Cardioprotective Potential of Cymbopogon citratus Essential Oil against Isoproterenol-induced Cardiomyocyte Hypertrophy: Possible Involvement of NLRP3 Inflammasome and Oxidative Phosphorylation Complex Subunits.

OBJECTIVE: Cymbopogon citratus (DC.) Stapf is a medicinal and edible herb that is widely used for the treatment of gastric, nervous and hypertensive disorders. In this study, we investigated the cardioprotective effects and mechanisms of the essential oil, the main active ingredient of Cymbopogon citratus, on isoproterenol (ISO)-induced cardiomyocyte hypertrophy. METHODS: The compositions of Cymbopogon citratus essential oil (CCEO) were determined by gas chromatography-mass spectrometry. Cardiomyocytes were pretreated with 16.9 µg/L CCEO for 1 h followed by 10 µmol/L ISO for 24 h. Cardiac hypertrophy-related indicators and NLRP3 inflammasome expression were evaluated. Subsequently, transcriptome sequencing (RNA-seq) and target verification were used to further explore the underlying mechanism. RESULTS: Our results showed that the CCEO mainly included citronellal (45.66%), geraniol (23.32%), and citronellol (10.37%). CCEO inhibited ISO-induced increases in cell surface area and protein content, as well as the upregulation of fetal gene expression. Moreover, CCEO inhibited ISO-induced NLRP3 inflammasome expression, as evidenced by decreased lactate dehydrogenase content and downregulated mRNA levels of NLRP3, ASC, CASP1, GSDMD, and IL-1ß, as well as reduced protein levels of NLRP3, ASC, pro-caspase-1, caspase-1 (p20), GSDMD-FL, GSDMD-N, and pro-IL-1ß. The RNA-seq results showed that CCEO inhibited the increase in the mRNA levels of 26 oxidative phosphorylation complex subunits in ISO-treated cardiomyocytes. Our further experiments confirmed that CCEO suppressed ISO-induced upregulation of mt-Nd1, Sdhd, mt-Cytb, Uqcrq, and mt-Atp6 but had no obvious effects on mt-Col expression. CONCLUSION: CCEO inhibits ISO-induced cardiomyocyte hypertrophy through the suppression of NLRP3 inflammasome expression and the regulation of several oxidative phosphorylation complex subunits.

Metabolic profiling of lysophosphatidylcholines in chlorpromazine hydrochloride- and N-acetyl-p-amino-phenoltriptolide-induced liver injured rats based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Chlorpromazine hydrochloride (CH) and N-acetyl-p-amino-phenoltriptolide (APAP) are typical acentral dopamine receptor antagonists and antipyretic analgesics in clinical applications, respectively. However, it has been reported that these 2 drugs could cause liver damage. Lysophosphatidylcholines (LPCs) have multiple physiological functions and are metabolized primarily in the liver, where it undergoes significant changes when the liver is damaged. In the study, 15 LPCs in the rat serum with CH- and APAP-induced liver injury were quantified based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry, and multivariate statistical analyses including principal component analysis (PCA) and orthogonal partial least squares discriminate analysis (OPLS-DA) were combined to understand CH- and APAP-induced liver injury from the perspective of LPC metabolic profiling. The quantitative results showed that there were significant changes in 10 LPCs and 5 LPCs after CH- and APAP-administration, separately. The results of PCA and OPLS-DA indicated that CH- and APAP-induced liver injury could be well distinguished by the LPC metabolic profiling, and 7 LPCs and 1 LPC biomarkers that could characterize CH- and APAP-induced liver damage in turn had been screened. This study will not only provide a new perspective for the clinical diagnosis of CH- and APAP-induced liver injury, but also offer a reference for further study of their hepatotoxicity mechanisms.

Chemosensitizing effect and mechanism of imperatorin on the anti-tumor activity of doxorubicin in tumor cells and transplantation tumor model.

Multidrug resistance of tumors has been a severe obstacle to the success of cancer chemotherapy. The study wants to investigate the reversal effects of imperatorin (IMP) on doxorubicin (DOX) resistance in K562/DOX leukemia cells, A2780/Taxol cells and in NOD/SCID mice, to explore the possible molecular mechanisms. K562/DOX and A2780/Taxol cells were treated with various concentrations of DOX and Taol with or without different concentrations of IMP, respectively. K562/DOX xenograft model was used to assess anti-tumor effect of IMP combined with DOX. MTT assay, Rhodamine 123 efflux assay, RT-PCR, and Western blot analysis were determined in vivo and in vitro. Results showed that IMP significantly enhanced the cytotoxicity of DOX and Taxol toward corresponding resistance cells. In vivo results illustrated both the tumor volume and tumor weight were significantly decreased after 2-week treatment with IMP combined with DOX compared to the DOX alone group. Western blotting and RT-PCR analyses indicated that IMP downregulated the expression of P-gp in K562/DOX xenograft tumors in NOD/SCID mice. We also evaluated glycolysis and glutamine metabolism in K562/DOX cells by measuring glucose consumption and lactate production. The results revealed that IMP could significantly reduce the glucose consumption and lactate production of K562/DOX cells. Furthermore, IMP could also remarkably repress the glutamine consumption, α-KG and ATP production of K562/DOX cells. Thus, IMP may sensitize K562/DOX cells to DOX and enhance the anti-tumor effect of DOX in K562/DOX xenograft tumors in NOD/SCID mice. IMP may be an adjuvant therapy to mitigate the multidrug resistance in leukemia chemotherapy.

Dampness-Heat Accelerates DMBA-Induced Mammary Tumors in Rats.

OBJECTIVE: To investigate the impact of dampness-heat (DH) on the development of mammary tumors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rats. METHODS: Forty rats were randomly divided into 3 groups in a randomized block design, including the control group (n=13), DMBA group (n=14), and DMBA plus DH group (n=13). Rats in the DMBA group and DMBA plus DH group were intragastrically administrated with DMBA (100 mg/kg) for twice, once per week, while rats in the control group were treated with equivalent volumes of sesame oil. After DMBA administration, rats in the DMBA plus DH group were exposed to a simulated climate chamber with ambient temperature (33.0±0.5°C) and humidity (90%±5%) for 8 weeks, 8 h per day. The body weight, time of tumor formation, and number of tumors were measured weekly to calculate tumor incidence, average latency period, average number of tumors, and average tumor weight. At the end of the experiment, the levels of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1) in serum, and the contents of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1ß in serum and tumor tissue were measured, respectively. Some tumor tissues were processed for hematoxylin-eosin staining to determine the histopathological changes. RESULTS: Compared with DMBA, DMBA plus DH significantly increased the average number of tumors, average tumor weight, levels of serum MMP-9, TIMP-1, TNF-α and IL-1ß, and contents of tumor tissue TNF-α and IL-1ß (P<0.05 or P<0.01). CONCLUSION: DH could accelerate the development of mammary tumors through increasing the expressions of MMP-9, TIMP-1, TNF-α and IL-1ß in DMBA-induced rats.

Protective effect of kaempferol on LPS plus ATP-induced inflammatory response in cardiac fibroblasts.

Inflammatory response is an important mechanism in the pathogenesis of cardiovascular diseases. Cardiac fibroblasts play a crucial role in cardiac inflammation and might become a potential therapeutic target in cardiovascular diseases. Kaempferol, a flavonoid commonly existing in many edible fruits, vegetables, and Chinese herbs, is well known to possess anti-inflammatory property and thus has a therapeutic potential for the treatment of inflammatory diseases. To date, the effect of kaempferol on cardiac fibroblasts inflammation is unknown. In this study, we investigated the anti-inflammatory effect of kaempferol on lipopolysaccharide (LPS) plus ATP-induced cardiac fibroblasts and explored the underlying mechanisms. Our results showed that kaempferol at concentrations of 12.5 and 25 µg/mL significantly suppressed the release of TNF-α, IL-1ß, IL-6, and IL-18 and inhibited activation of NF-κB and Akt in LPS plus ATP-induced cardiac fibroblasts. These findings suggest that kaempferol attenuates cardiac fibroblast inflammation through suppression of activation of NF-κB and Akt.

[Intervention effect of quercetin on inflammatory secretion of cardiac fibroblasts].

To establish neonatal rat cardiac fibroblast inflammatory secretion model by using LPS 100 microg x L(-1) combined with ATP 5 mmol x L(-1), in order to study the inhibitory effect of quercetin on the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts, further investigate the effect of quercetin on the protein expression of p-NF-kappaB p65 (S276) and p-Akt (S473) by western blot, and discuss the inhibitory effect of quercetin on the inflammatory secretion of cardiac fibroblasts. According to the findings, quercetin with the concentrations between 51.74 micromol x L(-1) and 827.81 micromol x L(-1) had no significant effect on the activity of cardiac fibroblasts. Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of TNF-alpha and IL-1beta induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.01 or P < 0.05). Quercetin with the concentrations of 82.78, 41.39 micromol x L(-1) could notably inhibit the increase of IL-6 induced LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.05), without any notable effect of quercetin with the concentration of 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20. 70 micromol x L(-1) could notably inhibit the NF-kappaB p65 (S276) activation induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 15 min, with the most significant effect in 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of p-Akt(473) expression induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 240 min (P < 0.05). Therefore, this study believes that quercetin could attenuate the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts by inhibiting the activation of NF-kappaB p65 (S276) and Akt (473).

Cardioprotective effect of protocatechuic acid on myocardial ischemia/reperfusion injury.

Protocatechuic acid (PCA), a phenolic compound and one of the main metabolites of complex polyphenols, has been found to possess various biological activities, and it may have a potential in the treatment of ischemic heart diseases. This study explored the cardioprotective effect of PCA on myocardial ischemia/reperfusion (MI/R) injury and the underlying mechanisms. In an in vivo rat model of MI/R injury, myocardial infarct size, serum TNF-a level, and platelet aggregation were measured. In a primary neonatal rat cardiomyocyte model of hypoxia/ reoxygenation (H/R) injury, the apoptotic rate, expressions of cleaved caspase-3, and phosphorylated Akt were observed. We found that PCA significantly reduced myocardial infarct size, serum TNF-a level, and platelet aggregation. In vitro experiments revealed that PCA significantly inhibited the apoptotic rate and the expression of cleaved caspase-3, and it upregulated the expression of phosphorylated Akt in cardiomyocytes subjected to H/R injury. Our results suggest that PCA can provide a significant protection against MI/R injury, which may be at least partially attributed to its inhibitions against injury induced by MI/R including the inflammatory response, platelet aggregation, and cardiomyocytes apoptosis.

Sodium tanshinone IIA silate inhibits high glucose-induced vascular smooth muscle cell proliferation and migration through activation of AMP-activated protein kinase.

The proliferation of vascular smooth muscle cells may perform a crucial role in the pathogenesis of diabetic vascular disease. AMPK additionally exerts several salutary effects on vascular function and improves vascular abnormalities. The current study sought to determine whether sodium tanshinone IIA silate (STS) has an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation and migration under high glucose conditions mimicking diabetes without dyslipidemia, and establish the underlying mechanism. In this study, STS promoted the phosphorylation of AMP-activated protein kinase (AMPK) at T172 in VSMCs. VSMC proliferation was enhanced under high glucose (25 mM glucose, HG) versus normal glucose conditions (5.5 mM glucose, NG), and this increase was inhibited significantly by STS treatment. We utilized western blotting analysis to evaluate the effects of STS on cell-cycle regulatory proteins and found that STS increased the expression of p53 and the Cdk inhibitor, p21, subsequent decreased the expression of cell cycle-associated protein, cyclin D1. We further observed that STS arrested cell cycle progression at the G0/G1 phase. Additionally, expression and enzymatic activity of MMP-2, translocation of NF-κB, as well as VSMC migration were suppressed in the presence of STS. Notably, Compound C (CC), a specific inhibitor of AMPK, as well as AMPK siRNA blocked STS-mediated inhibition of VSMC proliferation and migration. We further evaluated its potential for activating AMPK in aortas in animal models of type 2 diabetes and found that Oral administration of STS for 10 days resulted in activation of AMPK in aortas from ob/ob or db/db mice. In conclusion, STS inhibits high glucose-induced VSMC proliferation and migration, possibly through AMPK activation. The growth suppression effect may be attributable to activation of AMPK-p53-p21 signaling, and the inhibitory effect on migration to the AMPK/NF-κB signaling axis.

[Protective effect of succinic acid on primary cardiomyocyte hypoxia/reoxygenation injury].

To establish cardiomyocyte hypoxia/reoxygenation injury model by culturing primary cardiomyocytes from suckling SD rats, in order to study the effect of succinic acid on LDH leakage rate cardiomyocyte ischemia/reperfusion injury. Furthermore, flow cytometry and western blot were conducted to detect the effect of succinic acid on cardiomyocyte apoptosis, cleaved caspase-3 and p-Akt, and discuss the protective effect of succinic acid on primary cardiomyocyte hypoxia/reoxygenation injury of primary cardiomyocytes from neonatal SD rats. According to the findings of the study, succinic acid at the concentrations ranging between 31.25 mg x L(-1) and 500 mg x L(-1) had no significant effect on primary cardiomyocyte activity, and succinic acid at the concentrations of 400, 200, 100, 50 mg x L(-1) could notably reduce cardiomyocyte ischemia/reperfusion LDH leakage rate (P < 0.01 or P < 0.05, respectively). Succinic acid at the concentrations of 400 mg x L(-1) and 200 mg x L(-1) could significantly reduce the percentage of cardiomyocyte apoptosis (P < 0.05), and inhibit the protein expression of cleaved caspase-3 caused by cardiomyocyte ischemia/reperfusion (P < 0.05). Succinic acid at the concentration of 400 mg x L(-1) could remarkably increase the protein expression of cardiomyocyte Akt (P < 0.05), while succinic acid at the concentration of 200 mg x L(-1) had no obvious effect on the protein expression of cardiomyocyte Akt. Therefore, this study demonstrated that succinic acid could inhibit necrosis and apoptosis caused by cardiomyocyte hypoxia/reoxygenation by activating Akt phosphorylation.

[Effect of huoxueyiqimingmu capsule on retinal degenerative diseases].

OBJECTIVE: To investigate therapeutic effects of Huoxueyiqimingmu capsule on retinal degenerative diseases. METHODS: Sixty SD rats were randomly divided into three groups: negative control group, light damage model group, Huoxueyiqimingmu capsule group. The light damage model was used as retinal degenerative diseases animal model. Normal saline and Huoxueyiqimingmu capsule were respectively administrated after light damage. After 14 days post-treatment, the change of electroretinogram was observed, content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in rats retina were determined, and histopathologic changes were observed with transmission electron microscopy and light microscopy. RESULTS: After 14 days post-treatment, Huoxueyiqimingmu capsule had protective effects on fall of ERG a wave and b wave amplitude induced by light damage. The activity of SOD in Huoxueyiqimingmu capsule group was higher than that in light damage model group (P < 0.01), while the content of MDA in Huoxueyiqimingmu capsule group was lower than that in light damage model group (P < 0.01). The histopathologic injury of rat retina in Huoxueyiqimingmu capsule group was lighter than that in light damage model group by means of transmission electron microscopy and light microscopy. CONCLUSIONS: Huoxueyiqimingmu capsule is effective to treat retinal degenerative diseases.

[Effects of cytochrome P450 isozymes on aristolochic acid renal cytotoxicity].

OBJECTIVE: To investigate the effect of cytochrome P450 isozymes on aristolochic acid induced cytotoxicity on renal proximal tubular epithelial cell (cell line HK-2). METHOD: Human renal tubular cells (cell line HK-2), were treated with aristolochic acid (AA) alone or in combination with cytochrome P450 isozymes inhibitors, including alpha-naphthoflavone (CYP450 1A1 and 1 A2 inhibitors), ketoconazole (CYP450 3A4 inhibitor), sodium diethyldithiocarbamate (CYP450 2A6 and 2E1 inhibitors), quinidine (CYP450 2D inhibitor), alpha-lipoic acid (NADPH: P450 reductase inhibitor), sulfaphenazole (CYP450 2C inhibitor) in the presence or absence of liver microsome(S9). The inhibition of cell proliferation rate was studied by MTT assay and the lactate dehydrogenase release rate was determined with continuous monitoring method. RESULT: AA inhibits cell proliferation and promotes the release of LDH over the range of 12.5-100 mg x L(-1), in a dose-dependent manner. Addition of S9 into the culture system reduced AA cytotoxicity, with the cell proliferation inhibition reducing and the release of LDH decreasing (AA + S9 group vs the same concentration of AA alone group, P < 0.05). In the absence of S9, ketoconazole or alpha-naphthoflavone has no obvious effect on AA cytotoxicity, however,under the conditions of adding S9, ketoconazole or alpha-naphthoflavone enhances AA cytotoxicity. Other inhibitors of CYP450 isozymes has no distinct effect on AA cytotoxicity. CONCLUSION: The microsomal enzyme of Liver can reduce the AA cytotoxicity, and CYP450 3A, CYP450 1A may be the major cytochrome P450 isozymes which impact AA cytotoxicity.

[Protective effect of Yiqi Mingmu solution on rat's retina with ischemia reperfusion injury].

OBJECTIVE: To investigate the protective effect of the Yiqi Mingmu solution irrigated into rat on retinal injury of rat induced by ischemia reperfusion. METHODS: SD rats were divided into four groups randomly: negative control group, model control group, low and high dose group. Increasing the intraocular pressure with anterior chambers infused liquid induced the models of retinal ischemia reperfusion of rats. Different doses of Yiqi Mingmu solution were respectively irrigated into rats 7 days before ischemia in low and high dose group. Normal salire instead was used in normal and model control group. At 1, 3, 7, 15 days after the ischemia reperfusion injury, the content of MDA and activity of SOD in rat's retina were measured. The histopathologic changes were observed with transmission electron microscopy and light microscopy. RESULTS: At 3, 7, 15 days after the ischemia reperfusion injury, the activity of SOD in Yiqi Mingmu solution low and high dose group were higher than in the model control group, the content of MDA in Yiqi Mingmu solution high dose group were lower than in the model control group. The histopathologic injury of retina in the Yiqi Mingmu solution low and high dose group were less than in the model control group. CONCLUSION: Yiqi Mingmu solution has protective effect on rat's retina ischemia reperfusion injury, and it is an effective traditional Chinese recipe to treat the diseases of retina ischemia.