Luchen Shan

A novel Danshensu derivative confers cardioprotection via PI3K/Akt and Nrf2 pathways

BACKGROUND Danshensu (3-(3,4-dihydroxyphenyl) lactic acid, DSS) is one of the most promising cardioprotective components in the root of Salvia miltiorrhiza but its poor chemical stability poses hurdles in its therapeutic development. It is therefore desirable to enhance the stability of DSS by chemical modification to improve its activities. In the present study, a novel DSS derivative named ADTM was synthesized and characterized for its cardioprotective properties. METHODS Oxidative stress was induced in H9c2 cardiomyoblast cells by tert-butylhydroperoxide (t-BHP) and the protective effects of ADTM were evaluated. For in vivo study, adult rats were treated with vehicle, DSS, ADTM or amlodipine (n=6-8/group) for 24h before the induction of acute myocardial ischemia. At the end of each experiment, infarct size was measured. Underlying the mechanisms of the cardioprotective effects of ADTM were further investigated in H9c2 cells and rat myocardium by evaluating the effects of Nrf2 (NF-E2-related factor 2) and Akt/PI3K pathways. RESULTS ADTM was approximately 10 times more effective than DSS against t-BHP-induced cell injury in H9c2 cells. In rat myocardial ischemia model, ADTM treatment significantly alleviated myocardial infarction. Akt/PI3K and Nrf2 pathways were demonstrated to be involved in both in vitro and in vivo experiments. CONCLUSIONS These results demonstrated that ADTM displayed much better cardioprotective effects than its parent compounds both in vitro and in vivo. This cardioprotection is mediated, at least in part, through Akt/PI3K and Nrf2 pathways. This novel compound represents a promising candidate for the treatment of cardiovascular diseases (CVDs), particularly myocardial infarction.

A novel agent exerts antitumor activity in breast cancer cells by targeting mitochondrial complex II

The mitochondrial respiratory chain, including mitochondrial complex II, has emerged as a potential target for cancer therapy. In the present study, a novel conjugate of danshensu (DSS) and tetramethylpyrazine (TMP), DT-010, was synthesized. Our results showed that DT-010 is more potent than its parental compounds separately or in combination, in inhibiting the proliferation of MCF-7 and MDA-MB-231 cells by inducing cytotoxicity and promoting cell cycle arrest. It also inhibited the growth of 4T1 breast cancer cells in vivo. DT-010 suppressed the fundamental parameters of mitochondrial function in MCF-7 cells, including basal respiration, ATP turnover, maximal respiration. Treatment with DT-010 in MCF-7 and MDA-MB-231 cells resulted in the loss of mitochondrial membrane potential and decreased ATP production. DT-010 also promoted ROS generation, while treatment with ROS scavenger, NAC (N-acetyl-L-cysteine), reversed DT-010-induced cytotoxicity. Further study showed that DT-010 suppressed succinate-induced mitochondrial respiration and impaired mitochondrial complex II enzyme activity indicating that DT-010 may inhibit mitochondrial complex II. Overall, our results suggested that the antitumor activity of DT-010 is associated with inhibition of mitochondrial complex II, which triggers ROS generation and mitochondrial dysfunction in breast cancer cells.

Design, Synthesis, and Preliminary Cardioprotective Effect Evaluation of Danshensu Derivatives

A series of (R)‐3,4‐dihydroxyphenyllactic acid Danshensu (DSS) derivatives were synthesized, and their cardioprotective effects were evaluated in vitro and in vivo. Among the new derivatives, compound 14 showed significant protective effects in cultured myocardial cells and in the rat model of myocardial ischemia. The therapeutic efficacy of compound 14 was significantly higher than that of its parent compound DSS, and amlodipine, a first‐line treatment for angina pain. Compound 14 potently scavenged free radicals, significantly decreased the levels of LDH and MDA, and inhibited the leakage of CK in animal model of ischemia. We had previously found that compound 14 activated PI3K/Akt/GSK‐3β and Nrf2//Keap1/heme oxygenase‐1 (HO‐1) signaling pathways in H9c2 cells. These results suggest that compound 14 has a unique mechanism of action, that is, multifunctional. Compound 14 may be a new potential therapy for ischemic heart diseases.

Synthesis and biological evaluations of novel apocynin analogues.

We have designed and synthesized a series of novel apocynin analogues, and evaluated their biological activity. Compound 10, an apocynin dimer analogue, compound 12, the lipoic acid (LA) and apocynin conjugate, were the most potent in protecting cells from lipopolysaccharide (LPS)-induced cytotoxicity, had significant activity scavenging ROS induced by LPS, and greatly decreased LPS-induced P67(phox) protein expression. SAR analysis suggests that modification of apocynin can increase its activity. Our results demonstrate that arming apocynin with a powerful antioxidant such as lipoic acid is a valid strategy to design new apocynin analogues with enhanced biological activity.

Andrographolide derivative AL-1 ameliorates TNBS-induced colitis in mice: involvement of NF-кB and PPAR-γ signaling pathways

Andrographolide is a traditional herb medicine, widely used in Asia for conditions involving inflammation. The andrographlide-lipoic acid conjugate, AL-1, has been found being able to alleviate inflammation in our previous reports. Although the anti-inflammatory activity of AL-1 contributes to its cytoprotective effects, whether AL-1 can improve inflammatory bowel disease (IBD) and the underlying mechanisms of its action remain largely unknown. In this study, we investigated the anti-inflammatory effects of AL-1 in C57BL/6 mice with trinitrobenzenesulfonic acid (TNBS)-induced colitis. The body weight loss and length change of colon after TNBS instillation were more severe than those in normal mice. AL-1 treatment led to significant reductions in disease activity index (DAI), macroscopic score and colon mucosa damage index (CMDI) associated with TNBS administration. AL-1 inhibited the inflammatory response via lowering the level of inflammatory cytokines and myeloperoxidase (MPO) activity. AL-1 attenuated the expression of p-p65, p-IκBα and COX-2 in the colitis mice. The alleviation of colon injury by AL-1 treatment was also evidenced by the increased expression of PPAR-γ. These results indicated that AL-1 could protect intestinal tract from the injury induced by TNBS in mice, suggesting that AL-1 may have potential in treatment for IBD.

A Novel Danshensu Derivative Prevents Cardiac Dysfunction and Improves the Chemotherapeutic Efficacy of Doxorubicin in Breast Cancer Cells.

Doxorubicin (Dox) is an anthracycline antibiotic widely used in clinics as an anticancer agent. However, the use of Dox is limited by its cardiotoxicity. We have previously shown that a Danshensu (DSS) derivative, ADTM, displayed strong cardioprotective effects. With improved chemical stability and activity, a novel DSS derivative, D006, based on the structure of ADTM, was synthesized. In the present study, the protective effects of D006, indexed by attenuation of the cardiotoxicity induced by Dox as well as chemosensitizing effects that increase the antitumor activity of Dox, were investigated. Our results showed that D006 was more potent than either parental compound, or their use in combination, in ameliorating Dox‐induced toxicity in H9c2 cells. In our zebrafish model, D006, but not DSS, alone significantly preserved the ventricular function of zebrafish after Dox treatment. Moreover, D006 upregulated mitochondrial biogenesis and increased mtDNA copy number after Dox treatment of H9c2 cells. D006 promoted the expression of HO‐1 protein in a time‐dependent manner while the HO‐1 inhibitor, Znpp, reversed the protective effects of D006. In human breast tumor MCF‐7 cells, D006 enhanced Dox‐induced cytotoxicity by increasing apoptosis. In conclusion, our results indicate that a new DSS derivative exhibits promising protective effects against Dox‐induced cardiotoxicity both in vivo and in vitro, an effect at least partially mediated by induction of HO‐1 expression and the activation of mitochondrial biogenesis. Meanwhile, D006 also potentiated the anti‐cancer effects of Dox in breast tumor cells. J. Cell. Biochem. 117: 94–105, 2016.

Novel anti-thrombotic agent for modulation of protein disulfide isomerase family member ERp57 for prophylactic therapy

Protein disulfide isomerase (PDI) family members including PDI and ERp57 emerge as novel targets for anti-thrombotic treatments, but chemical agents with selectivity remain to be explored. We previously reported a novel derivative of danshensu (DSS), known as ADTM, displayed strong cardioprotective effects against oxidative stress-induced cellular injury in vitro and acute myocardial infarct in vivo. Herein, using chemical proteomics approach, we identified ERp57 as a major target of ADTM. ADTM displayed potent inhibitory effects on the redox activity of ERp57, inhibited the adenosine diphosphate (ADP)-induced expressions of P-selectin and αIIbβ3 integrin, and disrupted the interaction between ERp57 and αIIbβ3. In addition, ADTM inhibited both arachidonic acid (AA)-induced and ADP-induced platelet aggregation in vitro. Furthermore, ADTM significantly inhibited rat platelet aggregation and thrombus formation in vivo. Taken together, ADTM represents a promising candidate for anti-thrombotic therapy targeting ERp57.

Pharmacokinetic and Metabolic Studies of ADTM: A Novel Danshensu Derivative Confers Cardioprotection by HPLC-UV and LC-MS/MS.

(R)-(3,5,6-Trimethylpyrazinyl) methyl-2-acetoxy-3-(3,4-diacetoxyphenyl) propanoate (ADTM) is a novel Danshensu (DSS) derivative regarded as a potential new agent for the treatment of myocardial ischemia. A validated high performance liquid chromatography (HPLC) approach with a detection limit of 5 ng/mL was used for pharmacokinetic evaluation of ADTM in rat plasma. The intra- and interday precision in terms of relative standard deviation were <4.98 and 4.84%, respectively, at concentration levels of 0.02, 0.20 and 0.80 µg/mL. ADTMs absolute oral bioavailability value was 30.4% and t1/2 was 34.33 ± 11.51 and 29.94 ± 8.19 min after oral and intravenous administration of 20 mg/kg. In addition, the major metabolites both in vitro and in vivo were 2-hydroxymethy-3,5,6-trimethylpyrazin and DSS. The results indicated that the hydrolysis was the main metabolic pathway of ADTM, and carboxylesterase may play an important role in ADTMs metabolism. The present work provides basic information for ADTMs further preclinical research and DSSs chemical structure modification.

Relaxation effect of a novel Danshensu/tetramethylpyrazine derivative on rat mesenteric arteries.

Danshen (Radix Salviae miltiorrhizae) and ChuanXiong (Ligusticum wallichii) are two traditional herbal medicines commonly used in China for the treatment of cardiovascular diseases. The active components in Danshen and ChuanXiong are Danshensu (DSS, (R)-3, 4-dihydroxyphenyllactic acid) and tetramethylpyrazine (TMP), respectively. In the present study, a new compound named ADTM, which is a conjugation of DSS and TMP, was synthesized and its effect on the contractility of rat mesenteric arteries was examined. The relaxation effect of ADTM on rat mesenteric arteries was studied using myography. The effects of ADTM on Ca(2+) channels were measured by Ca(2+) imaging and patch-clamp techniques. The results showed that ADTM caused a concentration-dependent relaxation of rat mesenteric arteries. This relaxation effect was not affected by the removal of endothelium or inhibitors of nitric oxide synthase, cyclooxygenase, guanylyl cyclase and adenylyl cyclase. Potassium channel blockers including tetraethylammonium, iberiotoxin, apamin, 4-aminopyridine, BaCl2 and glibenclamide also failed to inhibit the relaxation response to ADTM. ADTM inhibited CaCl2-induced contractions and reduced the Ca(2+) influx in isolated mesenteric arterial muscle cells. Our results suggest that ADTM may be a novel relaxing agent. Its mechanism of action involves the direct blockade of voltage-gated Ca(2+) channels in vascular smooth muscle cells, resulting in a decrease in Ca(2+) influx into the cells.

Identification of disulfide isomerase ERp57 as a target for small molecule cardioprotective agents

We previously reported a novel danshensu analogue known as ADTM, which exhibited strong protective effects against oxidative stress-induced cellular injury and acute ischemic myocardial infarct in rat; however, the exact protein target of ADTM has not been fully characterized. In the present study, a biotin-conjugated ADTM analogue (BAA) was employed as molecular probe to identify its protein targets. BAA exhibited similar protective effect against oxidative stress-induced cell injury in H9c2 cardiomyoblast. A chemical proteomic approach identified ERp57 as the specific target for BAA. Further evaluation with Western blot and immunofluorescence staining assays confirmed the direct interactions between BAA and ERp57. Moreover, BAA displayed potent inhibitory effect on the catalytic activity of ERp57 in the insulin reduction assay. Molecular docking showed that BAA bound at the active site of ERp57. These data suggested that ERp57 is a potential target of cardioprotective danshensu analogues, and provided the basis for the further optimization of the cardioprotective compounds.