Objective To explore the role and mechanism of mammalian target of rapamycin (mTOR) in oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in vascular smooth muscle cells (VSMCs). Methods A model of oxLDL-induced VSMC ferroptosis was established. VSMCs were co-treated with either the mTOR inhibitor rapamycin or the autophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP), followed by detection of autophagy and ferroptosis-related indexes. Quantitative real-time PCR and Western blot were used respectively to analyze the expression of mTOR, glutathione peroxidase 4 (GPX4), sequestosome 1 (p62), and microtubule-associated protein 1 light chain 3 (LC3). Flow cytometry was employed to assess VSMC death. C11 BODIPY fluorescent staining was used to measure cellular lipid peroxidation levels. Colorimetric assays were performed to determine the contents of malondialdehyde (MDA), ferrous ion (Fe²⁺) and glutathione (GSH). Results oxLDL significantly upregulated mTOR expression in VSMCs, while increasing p62 expression and reducing LC3 expression, thereby suppressing VSMC autophagy. Compared with oxLDL treatment alone, rapamycin co-treatment reversed oxLDL-induced VSMC ferroptosis, as characterized by reduced VSMC death, increased GPX4 expression and GSH contents, along with decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Similarly, CCCP co-treatment activated autophagy characterized by reduced p62 expression and elevated LC3 expression, which subsequently alleviated oxLDL-induced ferroptosis, showing reduced VSMC death, increased GPX4 expressions and GSH contents, and decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Moreover, mTOR inhibition by rapamycin significantly reversed the oxLDL-induced upregulation of p62 and downregulation of LC3. Conclusion mTOR may promote oxLDL-induced VSMC ferroptosis by suppressing autophagy.
Ferroptosis/drug effects* ; Lipoproteins, LDL/metabolism* ; TOR Serine-Threonine Kinases/physiology* ; Autophagy/drug effects* ; Muscle, Smooth, Vascular/metabolism* ; Animals ; Rats ; Myocytes, Smooth Muscle/cytology* ; Cells, Cultured ; Lipid Peroxidation/drug effects* ; Sequestosome-1 Protein/genetics* ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism* ; Microtubule-Associated Proteins/genetics* ; Sirolimus/pharmacology*

OBJECTIVES: To investigate the synergistic mechanism of the traditional Chinese medicine Rosa laevigata Michx. (RLM) for treatment of pulmonary arterial hypertension (PAH). METHODS: Network pharmacological analysis was carried out to screen the active ingredients of RLM and PAH disease targets and construct the "component-target-disease" interaction network, followed by gene enrichment analysis and molecular docking studies. In the cell experiments, primary cultures of rat pulmonary arterial smooth muscle cells were exposed to hypoxia for 24 h and treated with solvent or 100, 200 and 300 mg/mL RLM, and the changes in cell proliferation were detected using Western blotting for PCNA and immunofluorescence staining. In the animal experiment, male SD rats were randomized into 5 control group, monocrotaline (MCT) solvent group, and MCT with RLM (100, 200 and 300 mg/mL) treatment groups. HE staining and immunofluorescence staining were used to observe histopathological changes in the pulmonary blood vessels of the rats. RESULTS: Seven core active ingredients (including β-sitosterol and kaempferol) in RLM and 39 key disease targets were identified, and molecular docking showed that SRC was a high-affinity target. KEGG enrichment analysis showed that the differential genes were significantly enriched in calcium signaling and PI3K-AKT pathways. In rat pulmonary arterial smooth muscle cells, hypoxic exposure significantly up-regulated cellular expression of PCNA and phosphorylation levels of Src and AKT1, which were obviously lowered by RLM treatment. In RLM-treated rat models, the mean pulmonary artery pressure and right ventricular hypertrophy index (Fulton index) were significantly reduced, the tricuspid annular plane systolic excursion (TAPSE) was improved, and pulmonary vascular wall thickening and fibrosis were obviously ameliorated. CONCLUSIONS RLM inhibits pulmonary arterial smooth muscle cell proliferation in rat models of hypertension possibly by regulating the Src-AKT1 axis, suggesting the potential of RLM as a new natural drug for treatment of pulmonary hypertension.
Animals ; Cell Proliferation/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats, Sprague-Dawley ; Pulmonary Artery/cytology* ; Male ; Rats ; Myocytes, Smooth Muscle/cytology* ; Hypertension, Pulmonary/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Signal Transduction/drug effects* ; Muscle, Smooth, Vascular/cytology* ; src-Family Kinases/metabolism* ; Cells, Cultured

BACKGROUND: We previously demonstrated that continuous exposure to nitrous acid gas (HONO) for 4 weeks, at a concentration of 3.6 parts per million (ppm), induced pulmonary emphysema-like alterations in guinea pigs. In addition, we found that HONO affected asthma symptoms, based on the measurement of respiratory function in rats exposed to 5.8 ppm HONO. This study aimed to investigate the dose-response effects of HONO exposure on the histopathological alterations in the respiratory tract of guinea pigs to determine the lowest observed adverse effect level (LOAEL) of HONO. METHODS: We continuously exposed male Hartley guinea pigs (n = 5) to four different concentrations of HONO (0.0, 0.1, 0.4, and 1.7 ppm) for 4 weeks (24 h/day). We performed histopathological analysis by observing lung tissue samples. We examined samples from three guinea pigs in each group under a light microscope and measured the alveolar mean linear intercept (Lm) and the thickness of the bronchial smooth muscle layer. We further examined samples from two guinea pigs in each group under a scanning electron microscope (SEM) and a transmission electron microscope (TEM). RESULTS: We observed the following dose-dependent changes: pulmonary emphysema-like alterations in the centriacinar regions of alveolar ducts, significant increase in Lm in the 1.7 ppm HONO-exposure group, tendency for hyperplasia and pseudostratification of bronchial epithelial cells, and extension of the bronchial epithelial cells and smooth muscle cells in the alveolar duct regions. CONCLUSIONS These histopathological findings suggest that the LOAEL of HONO is < 0.1 ppm.
Alveolar Epithelial Cells ; drug effects ; Animals ; Bronchi ; drug effects ; Dose-Response Relationship, Drug ; Emphysema ; chemically induced ; Epithelial Cells ; drug effects ; Guinea Pigs ; Hyperplasia ; chemically induced ; Inhalation Exposure ; adverse effects ; Lung ; drug effects ; pathology ; ultrastructure ; Male ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Myocytes, Smooth Muscle ; drug effects ; Nitrous Acid ; toxicity

The aim of this study was to investigate the inhibitory effect and the underlying mechanism of ethacrynic acid (EA) on the contraction in mice. BL-420S force measuring system was used to measure the tension of mouse tracheal rings. The whole cell patch clamp technique was utilized to record the channel currents of airway smooth muscle (ASM) cells. The calcium imaging system was used to determine the intracellular Ca concentration ([Ca]) in ASM cells. The results showed that EA significantly inhibited the high K (80 mmol/L) and acetylcholine (ACh, 100 µmol/L)-induced contraction of mouse tracheal rings in a dose-dependent manner. The maximal relaxation percentages were (97.02 ± 1.56)% and (85.21 ± 0.03)%, and the median effective concentrations were (40.28 ± 2.20) μmol/L and (56.22 ± 7.62) μmol/L, respectively. EA decreased the K and ACh-induced elevation of [Ca] from 0.40 ± 0.04 to 0.16 ± 0.01 and from 0.50 ± 0.01 to 0.39 ± 0.01, respectively. In addition, EA inhibited L-type voltage-dependent calcium channel (LVDCC) and store-operated calcium channel (SOCC) currents in ASM cells, and Ca influx. Moreover, EA decreased the resistance of the respiratory system (Rrs) in vivo in mice. These results indicated that EA inhibits LVDCC and SOCC, which results in termination of Ca influx and decreases of [Ca], leading to relaxation of ASM. Taken together, EA might be a potential bronchodilator.
Animals ; Calcium ; metabolism ; Calcium Channels, L-Type ; Enzyme Inhibitors ; pharmacology ; Ethacrynic Acid ; pharmacology ; Mice ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; Respiratory System ; cytology ; drug effects

Lotus( Nelumbo nucifera) is a traditional medicinal plant,and nowadays it is regarded both as medicine and food. It is widespread across China and rich in natural resources. Almost every part of N. nucifera could be used for medical or edible purpose,including seeds( Lianzi),black ripe fruits( Shilianzi),seed coats( Lianyi),green embryos of mature seed( Lianzixin),flowers( Lianhua),stamens( Lianxu),receptacles( Lianfang),leaves( Heye),leaf or flower stalks( Hegeng),leaf bases( Heyedi),rhizomes( Ou) and rhizome nodes( Oujie). Therefore,this plant is praised as a commercial crop with great economic values. Isoquinoline type alkaloids are the main chemical components of lotus. Smooth muscles usually exist in the digestive tract,respiratory tract and vascular,urinary,reproductive and other human systems. Dysfunction of smooth muscle contraction will induce many diseases including hypertension,asthma and gastrointestinal disorder,etc.,and most of current therapeutic strategies rely on relaxation of smooth muscle by drugs.Previous studies have shown that alkaloids of lotus have strong relaxation activity on smooth muscle. The present paper reviews phytochemistry and smooth muscle relaxation activity of 59 isoquinoline alkaloids from N. nucifera through accessing CNKI,PubMed and multiple databases for biomedical sciences.
Alkaloids/pharmacology* ; China ; Humans ; Isoquinolines/pharmacology* ; Muscle Relaxation ; Muscle, Smooth/drug effects* ; Nelumbo/chemistry* ; Phytochemicals/pharmacology* ; Plant Extracts

Calpain activation contributes to hyperglycemia-induced endothelial dysfunction and apoptosis. This study was designed to investigate the role of calpain inhibition in improving diabetic erectile dysfunction (ED) in mice. Thirty-eight-week-old male C57BL/6J mice were divided into three groups: (1) nondiabetic control group, (2) diabetic mice + vehicle group, and (3) diabetic mice + MDL28170 (an inhibitor of calpain) group. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg kg^-1 body weight for 5 consecutive days. Thirteen weeks later, diabetic mice were treated with MDL28170 or vehicle for 4 weeks. The erectile function was assessed by electrical stimulation of the cavernous nerve. Penile tissues were collected for measurement of calpain activity and the endothelial nitric oxide synthase (eNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining was used to evaluate apoptosis. Caspase-3 expression and activity were also measured to determine apoptosis. Our results showed that erectile function was enhanced by MDL28170 treatment in diabetic mice compared with the vehicle diabetic group. No differences in calpain-1 and calpain-2 expressions were observed among the three groups. However, calpain activity was increased in the diabetic group and reduced by MDL28170. The eNOS-NO-cGMP pathway was upregulated by MDL28170 treatment in diabetic mice. Additionally, MDL28170 could attenuate apoptosis and increase the endothelium and smooth muscle levels in corpus cavernosum. Inhibition of calpain could improve erectile function, probably by upregulating the eNOS-NO-cGMP pathway and reducing apoptosis.
Animals ; Apoptosis/drug effects* ; Calpain/antagonists & inhibitors* ; Cyclic GMP/biosynthesis* ; Diabetes Complications/drug therapy* ; Diabetes Mellitus, Experimental/complications* ; Dipeptides/therapeutic use* ; Endothelium/metabolism* ; Enzyme Inhibitors/therapeutic use* ; Erectile Dysfunction/etiology* ; In Situ Nick-End Labeling ; Male ; Mice ; Mice, Inbred C57BL ; Muscle, Smooth/metabolism* ; Nitric Oxide Synthase Type III/biosynthesis* ; Penis/enzymology* ; Up-Regulation

OBJECTIVETo investigate whether the methanol extract of Berberis amurensis Rupr. (BAR) augments penile erection using in vitro and in vivo experiments.

METHODSThe ex vivo study used corpus cavernosum strips prepared from adult male New Zealand White rabbits. In in vivo studies for intracavernous pressure (ICP), blood pressure, mean arterial pressure (MAP), and increase of peak ICP were continuously monitored during electrical stimulation of Sprague-Dawley rats.

RESULTSPreconstricted with phenylephrine (PE) in isolated endotheliumintact rabbit corus cavernosum, BAR relaxed penile smooth muscle in a dose-dependent manner, which was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one, a soluble guanylyl cclase inhibitor. BAR significantly relaxed penile smooth muscles dose-dependently in ex vivo, and this was inhibited by pretreatment with L-NAME H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one. BAR-induced relaxation was significantly attenuated by pretreatment with tetraethylammonium (TEA, P<0.01), a nonselective K channel blocker, 4-aminopyridine (4-AP, P<0.01), a voltage-dependent K channel blocker, and charybdotoxin (P<0.01), a large and intermediate conductance Ca sensitive-K channel blocker, respectively. BAR induced an increase in peak ICP, ICP/MAP ratio and area under the curve dose dependently.

CONCLUSIONBAR augments penile erection via the nitric oxide/cyclic guanosine monophosphate system and Ca sensitive-K (BK and IK) channels in the corpus cavernosum.

Animals ; Area Under Curve ; Berberis ; chemistry ; Blood Pressure ; drug effects ; Cyclic GMP ; metabolism ; Epoprostenol ; pharmacology ; In Vitro Techniques ; Indomethacin ; pharmacology ; Male ; Models, Biological ; Muscle Relaxation ; drug effects ; Muscle, Smooth ; drug effects ; physiology ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; metabolism ; Penile Erection ; drug effects ; Phenylephrine ; pharmacology ; Plant Extracts ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; metabolism ; Pressure ; Rabbits

OBJECTIVE: To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS). METHODS: VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected. RESULTS: Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01). CONCLUSIONS Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.
Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Dedifferentiation ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Homocysteine ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Rosuvastatin Calcium ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; X-Box Binding Protein 1 ; metabolism

The present study attempted to test a novel hypothesis that Ca(2+) sparks play an important role in arterial relaxation induced by tacrolimus. Recorded with confocal laser scanning microscopy, tacrolimus (10 µmol/L) increased the frequency of Ca(2+) sparks, which could be reversed by ryanodine (10 µmol/L). Electrophysiological experiments revealed that tacrolimus (10 µmol/L) increased the large-conductance Ca(2+)-activated K(+) currents (BKCa) in rat aortic vascular smooth muscle cells (AVSMCs), which could be blocked by ryanodine (10 µmol/L). Furthermore, tacrolimus (10 and 50 µmol/L) reduced the contractile force induced by norepinephrine (NE) or KCl in aortic vascular smooth muscle in a concentration-dependent manner, which could be also significantly attenuated by iberiotoxin (100 nmol/L) and ryanodine (10 µmol/L) respectively. In conclusion, tacrolimus could indirectly activate BKCa currents by increasing Ca(2+) sparks released from ryanodine receptors, which inhibited the NE- or KCl-induced contraction in rat aorta.
Animals ; Aorta ; cytology ; metabolism ; physiology ; Calcium Signaling ; Cells, Cultured ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Male ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; physiology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Norepinephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Ryanodine ; pharmacology ; Tacrolimus ; pharmacology ; Vasoconstriction

To investigate the effects and the underlying molecular mechanisms of curcumin on pulmonary artery smooth muscle cells in rat model with chronic obstructive pulmonary disease (COPD).A total of 75 male Wistar rats were randomly divided into control group (group CN), model group (group M), low-dose curcumin group (group CL), medium-dose curcumin group (group CM) and high-dose curcumin group (group CH). HE staining was used to observe the morphology of pulmonary artery. Proliferating cell nuclear antigen (PCNA), apoptosis-related protein Bcl-2 and Bax were detected by immunohistochemical staining. TUNEL kit was used to analyze the effects of curcumin on apoptosis of smooth muscle cells, and the protein expressions of SOCS-3/JAK2/STAT pathway in lung tissues were determined by western blot.Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVMI) in group M were significantly higher than those in group CN, group CH and group CM (all<0.05). HE staining and TUNEL kit test showed that the number of pulmonary artery smooth muscle cells had a significant increase in group M, while the pulmonary artery tube became thin, and the smooth muscle cells shrinked in group CM and group CH. Immunohistochemistry showed that PCNA and Bcl-2 in group M were significantly higher than those in group CN (all<0.05), while Bax expression was significantly lower than that in group CN (<0.05). PCNA in group CM and group CH were significantly lower than that in group M (all<0.05), while Bax expression was significantly higher than that in group M (<0.05). Western blot showed that SOCS-3 protein was significantly decreased in group M, while the p-JAK2, p-STAT1, p-STAT3 were significantly increased (all<0.05). Compared with group M, SOCS-3 protein in group CM and group CH were significantly increased (all<0.05), while the p-JAK2, p-STAT3 were significantly reduced (all<0.05).Curcumin could promote the apoptosis of smooth muscle cells in rats with COPD, and improve the mean pulmonary artery pressure and RVMI through stimulating SOCS-3/JAK2/STAT signaling pathway.
Animals ; Apoptosis ; drug effects ; physiology ; Arterial Pressure ; drug effects ; physiology ; Curcumin ; pharmacology ; Hypertrophy, Right Ventricular ; pathology ; physiopathology ; Janus Kinase 2 ; drug effects ; physiology ; Lung ; chemistry ; drug effects ; Male ; Myocytes, Smooth Muscle ; drug effects ; pathology ; Proliferating Cell Nuclear Antigen ; drug effects ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; drug effects ; metabolism ; Pulmonary Artery ; drug effects ; pathology ; Pulmonary Disease, Chronic Obstructive ; pathology ; physiopathology ; Rats ; Rats, Wistar ; STAT Transcription Factors ; Suppressor of Cytokine Signaling 3 Protein ; drug effects ; physiology ; Ventricular Pressure ; drug effects ; bcl-2-Associated X Protein ; drug effects ; metabolism