Gastrodin attenuates renal injury and collagen deposition via suppression of the TGF-β1/Smad2/3 signaling pathway based on network pharmacology analysis

Background: Gastrodin has been widely used clinically in China as an antihypertensive drug. However, its effect on hypertensive renal injury is yet to be elucidated. The current study aimed to investigate the effects of gastrodin on hypertensive renal injury and its underlying mechanisms by network pharmacology analysis and validation in vivo and in vitro.Methods: A total of 10 spontaneously hypertensive rats (SHRs) were randomly categorized into the following two groups: SHR and SHR + Gastrodin groups. Wistar Kyoto (WKY) rats were used as the control group (n = 5). The SHR + Gastrodin group was intragastrically administered gastrodin (3.5 mg/kg/day), and the rats in both WKY and SHR groups were intragastrically administered an equal amount of double-distilled water for 10 weeks. Hematoxylin-eosin, Masson’s trichrome, and Sirius red staining were used to detect the pathological changes and collagen content in the renal tissues. Network pharmacology analysis was performed to explore its potential targets and related pathways. In vitro, the CCK-8 assay was used to determine the cell viability. Immunohistochemistry and western-blotting analyses were employed to assess the protein expression associated with renal fibrosis and transforming growth factor-β1 (TGF-β1) pathway-related proteins in the renal tissues or in TGF-β1-stimulated rat kidney fibroblast cell lines (NRK-49F).Results: Gastrodin treatment attenuates renal injury and pathological alterations in SHRs, including glomerular sclerosis and atrophy, epithelial cell atrophy, and tubular dilation. Gastrodin also reduced the accumulation of collagen in the renal tissues of SHRs, which were confirmed by downregulation of α-SMA, collagen I, collagen III protein expression. Network pharmacology analysis identified TGFB1 and SMAD2 as two of lead candidate targets of gastrodin on against hypertensive renal injury. Consistently, gastrodin treatment downregulated the increase of the protein expression of TGF-β1, and ratios of both p-Smad2/Smad2 and p-Samd3/Smad3 in renal tissues of SHRs. In vitro, gastrodin (25–100 μM) treatment significantly reversed the upregulation of α-SMA, fibronectin, collagen I, as well as p-Smad2 and p-Smad3 protein expressions without affecting the cell viability of TGF-β1 stimulated NRK-49F cells.Conclusion: Gastrodin treatment significantly attenuates hypertensive renal injury and renal fibrosis and suppresses TGF-β1/Smad2/3 signaling in vivo and in vitro

Carbon dots-based dual-emission ratiometric fluorescence sensor for dopamine detection

The detection of Dopamine (DA) is significant for disease surveillance and prevention. However, the development of the precise and simple detection techniques is still at a preliminary stage due to their high tester requirements, time-consuming process, and low accuracy. In this work, we present a novel dual-emission ratiometric fluorescence sensing system based on a hybrid of carbon dots (CDs) and 7-amino-4-methylcoumarin (AMC) to quickly monitor the DA concentration. Linked via amide bonds, the CDs and AMC offered dual-emissions with peaks located at 455 and 505 nm, respectively, under a single excitation wavelength of 300 nm. Attributed to the fluorescence of the CDs and AMC in the nanohybrid system can be quenched by DA, the concentration of DA could be quantitatively detected by monitoring the ratiometric ratio change in fluorescent intensity. More importantly, the CDs-AMC-based dual-emission ratiometric fluorescence sensing system demonstrated a remarkable linear relationship in the range of 0–33.6 μM to detection of DA, and a low detection limit of 5.67 nM. Additionally, this sensor successfully applied to the detection of DA in real samples. Therefore, the ratiometric fluorescence sensing system may become promising to find potential applications in biomedical dopamine detection

MiR-223 promoted TRAIL-induced apoptosis through the mitochondria/ROS pathway.

<p>(A) The mitochondrial membrane potential (ΔΨ_m) of MDA-MB-231 CSCs and MDA-MB-435 CSCs cells treated with miR-223 and TRAIL was detected using JC-1 staining and flow cytometry. (B) ROS generation was detected using DHE staining and flow cytometry. (C) MDA-MB-231 CSCs and MDA-MB-435 CSCs cells were treated with miR-223 and TRAIL (10 ng/ml) in the presence or absence of 5 mM NAC. Cell viability was then detected by MTT assay. *<i>P</i><0.05 <i>vs</i>. the TRAIL+miR-NC group, ^&<i>P</i><0.05 <i>vs</i>. the TRAIL group, ^#<i>P</i><0.05 <i>vs</i>. the TRAIL+miR-223 group, t test.</p

MiR-223 expression levels in breast cancer cell lines.

<p>(A) QRT-PCR analysis showed that the decrease of miR-223 expression was more significant in TNBC cell lines (MDA-MB-231 and MDA-MB-435) than the non-TNBC cell lines (MCF-7 and SKBR3). *<i>P</i><0.05 <i>vs</i>. MCF-10A cells, t test, **<i>P</i><0.01 <i>vs</i>. MCF-10A cells, t test. (B) MiR-223 expression was significantly down-regulated in both MDA-MB-231 CSCs and MDA-MB-435 CSCs compared with their parental cells. *<i>P</i><0.05, t test, **<i>P</i><0.01, t test.</p

TNBCSC and non-CSC sensitivity to TRAIL treatment.

<p>(A) Sensitivity of MCF-10A and breast cancer stem cells and non-stem cells to TRAIL was determined via MTT assays. *<i>P</i><0.05, t test. (B) The IC50 of TRAIL was determined according to the cell viability curves calculated based on the MTT assay results. *<i>P</i><0.05, t test.</p

MiR-223 increased TRAIL sensitivity via a caspase-dependent apoptotic pathway.

<p>(A) MDA-MB-231 CSCs and MDA-MB-435 CSCs were transfected with miR-223 mimics and HAX-1 vector. Twenty-four hours after transfection, the cells were treated with TRAIL (10 ng/ml) for 48 h. Flow cytometry analysis was performed to measure the cell apoptosis. *<i>P</i><0.05 <i>vs</i>. the TRAIL+miR-NC group, ^&<i>P</i><0.05 <i>vs</i>. the TRAIL group, ^#<i>P</i><0.05 <i>vs</i>. the TRAIL+miR-223 group, t test. (B) MDA-MB-231 CSCs and MDA-MB-435 CSCs were transfected with miR-223 mimics and HAX-1 vector. Twenty-four hours after transfection, the cells were treated with TRAIL (10 ng/ml) for 48 h. Western blot analysis was performed to detect caspase-9, caspase-7 and caspase-3 activation cleavage.</p

TNBCSCs are sensitive to miR-223-mediated cell death induced by TRAIL.

<p>(A) The transfection efficiency of miR-223 mimics was evaluated by qRT-PCR. *<i>P</i><0.05, t test. (B) TNBCSCs were transfected with miR-223 mimics. Twenty-four hours after transfection, the cells were treated with TRAIL (10 ng/ml) for 48 h. Relative cell viability was determined by MTT assay. *<i>P</i><0.05 <i>vs</i>. the TRAIL+miR-NC group, t test. (C) TNBCSCs or non-CSCs were transfected with miR-223 mimics. Twenty-four hours after transfection, the cells were treated with different concentrations of TRAIL for 48 h, and then MTT assays were performed. The IC50 of TRAIL was determined according to the cell viability curves calculated based on the MTT assay results. *<i>P</i><0.05, t test. (D) The CD44⁺/CD24^-/low TNBCSC population was assessed by FACS. *<i>P</i><0.05 <i>vs</i>. the TRAIL+miR-NC group, t test.</p

MiR-223 enhances the anti-tumor effect of doxorubicin and cisplatin in TNBCSCs.

<p>(A) MDA-MB-231 CSCs and MDA-MB-435 CSCs were transfected with miR-223 mimics and HAX-1 vector. Twenty-four hours after transfection, the cells were treated with doxorubicin (2 μg/ml) for 48 h. Relative cell viability was determined by MTT assay. *<i>P</i><0.05 <i>vs</i>. the doxorubicin+miR-NC group, t test, ^#<i>P</i><0.05 <i>vs</i>. the doxorubicin+miR-223 group, t test. (B) MDA-MB-231 CSCs and MDA-MB-435 CSCs were transfected with miR-223 mimics and HAX-1 vector. Twenty-four hours after transfection, the cells were treated with cisplatin (2 μM) for 48 h. Relative cell viability was determined by MTT assay. *<i>P</i><0.05 <i>vs</i>. the cisplatin+miR-NC group, t test, ^#<i>P</i><0.05 <i>vs</i>. the cisplatin+miR-223 group, t test.</p