ABSTRACT
ObjectiveTo explore the role of transient receptor potential vanilloid 1 （TRPV1） channel in reducing cardiomyocyte toxicity of Aconiti Kusnezoffii Radix processed with Chebulae Fructus. MethodH9c2 cardiomyocytes cultured in vitro were used as a model to assess cell viability by methyl thiazolyl tetrazolium （MTT） assay， the expression of TRPV1 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the leakage rate of lactate dehydrogenase （LDH）， the changes of nucleus， reactive oxygen species （ROS）， mitochondrial membrane potential and Ca2+ contents were detected by enzyme linked immunosorbent assay （ELISA）. ResultCompared with the blank group， when the concentration was ≥0.5 g·L-1， the cell viability was significantly decreased （P<0.01）， the leakage rate of LDH， the release of ROS and Ca2+ were increased， the mitochondrial membrane potential was decreased， and the nucleus was pyknosis or even broken in raw Aconiti Kusnezoffii Radix and Aconiti Kusnezoffii Radix processed with Chebulae Fructus groups. When the concentration was ≥0.5 g·L-1， compared with the same mass concentration of raw Aconiti Kusnezoffii Radix group， the cell viability increased significantly （P<0.01）， the leakage rate of LDH， the release of ROS and Ca2+ decreased， the mitochondrial membrane potential increased， and the nuclear morphology improved in Aconiti Kusnezoffii Radix processed with Chebulae Fructus group. Application of the same mass concentration of raw Aconiti Kusnezoffii Radix to H9c2 cardiomyocytes pretreated with the TRPV1 inhibitor BCTC significantly increased cell viability， decreased leakage rate of LDH， ROS and Ca2+ release， increased mitochondrial membrane potential and improved nuclear pyknosis compared with untreated H9c2 cardiomyocytes. Application of the same mass concentration of Aconiti Kusnezoffii Radix processed with Chebulae Fructus to H9c2 cardiomyocytes pretreated with BCTC decreased cell viability， increased LDH leakage rate， ROS and Ca2+ release， reduced mitochondrial membrane potential compared with untreated H9c2 cardiomyocytes. Real-time PCR results showed that both raw Aconiti Kusnezoffii Radix and Chebulae Fructus decoction could increase the expression of TRPV1 mRNA in cardiomyocytes in a concentration dependent manner. ConclusionRaw Aconiti Kusnezoffii Radix can induce cardiomyocyte apoptosis and cardiotoxicity by activating TRPV1 channel， while Aconiti Kusnezoffii Radix processed with Chebulae Fructus can attenuate the toxicity through TRPV1 channel， which may be related to the synergistic effect of acid components in Chebulae Fructus and alkaloids in Aconiti Kusnezoffii Radix on TRPV1 channel.
ABSTRACT
Lipid raft nanodomains of plasma membrane are rich in saturated lipids‚ cholesterol‚ sphingolipids‚ functioning as multimolecular platforms to recruit signaling and trafficking proteins involved in an array of physiological processes‚ which are critical for regulating signal transduction in cell. The staggering complexity of cell membranes and the transient formation of nanodomains greatly hinder research on lipid rafts by traditional experimental means. Molecular dynamics simulations have provided important insight into the organizational principles of cell membranes recently. Simulated membrane systems are under a transition from simple membrane models to multicomponent systems‚ culminating in realistic models of various cell types. Coarse-grained models have been extensively adopted as a powerful tool to explore membrane organization and interactions between lipids and proteins‚ providing efficient computational speed and enabling complex systems. In this work‚ coarse-grained molecular dynamics simulations with MARTINI force field were performed to build a raft-forming membrane with mixed lipids‚ including negatively charged lipid PIP2. Mixed lipids in this model were spontaneously partitioned into binary-phase membrane during 5 μs simulations by low temperature (295 K) treatment‚ forming lipid ordered (Lo) and liquid-disordered (Ld) nanodomains. Results of membrane thickness‚ lipid distribution‚ membrane fluidity‚ order parameters of the acyl tails‚ radial distribution functions were consistent with simulation and experimental data. Addition of small amounts of PIP2 did not affect the raft formation‚ and it showed remarkable affinity to lipid raft nanodomains. Simulations of the signaling transmembrane protein CD3ε in our raft-forming membranes were further performed to study the protein-lipid interaction as well. Results showed that the cytoplasmic tail of CD3ε was recruited to the Lo/ Ld boundary due to PIP2 binding‚ and this binding was regulated by Ca