Advances in Research of Pharmacological Effects and Molecular Mechanisms of Daurisoline

Quan QUAN, Chenghao JIN

College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Abstract Daurisoline is an important bisbenzylisoquinoline alkaloid and mainly exists in the rhizome of Menispermum dauricum DC., a plant of the family Menispermaceae. Due to its pharmacological effects such as anti-arrhythmia, anti-ischemia and anti-cancer, daurisoline has attracted wide attention of researchers. In this paper, the pharmacological effects and action mechanism of daurisoline were reviewed in combination with relevant research literature, to provide a theoretical basis for the development and clinical application of daurisoline.

Key words Daurisoline, Pharmacological effects, Calcium regulation, Anticancer

1 Introduction

MenispermumdauricumDC. is a plant of the family Menispermaceae and has a wide geographical distribution, has no harsh requirements for growth conditions, and is often grown in shrubs. Daurisoline is a milky yellow powder at room temperature, mainly extracted from the rhizome ofM.dauricumDC. Its molecular formula is C37H42N2O6, and its molecular weight is 610.739. Daurisoline has biological activities such as anti-arrhythmic imbalance and anti-tumor, and also has certain effects in brain tissue protection, thus has broad medical prospects. We reviewed the pharmacological effects and action mechanism of action of daurisoline in recent years, to provide a theoretical basis for its clinical application.

2 Pharmacological effects and molecular mechanisms of daurisoline

2.1 Regulation of calcium channels and intracellular calciumHomeostasis is the precondition for the normal chemical reactions of the body. Inorganic salts are an indispensable component for maintaining homeostasis in the body. Ca2+is a constant inorganic salt in the body and is the secondary messenger in cells. Ca2+maintains the health of human bones, muscles, heart and other organs and tissues, and exerts an important influence on various physiological activities in the body. It has been reported that daurisoline can regulate intracellular calcium content and calcium signal in various ways.

Calmodulin (CaM) has biological activity under the premise of combining with Ca2+, and participates in various signal transduction processes in cells. Benzylisoquinoline alkaloids depend on the combination of Ca2+and calmodulin to change their conformation, indirectly inhibit the activity of phosphodiesterase (PDE), and then inhibit the activity of Calmodulin-dependent phosphodiesterase (CaM-PDE), so as to play a role in regulating calcium-dependent information transmission. Hu Zhuoyietal.[1]studied the pharmacological effects of benzylisoquinoline alkaloids on calmodulin through comparative experiments. They found that benzylisoquinoline alkaloids had little effect on phosphodiesterase activity, but could regulate its downstream enzyme activity by antagonizing calmodulin. The bisbenzylisoquinoline compound daurisoline has a strong inhibitory effect on calmodulin, and the inhibitory effect of bisbenzylisoquinolines on calmodulin is stronger than that of monobenzylisoquinolines as calmodulin inhibitors.

The absolute configuration of natural daurisoline is R,R-configuration. Through pharmacological experiments, Kong Rongzuetal.[2]studied the mechanism of daurisoline regulating the contraction of rabbit aorta induced by calcium chloride, potassium chloride and norepinephrine. The results showed that both the synthetic daurisoline and the three optical isomers inhibited the influx of Ca2+in synaptosomes caused by the opening of voltage-dependent calcium channels by high K+depolarization. The experiment further compared the calcium antagonistic activity of the four optical isomers. The results showed that the calcium antagonistic activity of the two optical isomers of the S, R-configuration and the R, S-configuration was stronger than that of the natural daurisoline.

When a large amount of glutamate accumulates outside the nerve cells, it will be toxic to the nerve cells. Under the condition ofinvitroischemia, the glutamate output of the hippocampal slices increases rapidly with the influx of Ca2+[3]. Lu Youmingetal.[4]stimulated rat hippocampus with quinolinic acid, and detected the level of extracellular Ca2+in rat nerve cells to observe the situation of rat epileptic seizures. The results showed that after the injection of quinolinic acid, the concentration of extracellular Ca2+decreased, and a large amount of Ca2+entered the cell, and the EEG of the rats was abnormal. The influx of extracellular Ca2+was significantly reduced after treatment with L-daurisoline, but the abnormal EEG activity of rats was not inhibited, and the concentration of glutamate only changed slightly. L-daurisoline can block NMDA-operated Ca2+channels, but has no inhibitory effect on NMDA receptors.

R,R-(-)-daurisoline was once thought to be an N-type Ca2+blocker. Waldmeieretal.[5]explored the specificity of R,R-(-)-daurisoline on two Ca2+channels through comparing the effects of R,R-(-)-daurisoline with the P-type Ca2+channel blocker OMEGA-agatoxin IVA and the N-type Ca2+channel blocker OMEGA-conotoxin GVIA. The experimental results indicated that both R,R-(-)-daurisolin and OMEGA-agatoxin IVA participate in the regulation of P-type Ca2+channels, suggesting that R,R-(-)-daurisolin has a blocking effect on P-type Ca2+channels, and the inhibitory effect of daurisolin on the release of 3H-gamma aminobutyric acid is stronger than OMEGA-agatoxin, also indicating that daurisolin also interacts with one or more additional voltage-sensitive Ca2+channels apart from acting on P-type Ca2+channels. Ding Jieetal.[6-7]studied the effects of daurisolin on the expression of L-type Ca2+channel Cav1.2 and T-type Ca2+channel Cav3.1 on human embryonic kidney cells. The results showed that daurisolin in a certain concentration range had a certain inhibitory effect on the expression of these two Ca2+channels, and the effect on T-type Ca2+was free from the potential difference. The above results reflect that daurisolin has different degrees of inhibitory effect on various intracellular Ca2+channels.

2.1.1Anti-arrhythmic imbalance. Clinically, arrhythmic imbalance is a common disease and factors that cause arrhythmic imbalance are complex and diverse. It has been reported that daurisolin, as a calcium antagonist, has a good anti-arrhythmic effect. The triggering activity caused by delayed post-depolarization is an important mechanism of arrhythmia, and the generation of triggering activity is related to the increase of Ca2+in cardiomyocytes[8]. Wang Zhenxinetal.[9-10]studied the effect of daurisolin on delayed depolarization and triggering activity of ventricular papillary muscle of Cavia porcellus. They found that within a certain concentration range, daurisolin can inhibit the triggering activities induced by isoproterenol, phenylephrine hydrochloride and K+and high Ca2+environment, eliminate triggering activity caused by ouabaine or caffeine and reduce the probability of delayed depolarization.

As a Ca2+channel inhibitor, daurisolin can effectively prevent the transmembrane transport trend of Ca2+, has a significant inhibitory effect on delayed post-depolarization and the triggering activity caused by it, and reduces the probability of arrhythmic imbalance. Du Zuohuaetal.[11]studied the antiarrhythmic activity of daurisolin through multiple animal models. They found that daurisolin can effectively inhibit the premature ventricular contractions, ventricular tachycardia, ventricular fibrillation and cardiac arrest caused by aconitine and ouabain inC.porcellus. Besides, daurisolin also hadobvious inhibitory effects on calcium chloride-acetylcholine-induced atrial fibrillation and atrial flutter in mice, electrical stimulation-induced ventricular fibrillation threshold in rabbits, and coronary reperfusion-induced arrhythmias in rats.

Liu Qiangni[12-13]studied the effects of daurisolin on ion channels related to acquired long QT syndrome. The results of patch-clamp experiments showed that daurisolin had a weak inhibitory effect on IK1 and Ito, and also had a certain inhibitory effect on IKr and IKs tail currents. They used patch-clamp technique to record HERG channel currents expressed in human embryonic kidney cells (HEK293), and studied the effect of daurisolin on the electrophysiological properties of HERG channels. The results showed that daurisolin inhibited HERG channels in a voltage-dependent manner and reduced the half-inactivation voltage of HERG channels. The results of Western blot showed that the expression of HERG protein in HEK293-HERG cells was significantly decreased after incubation with 30 μmol/L daurisolin for 24 h. When the concentration of daurisolin was lower than 30 μmol/L, the expression and function of HERG protein were not affected, but daurisolin could block HERG channels to a certain extent.

2.1.2Anti-cerebral hypoxia-ischemia effect. In the early stage of nerve cell damage caused by cerebral hypoxia-ischemia, a large amount of calcium influx and release of calcium in the cell lead to a sharp increase in the concentration of intracellular Ca2+. There are a variety of calcium-dependent enzymes in cells, and excessive intracellular Ca2+concentration will activate the activity of these enzymes, promote the hydrolysis of membrane phospholipids and damage cells[14]. Through experiments on rat adrenal pheochromocytoma cell line (PC12) cells, He Lingetal.[15]found that daurisolin not only inhibited the influx of extracellular Ca2+caused by the opening of voltage-dependent Ca2+channels, but also inhibited the release of internal calcium caused by caffeine and sarcoplasmic reticulum calcium pump inhibitors, thereby avoiding cell damage due to increased Ca2+concentration, which may be an important mechanism for its anti-cerebral hypoxia and anti-ischemic abilities. Through cell ischemia model experiments, Liu Jinggenetal.[16]found that daurisolin and its three optical isomers can inhibit the increase of intracellular Ca2+concentration in PC12 cells, thereby alleviating cell ischemic damage. Through experiments with six injury models, Wang Tingetal.[17-18]found that daurisolin can maintain cell homeostasis and reduce the damage caused by various pathways to PC12 cells, and the mechanism is related to inhibiting the increase of intracellular Ca2+concentration. The experimental results of the rat cerebral ischemia injury model and the double-arterial and four-arterial ligation models show that O,O-acetyldaurisoline, a derivative of daurisolin, can inhibit the increase of Ca2+concentration in cells and reduce the lack of energy, reduce the content of lipid peroxides and increase the activity of superoxide dismutase in ischemic brain tissue, and has the effect of anti-oxidative free radicals and protection of nerves.

Shao Jiaetal.[19]studied the effects of daurisoline on the metabolic activity of vascular endothelial cells damaged by hypoxia by MTT method, they found that daurisoline has a good anti-hypoxia effect. Through the rat ischemia-reperfusion model, Wan Guangruietal.[20]found that in the pure ischemia group, the pyramidal neurons in the left parietal cortex were severely damaged after the blood supply was lost, and cells showed symptoms of edema, the cytoplasmic concentration decreased, vacuoles of different sizes appeared inside the cells, and the nuclear membrane and other cell structures were severely damaged. However, the nerve cells of the rats in the daurisoline treatment group were only slightly damaged, and the morphology of the nucleus was only slightly irregular, and there were only a few small vacuoles in the cytoplasm, which proved that daurisoline can reduce the damage of nerve cells caused by focal cerebral ischemia-reperfusion from submicroscopic structure. Through the control experiment, Guo Hualietal.[21]studied the inhibitory effect of daurisoline on bradykinin-induced increase of Ca2+concentration in rat cortical neurons and damaged cells. MTT results showed that the survival rate of neurons treated with daurisoline was higher than that of the control group treated with bradykinin alone. The results of dual-wavelength calcium imaging showed that daurisoline had a certain inhibitory effect on the increase of intracellular Ca2+concentration, and protected nerve cells damaged due to the imbalance of intracellular Ca2+concentration. Through TTC staining, Hu Defengetal.[22]studied the protective effect of daurisoline on rat nerve cells from oxygen-glucose deprivation injury. The results showed that daurisoline inhibited the release of lactate dehydrogenase in nerve cells in a dose-dependent manner, and the injury rate of cells was significantly reduced. The above results show that daurisoline and its derivatives have anti-hypoxic-ischemia and protective effects on brain nerve tissue, and its mechanism is related to inhibiting the increase of intracellular Ca2+concentration.

2.2 Anti-tumor effectAfter normal cells become cancerous, they can proliferate to form tumors, and it is difficult to completely remove cancer cells by ordinary medical methods. Therefore, it is urgent to develop efficient and safe anticancer drugs. Studies have found that daurisoline not only has anti-cancer effect, but also has the effect of improving the efficacy of other anti-cancer drugs and reversing the drug resistance of cancer cells.

2.2.1Inhibition of autophagy. In cells that normally undergo autophagy, autophagosomes bind to lysosomes, lysosomes digest damaged organelles, and the hydrolyzed products are utilized or excreted by cells. This process is called autophagy. Autophagy plays an important role in the defense of cells against adverse environmental influences. According to many studies, the inhibition of autophagy can greatly improve the chemotherapy efficiency of hepatocellular carcinoma (HCC). Therefore, many researchers have started to try to find effective autophagy inhibitors as new anticancer drugs[23]. Xue Legangetal.[24]studied the inhibitory effect of daurisoline on autophagy. The results of co-localization experiments showed that autophagosomes and lysosomes in HCC cells could combine to form autophagolysosomes after daurisoline treatment. However, the levels of autophagy-related proteins LC3-II and p62 in cells continued to rise, the autophagy flux was disrupted, and autophagosomes such as damaged mitochondria were not degraded. The results of acridine orange staining showed that daurisoline could inhibit lysosomal acidification, hinder the maturation of hydrolases such as cathepsin B and cathepsin D, prevent the extracellular matrix from being decomposed, and reduce the invasion and metastasis ability of cancer cells. Wu Mingyueetal.[25]explored the effect of daurisoline on autophagy in Hela cells, and found that daurisoline could increase lysosomal pH and inhibit the activity of lysosomal V-type ATPase, resulting in a large number of autophagic vacuoles in cells, lipidation of LC3, and inhibition of protective autophagy in cancer cells.

Cisplatin is a commonly used anticancer drug in clinical practice, but cisplatin can induce lung cancer cells to produce autophagy activity, trigger protective autophagy of lung cancer cells, and gradually promote lung cancer cells resistant to cisplatin. Therefore, it is a new idea for cancer treatment to find drugs that can inhibit autophagy combined with cisplatin for adjuvant therapy. Daurisoline, which has autophagy inhibitory effect, has broad medical research prospects. Wang Kunetal.[26]studied the mechanism by which daurisoline enhanced the anticancer effect of cisplatin, and found that the combined use of daurisoline and cisplatin had a stronger therapeutic effect on lung cancer than cisplatin alone. They used Western blot experiments and immunofluorescence staining to detect the levels of p62 and LC3 and the number of autophagosomes in the cells, and found that daurisoline could up-regulate the levels of p62 and LC3-II, and significantly increase the number of autophagosomes in lung cancer cells. However, the results of fluorescence colocalization experiments showed that daurisoline did not block the mutual binding of autophagosomes and lysosomes in cells. The results of Lyso Traker experiment and acridine orange staining showed that daurisoline blocked the autophagy process of cancer cells by inhibiting the formation of hydrolase in lysosomes. Transcriptomic sequencing showed that daurisoline could increase the intracellular Ca2+concentration in lung cancer cells by activating ITPR2 and TPC2, thereby inhibiting the changes of lysosomal acidity. On the contrary, down-regulation of ITPR2 and TPC2 activity reversed this effect, suggesting that daurisoline interferes with autophagy by hindering the maturation pathway of acid hydrolase. The experiment also explored the effect and specific mechanism of daurisoline combined with cisplatin in the treatment of cancer. Electron microscopy and immunofluorescence experiments showed that daurisoline combined with cisplatin can increase the number of mitochondria in cancer cells, and mitochondrial autophagosomes accumulate in cells, and then increase the level of ROS in cells and kills lung cancer cells through the ROS-JNK signaling pathway. If the intracellular ROS level is reduced, the synergistic effect of daurisoline on the killing of lung cancer cells by cisplatin can be eliminated. The detection results of Annexin V/PI double staining experiment and CCK-8 experiment showed that the therapeutic effect of the combination of the two was significantly stronger than that of cisplatin alone, and inhibition of JNK signaling pathway could reduce the synergistic effect of daurisoline on cisplatin-killing lung cancer cells, which suggests that daurisoline can enhance cisplatin-mediated apoptosis of lung cancer cells.

Tian Huietal.[27]studied the effects of daurisoline on MCF-7/Adr-acquired drug-resistant human breast cancer cells, KBV200-acquired drug-resistant oral cancer cells, BEL-7402 naturally drug-resistant liver cancer cells and other drug-resistant cell lines. The results showed that daurisoline had a dose-dependent reversal of doxorubicin resistance in P-pg positive MCF-7/Adr cells; reversed vincristine resistance of P-pg positive KBV200 cells; reversing the effect of vincristine resistance of BEL-7402 cells significantly enhanced the drug sensitivity of cancer cells, and improved the inhibitory effect of drugs on cancer cells. In summary, daurisoline can improve the effect of chemotherapy by interfering with autophagy, and enhance the killing power of various drugs such as cisplatin, vinblastine and adriamycin on cancer cells.

2.2.2Cell cycle arrest. The process of a cell from the end of one division to the completion of the next division is called the cell cycle, which is of great significance to the growth and development of organisms. The disruption of the cell cycle regulation mechanism will lead to uncontrolled cell proliferation. Huang Xiaohuietal.[28]found that daurisoline has cell cycle arresting effect, the lung cancer cell cycle after daurisoline treatment will be arrested in G1 phase, cell proliferation is blocked, migration and invasion of cancer cells are inhibited, and there are no obvious side effects. In addition, daurisoline has the effect of inhibiting the expression of cell division-related transcription factor β-catenin and its downstream targetsc-mycgene and cyclin D1, the cancer cells cannot complete the normal cell cycle, causing their division to be blocked and unable to proliferate rapidly. The results of target stability and isothermal titration quantitative calorimetry analysis of drug affinity reaction showed that daurisoline can directly target the heat shock protein HSP90, making it unable to interact with β-catenin normally and increasing ubiquitin-mediated β-catenin degradation of proteins, thereby interfering with the cell cycle process of cancer cells, to achieve the purpose of anti-cancer[29].

2.3 Other pharmacological effectsLiu Yuetal.[30]studied the effects of daurisoline and dauricine on the aggregation and adhesion of human platelets through comparative experiments. The results showed that daurisoline and dauricine dose-dependently inhibited platelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (ARA) and collagen. In addition, daurisoline can also increase the content of cyclic adenosine monophosphate in platelets, which may be the mechanism of daurisoline’s anti-platelet adhesion, but whether daurisoline can be used as an antithrombotic drug remains to be further studied. Throughinvitroandinvivoexperiments, Wangetal.[31]found that daurisoline also has a certain effect on alleviating oxygen toxicity in the central nervous system, and its mechanism may also be related to the regulating ability of Ca2+. Liu Xinxiaetal.[32]studied the effect of daurisoline on cytochrome P450 enzymes in rat liver and found that daurisoline could increase the expression and catalytic activity ofCYP1A1in rat liver, and inhibit the transcription and activity ofCYP2B1,CYP2D1andCYP3A1at the transcriptional level. Furthermore, studies have also reported that daurisoline has anti-colitis[33], improves the brain hippocampal structure of Alzheimer’s mice, regulates learning and memory ability[34], and inhibits the proliferation of COVID-19 in cells[35].

3 Conclusions and prospects

Daurisoline is a bisbenzylisoquinoline compound that has wide sources. In December 2019, the widespread spread of COVID-19 triggered a global health crisis. It has been reported that daurisoline, an autophagy inhibitor, can also effectively inhibit virus replication in host cells. It has broad research value and drug development potential and is helpful for the research of targeted drugs. However, this is only the result of research at the cellular level, and its actual effect needs to be further tested in animal experiments.