HCC tissues. Intriguingly, HULC sequestered miR-107, which targeted E2F1 mRNA 3'UTR, by complementary base pairing. Functionally, si-SPHK1 remarkably abolished the HULC-enhanced tumor angiogenesis in vitro and in vivo. Taken together, we conclude that HULC promotes tumor angiogenesis in liver cancer through miR-107/E2F1/SPHK1 signaling. Our finding provides new insights into the mechanism of tumor angiogenesis.

Long non-coding RNA HULC promotes tumor angiogenesis in liver cancer by up-regulating sphingosine kinase 1 (SPHK1).

5-Aminosalicylic acid (5-ASA), as an anti-inflammatory drug, has been extensively used for the treatment of mild to moderate active ulcerative colitis (UC), but the possible mechanisms of action remain unclear.

5-Aminosalicylic acid inhibits inflammatory responses by suppressing JNK and p38 activity in murine macrophages.

This experiment researched on three kinds of Perilla frutescens including the widespread PK, PA and rare PL chemotype. The Perilla samples were the mature leaves collected in nutrition, flowering and frutescence three different phenological periods, and at 7 am, 12 pm and 6 pm three day time. The volatile oil was extracted by steam distillationand analyzed by GC-MS, as a result, the three chemotype samples'volatile oil yield was between 0.08% and 0.96%; volatile oil yield of different growth period was as follow: nutrition>flowering>fructescence, and the volatile oil yield of nutrition period: PA type>PK type>PL type. Each chemotype was not affected by the growth and development, indicating that the chemotype is determined by genetic factors. Characteristic and main components of PA and PK type are relatively stable, and the characteristic components of PL type are significantly decreased with the growth. There are still a large number of upstream metabolism components, and the chemical type may have their primitiveness and changeability. The relative content of perillaldehyde, characteristic components of PA type, is basically decreased from morning to night, in all the period. The relative content of perillaketone, characteristic components of PK type, in nutrition and flowering period, when samples were collected at 12 noon is relatively higher than that at 7 am and 6 pm, and contrary to samples collected in frutescence period. The relative content of perillene, characteristic components of PL type, in nutrition and frutescence period are highest at 12 noon, while in flowering period is highest at 6 pm. According to the volatile oil yield and relative content of maincomponents, the best harvest time of PA type is in the morning of the nutrition period; the best harvest time of PK type is in the morning of all the period; and the best harvest time of PL type is at dusk of the nutrition period.

[Research on effects of chemotype and components of Perilla frutescens leaf volatile oil I: different phenological periods].

Luteolin has been shown to be beneficial to cardiovascular tissues and organs. We aimed to study its vasospasmolytic effects against various vasoconstrictors in the isolated rat coronary arteries (RCAs) and its electrophysiological effects on K currents via voltage-gated potassium (Kv) channels and inward rectifier potassium (Kir) channels in freshly isolated rat coronary arterial smooth muscle cells (RCASMCs).

Luteolin-induced coronary arterial relaxation involves activation of the myocyte voltage-gated K channels and inward rectifier K channels.

The vasorelaxant effect of apigenin (API) has been demonstrated in a number of vascular beds. We aimed to study the possible involvement of Cl channels and K channels in API-induced vasorelaxation in intrarenal arteries. The vascular tone of isolated rat intrarenal arteries (RIRAs) was measured with a myograph. The myocyte transmembrane Cl currents through Ca activated Cl channels (CaCCs) and the K currents through voltage-dependent (Kv) K channels were recorded using a patch clamp in the single arterial smooth muscle cells (ASMCs) isolated freshly from RIRAs. Preincubation with API (10-100 μM) concentration-dependently depressed the contractions induced by KCl, thromboxane A analog U46619, phenylephrine and vasopressin. The IC values were 13.27-26.26 μM. Instant application of API elicited immediate relaxations in RIRAs precontracted with these vasoconstrictors. The RC values were 5.80-24.33 μM. Chloride deprivation, Cl channel blockers, Kv blocker and nitric oxide synthase inhibitor attenuated API-induced RIRA relaxation. At 10-100 μM, API depressed CaCC currents, but augmented Kv currents. Taken together, the present study demonstrated that API depresses contractions induced by various vasoconstrictors in RIRAs, suppresses CaCC currents and augments Kv currents in RIRA ASMCs.

Apigenin relaxes rat intrarenal arteries, depresses Ca-activated Cl currents and augments voltage-dependent K currents of the arterial smooth muscle cells.

Insulin resistance (IR) is known to be a critical factor, which can lead to the onset of type 2 diabetes. Traditional Chinese medicine (TCM) has special advantages in treating IR, but the active components and action mechanisms of most TCM remain unclear. Therefore, the elucidation of the potential mechanisms is a major challenge in TCM research. In the study, we tried to elucidate the potential pharmacological efficacy and mechanism of breviacapine for improving IR through network analysis and validate the possible biological target for its quality evaluation. We computationally recognized the active components, potential targets, and the targets closely related to IR by using integrative analysis based on network pharmacology approach. We also established the active components-targets network, protein interactions network and analyzing the biological functions and pathways of targets to evaluate the links between components and pharmacological actions to help explain the action mechanisms of breviscapine. Based on the network analysis, our experimental data preliminarily confirmed that breviscapine could improve IR in HepG2 cells, which may be associated with the dynamic regulation of the PTP1B. This study combined network pharmacology with partial experiment validation to clarify the underlying mechanism of breviscapine in improving IR and thus laid the experimental foundation for the depth exploration of its functional mechanism.

A Preliminary Study on the Effect and Mechanism of Breviscapine for Improving Insulin Resistance in HepG2 Cells.

A method was established for content determination of two kinds of phenolic acids, including rosmarinic acid)(RA) and caffeic acid(CA), and six kinds of flavonoids including scutellarein-7-O-diglucuronide(SDG), luteolin-7-O-diglucuronide(LDG), apigenin-7-O-diglucuronide(ADG), scutellarin-7-O-glucuronide(SG), luteolin-7-O-glucuronide(LG), and apigenin-7-O-glucuronide(AG) in Perilla frutescens leaves. The content of eight chemical components was measured based on ten P. frutescens germplasms of different chemotypes of volatile oil, different cultivated years, and different harvesting periods. The results showed that there was a great difference between the two kinds of constituents of different germplasms. The total content of the two phenolic acids was 2.24-34.44 mg·g~(-1), and the total content of the six flavonoids was 11.55-34.71 mg·g~(-1). Then according to content from most to least, the order of each component was RA(2.13-33.97 mg·g~(-1)), LDG(1.31-14.80 mg·g~(-1)), SG(1.97-8.45 mg·g~(-1)), ADG(2.68-7.60 mg·g~(-1)), SDG(1.16-5.87 mg·g~(-1)), LG(0.78-1.91 mg·g~(-1)), AG(0.56-1.00 mg·g~(-1)), and CA(0.11-0.68 mg·g~(-1)). The chemical contents of the 5 PA-type germplasms in 2017 were mostly higher than those in 2018 showing a large variation with the cultivation years. These contents of two kinds of phenolic acids of 9 germplasms fluctuated with the harvesting time. The content decreased before early flower spike(the 3~(rd) to 18~(th) in August) at first and began to increase in flowering and fruiting period(the 18~(th) in August to 2~(nd) in September). However, these contents had slowly decreasing trend after 2~(nd) in September till 17~(th) in the same month. Interestingly, the content raised again in the maturity of fruits. The variation tendency of contents in six kinds of flavonoids components was inconsistent in different germplasms with the variation of harvesting time. The content of flavonoids in part of germplasms was negatively correlated with the fluctuation of phenolic acids. There was no correlation between phenolic acids and chemical type of the volatile oil. This paper may provide a reference for the high-quality germplasm of P. frutescens cultivation.

[Contents determination of eight phenolic compounds in Perilla frutescens leaves of different cultivation years and harvesting periods].

Ischemic stroke is regarded one of the most common causes of brain vulnerability. Silibinin (SIL), extracted from the seeds of Silybinisus laborinum L., has been found to exhibit obvious therapeutic effects on neurodegenerative diseases. GAS6 has been proven to have significant neuroprotective effects; however, the role of SIL and GAS6 in ischemic stroke remains unclear. This study aimed to investigate the protective effects of SIL against cerebral ischemia-reperfusion injury in neuroblastoma N2a cells, as well as the mechanisms involved. Firstly, the toxicity of SIL was evaluated, and safe concentrations were chosen for subsequent experiments. Then, SIL exerts significant neuroprotection against hypoxia/reoxygenation (HR) injury in N2a cells, as manifested by increased cell viability, decreased apoptotic rate, LDH, and ROS generation. Additionally, SIL was found to inhibit HR-induced apoptosis, mitochondria dysfunction, and oxidative stress. However, silencing of GAS6 inhibited the neuroprotective effects of SIL. To sum up, these results suggest that SIL may be a promising therapeutic agent for the treatment of ischemic stroke.

Silibinin exerts neuroprotective effects against cerebral hypoxia/reoxygenation injury by activating the GAS6/Axl pathway.

Obesity is associated with a wide variety of metabolic disorders that impose significant burdens on patients and society. The "browning" phenomenon in white adipose tissue (WAT) has emerged as a promising therapeutic strategy to combat metabolic disturbances. However, though the anti-diabetic drug dapagliflozin (DAPA) is thought to promote "browning," the specific mechanism of this was previously unclear.

Dapagliflozin promotes browning of white adipose tissue through the FGFR1-LKB1-AMPK signaling pathway.

Despite limited research on refractory and/or endocrine therapy failure in elderly metastatic breast cancer (MBC) patients, a prior study showed that low-dose oral cyclophosphamide (CY) can improve the overall survival rate of MBC patients, possibly through the immunoregulation of regulatory T cells (Tregs). We preliminarily investigated the combination of endocrine therapy (ET) with oral low-dose CY as salvage therapy in elderly patients via peripheral blood regulatory T-cell analyses. In addition, we evaluated the associations of tumor tertiary lymphoid structures (TLSs) with therapeutic outcomes. HR+/HER2- advanced breast cancer patients who received low-dose CY combined with ET or ET only from April 2015 to August 2021 were enrolled in this retrospective study. The primary outcome was the clinical control rate (CCR), and the secondary outcome was progression-free survival (PFS). Circulating T lymphocyte subpopulations represented by Tregs were monitored during treatment by flow cytometry methods. TLSs wereconfirmed by hematoxylin-eosin staining of pretreatment specimens, and CD3, CD4, and Foxp3 were detected using Opal multicolor immunofluorescence. A total of 85 patients who received CY + ET and 50 patients who received ET only were enrolled, the percentage of patients who received CCR was 73% (62/85) vs. 70% (45/50), and the objective response rate (ORR) was 28% (24/85) vs. 24% (12/50). No deaths occurred during the study period. The mean PFS time was 13 vs. 11 months (P = 0.03). In the CY + ET group, decreases in CD4+/CD25+/Foxp3+ T cells (P < 0.001) were favorable for both clinical control and prolonged PFS (P < 0.001). Compared with patients without TLSs, those with TLSs were more likely to have better clinical control and PFS (mean time = 6 months), and a greater number of Treg cells during TLS pretreatment correlated with longer PFS (P = 0.043). Oral low-dose CY combined with standard ET exerts immunological effects by decreasing Treg levels to achieve improved clinical responses. Moreover, patients with TLSs might benefit more from such therapy than those without TLSs, and a high Treg cell count in TLSs before treatment predicts better therapeutic efficacy.

Combined oral low-dose cyclophosphamide endocrine therapy may improve clinical response among patients with metastatic breast cancer via Tregs in TLSs.

Article	Year
Dantrolene enhances the protective effect of hypothermia on cerebral cortex neurons. Neural regeneration research\| PubMed ID: 26487856	2015
Long non-coding RNA HULC promotes tumor angiogenesis in liver cancer by up-regulating sphingosine kinase 1 (SPHK1). Oncotarget\| PubMed ID: 26540633	2016
5-Aminosalicylic acid inhibits inflammatory responses by suppressing JNK and p38 activity in murine macrophages. Immunopharmacology and immunotoxicology\| PubMed ID: 28071183	2017
[Research on effects of chemotype and components of Perilla frutescens leaf volatile oil I: different phenological periods]. Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica\| PubMed ID: 28959842	2017
Luteolin-induced coronary arterial relaxation involves activation of the myocyte voltage-gated K channels and inward rectifier K channels. Life sciences\| PubMed ID: 30771310	2019
Apigenin relaxes rat intrarenal arteries, depresses Ca-activated Cl currents and augments voltage-dependent K currents of the arterial smooth muscle cells. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie\| PubMed ID: 31079004	2019
A Preliminary Study on the Effect and Mechanism of Breviscapine for Improving Insulin Resistance in HepG2 Cells. Journal of cardiovascular pharmacology\| PubMed ID: 32398476	2020
[Contents determination of eight phenolic compounds in Perilla frutescens leaves of different cultivation years and harvesting periods]. Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica\| PubMed ID: 33645021	2021
Silibinin exerts neuroprotective effects against cerebral hypoxia/reoxygenation injury by activating the GAS6/Axl pathway. Toxicology\| PubMed ID: 37544575	2023
Dapagliflozin promotes browning of white adipose tissue through the FGFR1-LKB1-AMPK signaling pathway. Molecular biology reports\| PubMed ID: 38644407	2024
Combined oral low-dose cyclophosphamide endocrine therapy may improve clinical response among patients with metastatic breast cancer via Tregs in TLSs. Scientific reports\| PubMed ID: 38862586	2024

Weiping Li

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