Evidences for the mechanism of Shenmai injection antagonizing doxorubicin-induced cardiotoxicity.

BACKGROUND: Doxorubicin (DOX) is a widely used antitumor drug. However, its clinical application is limited for its serious cardiotoxicity. The mechanism of DOX-induced cardiotoxicity is attributed to the increasing of cell stress in cardiomyocytes, then following autophagic and apoptotic responses. Our previous studies have demonstrated the protective effect of Shenmai injection (SMI) on DOX-induced cardiotoxicity via regulation of inflammatory mediators for releasing cell stress. PURPOSE: To further investigate whether SMI attenuates the DOX-induced cell stress in cardiomyocytes, we explored the mechanism underlying cell stress as related to Jun N-terminal kinase (JNK) activity and the regulation of autophagic flux to determine the mechanism by which SMI antagonizes DOX-induced cardiotoxicity. STUDY DESIGN: The DOX-induced cardiotoxicity model of autophagic cell death was established in vitro to disclose the protected effects of SMI on oxidative stress, autophagic flux and JNK signaling pathway. Then the autophagic mechanism of SMI antagonizing DOX cardiotoxicity was validated in vivo. RESULTS: SMI was able to reduce the DOX-induced cardiomyocyte apoptosis associated with inhibition of activation of the JNK pathway and the accumulation of reactive oxygen species (ROS). Besides, SMI antagonized DOX cardiotoxicity, regulated cardiomyocytes homeostasis by restoring DOX-induced cardiomyocytes autophagy. Under specific circumstances, SMI depressed autophagic process by reducing the Beclin 1-Bcl-2 complex dissociation which was activated by DOX via stimulating the JNK signaling pathway. At the same time, SMI regulated lysosomal pH to restore the autophagic flux which was blocked by DOX in cardiomyocytes. CONCLUSION: SMI regulates cardiomyocytes apoptosis and autophagy by controlling JNK signaling pathway, blocking DOX-induced apoptotic pathway and autophagy formation. SMI was also found to play a key role in restoring autophagic flux for counteracting DOX-damaged cardiomyocyte homeostasis.

Protective effect of Shenmai injection on doxorubicin-induced cardiotoxicity via regulation of inflammatory mediators.

BACKGROUND: Doxorubicin (DOX) is a chemotherapy drug for malignant tumors. The clinical application of DOX is limited due to its dosage relative cardiotoxicity. Oxidative damage and cardiac inflammation appear to be involved in DOX-related cardiotoxicity. Shenmai injection (SMI), which mainly consists of Panax ginsengC.A.Mey.and Ophiopogon japonicus (Thunb.) Ker Gawl, is widely used for the treatment of atherosclerotic coronary heart disease and viral myocarditis in China. In this study, we investigated the protective effect of Shenmai injection on doxorubicin-induced acute cardiac injury via the regulation of inflammatory mediators. METHODS: Male ICR mice were randomly divided into seven groups: control, DOX (10 mg/kg), SMI (5 g/kg), DOX with pretreatment with SMI (0.5 g/kg, 1.5 g/kg or 5 g/kg) and DOX with post-treatment with SMI (5 g/kg). Forty-eight hours after the last DOX administration, all mice were anesthetized for ultrasound echocardiography. Then, serum was collected for biochemical and inflammatory cytokine detection, and heart tissue was collected for histological and Western blot detection. RESULTS: A cumulative dose of DOX (10 mg/kg) induced acute cardiotoxicity in mice manifested by altered echocardiographic outcome, and increased tumor necrosis factor, interleukin 6 (IL-6), monocyte chemotactic protein 1, interferon-γ, and serum AST and LDH levels, as well as cardiac cytoplasmic vacuolation and myofibrillar disarrangement. DOX also caused the increase in the expression of IKK-α and iNOS and produced a large amount of NO, resulting in the accumulation of nitrotyrosine in the heart tissue. Pretreatment with SMI elicited a dose-dependent cardioprotective effect in DOX-dosed mice as evidenced by the normalization of serum inflammatory mediators, as well as improve dcardiac function and myofibril disarrangement. CONCLUSIONS: SMI could recover inflammatory cytokine levels and suppress the expression of IKK-α and iNOS in vivo, which was increased by DOX. Overall, there was evidence that SMI could ameliorate DOX-induced cardiotoxicity by inhibiting inflammation and recovering heart dysfunction.

Simultaneous determination and pharmacokinetics of eight ginsenosides by LC-MS/MS after intravenously infusion of 'SHENMAI' injection in dogs.

SHENMAI injection, a prescription comprised of Panax ginseng and Ophiopogon japonicas, is being extensively applied in the field of cardio-protection and immune-modulation in China. Ginsenosides are the main active components in SHENMAI injection. In order to capture and analyze the pharmacokinetic profile of major ginsenosides of SHENMAI injection in Beagle dogs, liquid chromatography equipped with electro-spray ionization and tandem mass spectrometry method was applied in simultaneous determination for protopanaxatriol type ginsenoside (Re, Rf, Rg1), protopanaxadiol type ginsenoside (Rb2, Rb1, Rd, Rc) and oleanolic acid type ginsenoside (Ro). A C18 column (150 × 2.1mm, 5µm) and a linear gradient program were used to achieve chromatographic separation, with 0.02% acetic acid solution and acetonitrile. I.S. and ginsenosides were detected by LC-MS/MS in selective reaction mode. Good linearity spanning 5- 1500ng/mL was achieved with the R2 values higher than 0.99 for all analytes. Limit of quantification of all analytes were 3ng/mL. Intra- and inter-day precisions ranges from 0.47 to15.68 % and accuracies were within the range of 85.27-117.57%. Validated analyzing method was then used in the pharmacokinetic experiment for SMI in dogs. The results showed that the pharmacokinetic profile of protopanaxadiol, protopanaxatriol and oleanolic acid type ginsenoside were significant difference in dogs. Protopanaxadiol type ginsenosides exhibited an extremely higher level of exposure and a much slower elimination process. Whereas protopanaxatriol type ginsenosides were quickly eliminated. We concluded that 20 (S) - protopanaxadiol type ginseno sides could be a potential pharmacokinetic marker of SHENMAI injection.

Potential accumulation of protopanaxadiol-type ginsenosides in six-months toxicokinetic study of SHENMAI injection in dogs.

SHENMAI injection (SMI), derived from famous Shen Mai San, is a herbal injection widely used in China. Ginsenosides are the major components of SMI. To monitor the exposure level of SMI during long-term treatment, a 6-month toxicokinetic experiment was performed. Twenty-four beagle dogs were dived into four groups (n = 6 in each group): a control group (0.9% NaCl solution) and three SMI groups (2, 6 or 3 mg/kg). The dogs were i.v. infused with vehicle or SMI daily for 180 d. Blood samples for analysis were collected at specific time points as follows: pre-dose (0 h); at 10, 30, and 60 min during infusion; and at 10, 30, 60, 90, 120, 240, and 300 min post-administration. Concentrations of ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 in the plasma were determined simultaneously by liquid chromatography-tandem mass spectrometry. Non-compartmental parameters were further calculated and analyzed. Significant differences were found between the kinetic behavior of 20(S)-protopanaxadiol-type (PPD-type) and 20(S)-protopanaxatriol-type (PPT-type) ginsenosides. Increasing in the exposure level of PPD-type ginsenosides was observed in dogs during the experiment. Therefore, PPD-type ginsenosides are closely related to the immunity modulation effect of SMI. Increased PPD-type ginsenoside exposure level may present potential toxicity and induce drug-drug interaction risks during SMI administration. As such, PPD-type ginsenoside accumulation must be carefully monitored in future SMI research.

Resveratrol alleviates vascular inflammatory injury by inhibiting inflammasome activation in rats with hypercholesterolemia and vitamin D2 treatment.

OBJECTIVE: Atherosclerosis (AS) is an inflammatory disease involved in vascular inflammatory injury. The inflammasome is an important part of inflammatory diseases and participates in the vascular inflammatory injury. Resveratrol (RSV) possesses anti-inflammatory activities, but its effects on inflammasomes during vascular injury remain unclear. This study focused on the effects and mechanisms of RSV on inflammasomes during vascular injury. METHODS: Male Sprague-Dawley rats were treated with a purified diet or cholesterol-enriched diet combined with vitamin D2 (VD; 1.8 million units/kg/days, Po) and saline or RSV (50 mg/kg/days, Po) daily for 5 weeks. The concentrations and enzyme activities of related indicators were measured by a spectrophotometer or ELISA kit. Their gene and protein expression levels were analyzed by reverse transcription-polymerase chain reaction and Western blot, respectively. RESULTS: Upon administration with RSV, rats with combined hyper cholesterol and VD demonstrated the following changes: the vascular histopathological changes were relieved, and the level of the von Willebrand factor decreased. The level of serum IL-1ß, a marker of inflammasome activation, significantly decreased. The mRNA and protein expression levels of the three components of inflammasomes, namely, NOD-like receptor pyrin domain containing 3, apoptosis-associated speck-like protein containing a caspase-recruitment domain, and caspase-1, were downregulated. The effects of RSV were closely related to hypolipidemia (decrease in the levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol combined with the expression of the lectin-like ox-LDL receptor and increase in high-density lipoprotein cholesterol), antioxidation (decrease in MDA levels and increase in SOD and GPx activities), and anti-inflammation (downregulation of the expression of IL-1ß, intracellular adhesion molecule-1, and monocyte chemotactic protein-1). The mechanisms for the downregulation of NF-κB p65 and p38 MAPK expression, as well as the upregulation of SIRT1 expression, were analyzed. CONCLUSION: This study proved that RSV inhibited inflammasome activation to protect vascular injury in vivo. RSV exhibited therapeutic potential in the treatment of vascular injury.

One-month toxicokinetic study of SHENMAI injection in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: 'SHENMAI' injection (SMI) has been widely used in cardioprotection and modulation of the immune system because of its great efficacy. SMI primarily comprises the saponins from Panax ginseng and Ophiopogon japonicas. The profiles of saponins in SMI during long-term toxicokinetics remain unclear. MiR-146a possesses excellent sensitivity as a bio-marker in the innate immunity modification effect of SMI. AIM OF THE STUDY: Is to monitor the exposure level of SMI during a one-month toxicokinetic experiment, an analytical method involving ESI-LC-MS/MS technology was developed to determine 20 (S)-protopanaxadiol-type ginsenoside (Rb1, Rb2, Rc, Rd), 20 (S)-protopanaxatriol-type ginsenoside (Rg1, Re, Rf), oleanolic acid-type ginsenoside (Ro), and ophiopogonin D in rats. The levels of AST, CK, ALT, SOD, GSH-pX, MDA, miR-146a, and ECG were measured to explore the effects of SMI in cardiologic function and immune activity. RESULTS: Results show that the levels of AST, CK, and MDA decreased upon the administration of SMI. The level of miR-146a increased upon the administration of SMI dosage. During the administration of SMI, increasing exposure levels of 20 (S)-protopanaxadiol-type ginsenosides were also observed. CONCLUSION: The 20 (S)-protopanaxadiol-type ginsenosides were considered potential PK/TK markers because of their high exposure levels that continuously increased. Oxidative stress was slightly alleviated during the toxicokinetic study. Based on the level of miR-146a, negatively regulated innate immunity was observed. The regulation became more serious with increasing exposure levels of 20 (S)-protopanaxadiol-type ginsenosides. Negatively regulated innate immunity could be induced by long-term administration of SMI (>0.4g/kg).

Biological implications of oxidation and unidirectional chiral inversion of D-amino acids.

Recent progress in chiral separation of D- and L-amino acids by chromatography ascertained the presence of several free Damino acids in a variety of mammals including humans. Unidirectional chiral inversion of many D-amino acid analogs such as exogenous NG-nitro-D-arginine (D-NNA), endogenous D-leucine, D-phenylanine and D-methionine have been shown to take place with inversion rates of 4-90%, probably dependent on various species D-amino acid oxidase (DAAO) enzymatic activities. DAAO is known to catalyze the oxidative deamination of neutral and basic D-amino acids to their corresponding α-keto acids, hydrogen peroxide and ammonia, and is responsible for the chiral inversion. This review provides an overview of recent research in this area: 1) oxidation and chiral inversion of several D-amino acid analogs in the body; 2) the indispensable but insufficient role of DAAO particularly in the kidneys and brain for the oxidation and chiral inversion of D-amino acids analogs; and 3) unidentified transaminase(s) responsible for the second step of chiral inversion. The review also discusses the physiological significance of oxidation and chiral inversion of D-amino acids, which is still a subject of dispute.

Protective effect of Lycium barbarum polysaccharides against doxorubicin-induced testicular toxicity in rats.

The present study aimed to investigate whether Lycium barbarum polysaccharides (LBP) would protect against doxorubicin (DOX)-induced testicular toxicity. Male Sprague-Dawley rats were treated with distilled water (4 mL/kg) or LBP (200 mg/kg, p.o.) daily for 10 days and followed by saline (0.9 %, 10 mL/kg) or DOX (10 mg/kg) intravenous injection at day 7. Pretreatment with LBP ameliorated DOX-induced reduction in the testicular weights, sperm concentrations and percentage of motile sperms, as well as the increase in abnormal sperm rate. LBP administration to DOX-treated rats successfully reversed the changes in MDA and GHS-Px levels. Compared with the control, pretreatment with LBP significantly increased the plasma testosterone level in the LBP + DOX group. The histopathology examinations further confirmed that LBP effectively attenuated DOX-induced severe degenerative changes of seminiferous tubules. This study illustrated the capability of LBP in attenuating testicular oxidative stress and protecting testis-specific toxicity in DOX-exposed rats.

Alleviation of the acute doxorubicin-induced cardiotoxicity by Lycium barbarum polysaccharides through the suppression of oxidative stress.

The present study aims to investigate whether Lycium barbarum polysaccharides (LBP) could protect against acute doxorubicin (DOX)-induced cardiotoxicity. Rats received daily treatment of either distilled water (4 ml/kg) or LBP (200mg/kg) for 10 days and then followed by an intravenous injection at day 7 of either saline (10 ml/kg) or DOX (10 mg/kg). DOX induced significantly myocardial damage in rats, which were characterized as conduction abnormalities, decreased heart-to-body weight ratio, increased serum CK, and myofibrillar disarrangement. DOX treatment also increased MDA and decreased SOD and GSH-Px activity in cardiac tissues. Pretreatment with LBP significantly reduced DOX-induced oxidative injury in cardiac tissue, suggesting by the fact that LBP significantly attenuated DOX-induced cardiac myofibrillar disarrangement and LBP was effective in decreasing the levels of serum CK and thus improving conduction abnormalities caused by DOX. LBP treatment significantly increased SOD and GSH-Px activity and decreased the MDA level of heart tissues damaged by DOX exposure in rats. Furthermore, the cytotoxic study showed that LBP protect against cytotoxicity of DOX in cardiac myoblasts H9c2 but dose not attenuate the anti-tumor activity of DOX. In summary, our evidence indicates that LBP elicited a typical protective effect on DOX-induced acute cardiotoxicity via suppressing oxidative stress.

Stereospecific pharmacodynamics and chiral inversion of NG-nitro-arginine in the beagle dogs.

In the present study, we analyzed the stereospecific pharmacodynamics and inversion of N(G)-nitro-arginine by an intravenous blous injection of L-N(G)-nitro-arginine (L-NNA) or D-N(G)-nitro-arginine (D-NNA) (10 mg/kg) in beagle dogs. Significant pressor responses were observed for both substances, though a similar maximum response induced by L-NNA was reached at 120 min slower as compared with D-NNA. The rise in mean arterial pressure (MAP) of D-NNA dogs was also shown to be slower than the L-NNA group. Our data showed that D-NNA had no impact on MAP within 60 min after its injection. Plasma L-NNA started to appear after 45 min posterior to the i.v. bolus injection of D-NNA. This chiral inversion is unidirectional because no D-NNA was not produced from L-NNA. The pressor response in the D-NNA-injected dogs was well parallel to the plasma L-NNA concentration. Similar disposition of N(G)-nitro-arginine enantiomers and 4% of chiral inversion ratio from D-NNA to L-NNA was found in the beagle dogs. Given that D-amino acid oxidase (DAAO) is the essential enzyme in chiral inversion of D-NNA, we further compared the enzymatic activity of the renal DAAO between dogs and rats. Our data showed that dogs had a significantly lower enzymatic activity than rats, thus supported a lower inversion ratio of D-NNA in dogs.

Indispensable but insufficient role of renal D-amino acid oxidase in chiral inversion of NG-nitro-D-arginine.

Unidirectionally chiral inversion of N(G)-nitro-D-arginine (D-NNA) to its L-enantiomer (L-NNA) occurred in rats, and it was blocked markedly (ca. 80%) by renal vascular ligation, and entirely (100%) by the D-amino acid oxidase (DAO) inhibitor sodium benzoate, suggesting that renal DAO is essential for the inversion. However, the doses of sodium benzoate administrated were extremely high (e.g., 400 mg/kg) due to its low potency. It is thus possible that sodium benzoate-mediated blockade of D-NNA inversion might be due to its nonspecific (or non-DAO-related) effects. In addition, after D-NNA was incubated with the pure enzyme of DAO in vitro without tissue homogenates, L-NNA was not produced, even though D-NNA was disposed. We propose that this occurred because D-NNA was first converted to its corresponding alpha-keto acid by DAO and then to L-NNA by transaminase(s); however, there was no direct evidence for this process. The goal of this study is to further elucidate the process of D-NNA chiral inversion both in vivo and in in vitro tissue homogenates by comparing mutant ddY/DAO(-/-) mice that lack DAO activity entirely compared to normal ddY/DAO(+/+) mice and Swiss mice. Furthermore, the ability to produce L-NNA from D-NNA-corresponding alpha-keto acids (N(G)-nitroguanidino-2-oxopentanoic acid) produced by porcine kidney-derived DAO (pkDAO) was also studied in the DAO inhibitor-pretreated rats. We found that D-NNA chiral inversion occurred in Swiss mice and ddY/DAO(+/+) mice both in vivo and in in vitro kidney homogenates, but not in ddY/DAO(-/-) mice, correlated to their DAO activities. The alpha-keto acid (N(G)-nitro-guanidino-2-oxopentanoic acid) from D-NNA was able to produce L-NNA, and subsequent vasoconstriction and pressor responses. These results indicate that the role of renal DAO is indispensible but insufficient for chiral inversion of D-NNA and other neutral and polar D-amino acids, and unidentified aminotransferase(s) are involved in a subsequent mechanism for the process of chiral inversion.

Angelica sinensis: a novel adjunct to prevent doxorubicin-induced chronic cardiotoxicity.

Doxorubicin is an anthracycline antibiotic agent used in the treatment of a variety of solid and haematopoietic tumours, but its use is limited by formation of metabolites that induce acute and chronic cardiac toxicities. Angelica sinensis has been widely used to treat cardiovascular and cerebrovascular diseases in China. In the present study, we used an in vivo mouse model to explore whether A. sinensis could protect against doxorubicin-induced chronic cardiotoxicity. Male ICR mice were treated with distilled water or water extraction of A. sinensis (15 g/kg, orally) daily for 4 weeks, followed by saline or doxorubicin (15 mg/kg, intravenously) treatments weekly. Cardiotoxicity was assessed by electrocardiograph, antioxidant activity in cardiac tissues, serum levels of creatine kinase, aspartate aminotransferase (AST) and histopathological change in cardiac tissues. A cumulative dose of doxorubicin (60 mg/kg) caused animal death and myocardial injury characterized by increased QT interval and decreased heart rate in electrocardiograph, decrease of heart antioxidant activity, increase of serum AST, as well as myocardial lesions. Pre-treatment with A. sinensis significantly reduced mortality and improved heart performance of the doxorubicin-treated mice as evidenced from normalization of antioxidative activity and serum AST, preventing loss of myofibrils as well as improving arrhythmias and conduction abnormalities. Furthermore, the in vitro cytotoxic study showed that A. sinensis did not compromise the antitumour activity of doxorubicin. These results suggested that A. sinensis elicited a typical cardioprotective effect on doxorubicin-related oxidative stress, and could be a novel adjunct in the combination with doxorubicin chemotherapy.

Protective effect of Lycium barbarum on doxorubicin-induced cardiotoxicity.

The objective of this work was to explore the hypothesis that Lycium barbarum (LB) may be protective against doxorubicin (DOX)-induced cardiotoxicity through antioxidant-mediated mechanisms. Male SD rats were treated with distilled water or a water extract of LB (25 mg/kg, p.o.) daily and saline or DOX (5 mg/kg, i.v.) weekly for 3 weeks. Mortality, general condition and body weight were observed during the experiment. DOX-induced cardiotoxicity was assessed by electrocardiograph, heart antioxidant activity, serum levels of creatine kinase (CK) and aspartate aminotransferase (AST) and histopathological change. The DOX group showed higher mortality (38%) and worse physical characterization. Moreover, DOX caused myocardial injury manifested by arrhythmias and conduction abnormalities in ECG (increased QT and ST intervals and ST elevation), a decrease of heart antioxidant activity, an increase of serum CK and AST, as well as myocardial lesions. Pretreatment with LB significantly prevented the loss of myofibrils and improved the heart function of the DOX-treated rats as evidenced from lower mortality (13%), normalization of antioxidative activity and serum AST and CK, as well as improving arrhythmias and conduction abnormalities. These results suggested that LB elicited a typical cardioprotective effect on DOX-related oxidative stress. Furthermore, in vitro cytotoxic study showed the antitumor activity of DOX was not compromised by LB. It is possible that LB could be used as a useful adjunct in combination with DOX chemotherapy.

[Purification of human VLDL apolipoproteins by middle pressure liquid chromatography].

OBJECTIVE: To purify human VLDL apolipoproteins by middle-pressure liquid chromatography. METHODS: Human VLDLs were isolated by one step density ultracentrifugation. Delipided human VLDL was separated by Sephacryl S-200 molecular sieve chromatography. ApoE was purified by heparin Sepharose CL-6B affinity chromatography. ApoC I ,C II and C III were purified from apoC. fraction by DEAE-Sephacel ion exchange chromatography: RESULTS: Purified apoE, apoC I, apoC II and apoC III were obtained. SDS-PAGE and immunodiffusion tests indicated the isolated proteins were pure. CONCLUSION: We have established a purification procedure for human VLDL apolipoproteins with highly efficiency and simplicity by MPLC.

[Purification of human serum apolipoprotein A I and A II by middle-pressure liquid chromatography].

OBJECTIVE: This study is made for the purification of human HDL apolipoproteins by middle-pressure liquid chromatography (MPLC). METHODS: Human HDL was isolated by one step density ultracentrifugation. Delipided human HDL was separated by Sephacryl S-200 molecular sieve chromatography. Sephadex G-75 molecular sieve chromatography was used to separate apoA I and apoA II. RESULTS: Purified apoA I and apoA II were obtained and SDS-PAGE and immunodiffusion test indicated that the proteins are the same as those theoretically predicted. CONCLUSION: We have established a purification procedure for human HDL apolipoproteins with high efficiency and simplicity by MPLC. It could serve as a base for preclinical and clinical trials of HDL apolipoproteins.

Renal D-amino acid oxidase mediates chiral inversion of N(G)-nitro-D-arginine.

N(G)-nitro-d-arginine (d-NNA), i.v. injected into rats, produced a pressor response, and was presumed to act via chiral inversion into N(G)-nitro-l-arginine (l-NNA), an inhibitor of nitric oxide synthase. We examined the possible role of renal d-amino acid oxidase (DAAO) in the chiral inversion of d-NNA to l-NNA. In pentobarbital-anesthetized rats, l-NNA was detected via capillary electrochromatography in the blood immediately after i.v. injection of d-NNA. The time course of appearance of l-NNA paralleled the increase in blood pressure elicited by d-NNA. Unilateral renal ligation partially, and bilateral ligation completely, blocked the pressor response as well as the conversion of d-NNA to l-NNA. Furthermore, injection into conscious rats of sodium benzoate, a selective DAAO inhibitor, completely blocked the pressor response to naive d-NNA, but not pressor response to d-NNA preincubated with homogenates of the kidney. Homogenates of the kidneys, liver (lesser degree), and brain (much lesser degree) converted d-NNA to l-NNA, and the chiral inversion was blocked by the addition of benzoate. Moreover, d-NNA chiral inversion correlates with the activity of DAAO. Our results reveal a novel pathway of chiral inversion of d-amino acids where the renal DAAO plays an essential role that accounts for the biological activity of d-NNA.