Realgar Treatment Research Articles

Xanthine oxidase, a therapeutic target of realgar for non-small cell lung cancer

BackgroundThe effects of realgar against non-small cell lung cancer (NSCLC) have been massively studied, but the direct therapeutic targets of realgar remain unclear. This study aimed to identify the molecular targets of realgar against NSCLC and explore their therapeutic mechanisms based on a network pharmacology approach and experimental validations. MethodsThe BATMAN-TCM and Digsee databases were used to predict realgar targets and NSCLC-related genes, respectively. A protein-protein interaction network was constructed for each gene set, and the overlapping genes were identified as potential targets of realgar against NSCLC. The correlation between potential targets and NSCLC was analyzed using The Cancer Genome Atlas and International Cancer Genome Consortium databases, and the key target was validated by in-silico and in-vitro experiments. ResultsTwenty-three overlapping genes, including xanthine oxidase (XO), were identified as potential targets of realgar against NSCLC. XO was selected as the key target for validation, as it was found to be upregulated in NSCLC tumor tissue, which correlated with poor overall survival. A possible interaction between realgar and XO was revealed by molecular docking which was further validated experimentally. Realgar treatment suppressed the activity of XO in NSCLC cells, as demonstrated by the unchanged XO protein levels. Finally, the mechanism of action of XO as a target against NSCLC through the cell-cell junction organization pathway was investigated. ConclusionsOverall, this study proposes a potential molecular mechanism illustrating that XO is a target of realgar against NSCLC and highlights the usefulness of XO as a therapeutic target for NSCLC.

Realgar Alleviated Neuroinflammation Induced by High Protein and High Calorie Diet in Rats via the Microbiota-Gut-Brain Axis.

Purpose: Gastrointestinal heat retention syndrome (GHRS) often occurs in adolescents, resulting into nervous system injury. Realgar, an arsenic mineral with neuroprotective effect, has been widely used to treat GHRS. However, its mechanism of action remains unknown. Methods: A GHRS rat model was established using a high protein and high calorie diet. We performed macroscopic characterization by assessing bowel sounds, hot/cold preference, anal temperature, and fecal features. Atomic fluorescence spectroscopy was employed to evaluate brain arsenic level while hippocampal ultrastructural changes were analyzed using transmission electron microscopy. In addition, inflammatory cytokines and BBB breakdown were analyzed by western blotting, immunofluorescence assays, and immunohistochemistry staining. We also evaluated hippocampal metabolites by LC-MS while fecal microorganisms were assessed by 16S rDNA sequencing. Results: Our data showed that the high protein and high calorie diet induced GHRS. The rat model depicted decreased bowel sounds, increased fecal characteristics score, preference for low temperature zone, and increased anal temperature. In addition, there was increase in inflammatory factors IL-6, Iba-1, and NF-κB p65 as well as reduced BBB structural protein Claudin-5 and Occludin. The data also showed appearance of hippocampus metabolites disorder and fecal microbial imbalance. Realgar treatment conferred a neuroprotective effect by inhibiting GHRS-specific characteristics, neuroinflammatory response, BBB impairment, metabolites disorder, and microbial imbalance in the GHRS rat model. Conclusion: Taken together, our analysis demonstrated that realgar confers a neuroprotective effect in GHRS rats through modulation of the microbiota-gut-brain axis.

Realgar (As4S4), a traditional Chinese medicine, induces acute promyelocytic leukemia cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway in vitro.

Acute promyelocytic leukemia (APL) is a specific subtype of acute myelogenous leukemia (AML) characterized by the proliferation of abnormal promyelocytes. Realgar, a Chinese medicine containing arsenic, can be taken orally. Traditional Chinese medicine physicians have employed realgar to treat APL for over a thousand years. Therefore, realgar may be a promising candidate for the treatment of APL. Nevertheless, the underlying mechanism behind realgar therapy is largely unclear. The present study aimed to investigate the effect of realgar on cell death in the APL cell line (NB4) in vitro and to elucidate the underlying mechanism. In this study, after APL cells were treated with different concentrations of realgar, the cell survival rate, apoptotic assay, morphological changes, ATP levels and cell cycle arrest were assessed. The expression of Bcl-2, Bax, Cytochrome C (Cyt-C) and apoptosis-inducing factor (AIF) at the mRNA and protein levels were also measured by immunofluorescence, quantitative PCR (qPCR) and Western blotting. We found that realgar could significantly inhibit APL cell proliferation and cell death in a time- and dose-dependent manner. Realgar effectively decreased the ATP levels in APL cells. Realgar also induced APL cell cycle arrest at the S and G2/M phases. Following realgar treatment, the mRNA and protein levels of Bcl-2 were significantly downregulated, whereas the levels of Bax, Cyt-C, and AIF were significantly upregulated. In summary, realgar can induce APL cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway, suggesting that realgar may be an effective therapeutic for APL.

Sub-chronic exposure to realgar induces liver injury via upregulating the TXNIP/NLRP3 pathway and disturbing bile acid homeostasis in mice

Ethnopharmacological relevanceRealgar is a traditional Chinese medicine used in China for a long history. Long-time or excessive use of realgar causes liver injury. However, its underlying mechanism is not fully clarified. Aim of the studyIn this study, we investigated the toxic effect of sub-chronic exposure to realgar on mice liver, and further revealed its underlying mechanism focused on the TXNIP/NLRP3 pathway and bile acid homeostasis. Material and methodsMice were divided into control and different doses of sub-chronic realgar exposed groups. Total arsenic levels in the blood and liver were determined by atomic fluorescence spectrometry. The effect of realgar on liver function was evaluated by biochemical analysis and histopathological examination. Assay kits were applied for the measurement of oxidative stress indexes, MPO and plasma inflammatory cytokines. The mRNA and proteins involved in the TXNIP/NLRP3 and NF-κB pathways were determined by RT-qPCR, western blot, Immunofluorescence and Immunohistochemistry. UHPLC/MS/MS was used for the quantitative analysis of bile acids (BAs) in mice plasma, liver and urine. The genes related to BAs metabolism were measured by RT-qPCR. ResultsSub-chronic exposure to realgar led to arsenic accumulation and caused oxidative damage and inflammatory infiltration in mouse liver, finally resulting in liver injury. Realgar treatment activated the NF-κB pathway and significantly upregulated the TXNIP/NLRP3 pathway in mouse liver. Realgar altered the metabolic balance of BAs, which is related to the abnormal expression of BAs transporters and enzymes. ConclusionSub-chronic exposure to realgar caused liver injury in mouse, and the mechanism may involve the upregulation of the TXNIP/NLRP3 pathway and disordered BAs homeostasis.

Realgar increases defenses against infection by Enterococcus faecalis in Caenorhabditis elegans

Ethnopharmacological relevanceRealgar has been used in traditional remedies for a long history in China and India. It is clinically used to treat diverse cancers, especially acute promyelocytic leukemia (APL), chronic myelogenous leukemia (CML) in China. However, paradoxic roles of realgar to increase or decrease immunity are reported. It is urgent to address this question, due to immune depression can be strongly benefit to cancer development, but detrimental to patients. Aim of the studyThis present work is to explore whether realgar promote or suppress immune responses, and shed light on its mode of action. Our results should provide cues for rational strategy to explore realgar for clinical use. Material and methodsInfection model in vivo was established by using Enterococcus faecalis to attack Caenorhabditis elegans, then realgar was used to treat the infected worms to investigate its effects on infectivity and the underlying mechanism. Killing analysis was carried out to test whether realgar can mitigate worm infection. Thermotolerance resistance analysis was used to evaluate if realgar functions hormetic effect. Quantification of live E. faecalis in nematode intestine was employed to ascertain if realgar alleviate the bacterial load in worm gut. Quantitative real-time PCR (qRT-PCR) was used to test the expression of antibacterial effectors. Western blot was used to test the effect of realgar on the expressions of p38 and phospho-p38 in worms infected by E. faecalis. ResultsRealgar alleviated the infected worms in strains of N2, glp-4, and daf-2, but failed in sek-1, glp-4; sek-1, and daf-2; daf-16 when p38 MAPK or daf-16 was blocked or inactivated. Western blot assay demonstrated that realgar increased the expression of phosph-p38. Thermotolerance assay showed that realgar played a hormetic role on nemtodes, triggered protective response and reduced bacterial load after realgar treatment for 120 h qRT-PCR demonstrated that realgar significantly increased antibacterial effectors, thus leading to pathogen elimination. ConclusionRealgar increased defenses against E. faecalis in C. elegans by inducing both immune responses and protective responses. It was regulated by p38 MAPK pathway and DAF-16.

Study of the accumulation and distribution of arsenic species and association with arsenic toxicity in rats after 30 days of oral realgar administration.

Because the toxicity and efficacy of arsenic is closely related to its chemical species, we conducted examinations of arsenic species accumulation and distribution in the rat body after one-time and 30-day realgar administration and then elucidated the probable roles of different arsenic species in the short-term toxicity of realgar. According to ICH M3 guidelines for non-clinical repeated dose toxicity studies and OECD Test guideline TG407 "Repeated Dose 28-Day oral Toxicity Study in Rodents, the doses of realgar set were 10.6 mg/kg, 40.5 mg/kg and 170 mg/kg. Rats were orally administered with realgar for one-tme and 30 days, respectively. Thereafter, biological samples (plasma, urine, liver, kidney, and brain) were obtained from rats and analyzed using high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) to determine realgar metabolism, arsenic species accumulation and distribution. Additionally, the toxicity of realgar in rats was evaluated. The absorption, distribution and elimination half-life of total arsenic species in realgar were 3.33 hs, 16.08 hs and 24.65 hs, respectively. After 30 days of oral administration of realgar in rats, no significant drug-related toxicity occurred in the rats. Dimethylarsenic acid (DMA) is the most abundant arsenic species. The DMA contents of the liver and kidney of the high-dose realgar group were approximately 40-fold and 50-fold higher than those in the corresponding tissues of the control group, respectively. The arsenic species (III) was mainly detected in the liver and its content was about 40-fold higher than that of the control group. MMA was mainly detected in rat kidney, and the MMA content of the realgar treatment group was more than 2000 times higher than that of the control group. Arsenic is rapidly absorbed and distributed over the liver, kidneys and brain, and the distribution and elimination of arsenic in the blood is slow. The realgar doses corresponded to human equivalent doses (HED) of 1.7, 6.4 and 27.2 mg/kg, respectively. Considering that humans are 10 times more sensitive than animals, the realgar dose is equivalent to 0.17, 0.64 and 2.7 mg/kg HED. It can be considered that if patients take no more than 2.7 mg/kg realgar for 2 weeks, there will be no adverse reactions.

UPLC/Q-TOFMS-Based Metabolomics Approach to Reveal the Protective Role of Other Herbs in An-Gong-Niu-Huang Wan Against the Hepatorenal Toxicity of Cinnabar and Realgar.

An-Gong-Niu-Huang Wan (AGNH) is a well-known traditional Chinese medicine (TCM) recipe containing cinnabar (HgS) and realgar (As2S2). However, the application of AGNH is limited by the hepato- and nephrotoxicity of cinnabar and realgar. It should be noted that cinnabar and realgar in AGNH are not used alone, but rather combined with other herbs as formula to use. In this study, the protective effects and mechanisms of the other herbs in AGNH against the hepatorenal toxicity induced by cinnabar and realgar were investigated. The combination use of the other herbs in AGNH alleviated inflammatory cell infiltration and damage in the liver and kidney and restored the disturbed serum metabolic profile induced by cinnabar and realgar insults. By UPLC/Q-TOFMS combined with pattern recognition approaches, we identified 41 endogenous metabolites in the sera of mice that were related to the hepatorenal toxicity of cinnabar and realgar, 36 of which were restored to normal levels when various kinds of herbs were combined as compound recipe. These metabolites function as modulators in inflammation-associated glycerophospholipid, arachidonic acid, linoleic acid, sphingolipid, and ether lipid metabolic pathways. Notably, lysophosphatidylcholines (LysoPCs) were the most elevated among all of the metabolites detected after cinnabar and realgar treatment, while these LysoPCs did not show overt differences between the AGNH and saline control groups, which was associated with relatively unaffected or even up-regulated expression of lysophosphatidylcholine acyltransferase 1 (LPCAT1) and autotaxin (ATX). These findings indicated that other herbs in AGNH could have a protective effect against cinnabar- and realgar-induced hepatic and renal damage via modulating the disordered homeostasis of the glycerophospholipid, arachidonic acid, linoleic acid, ether lipid, and sphingolipid metabolism.

Realgar (As4S4) nanoparticles and arsenic trioxide (As2O3) induced autophagy and apoptosis in human melanoma cells in vitro.

The aim of the present study was to compare the effect of realgar nanoparticles and arsenic trioxide (ATO) on viability, DNA damage, proliferation, autophagy and apoptosis in the human melanoma cell lines BOWES and A375. The application of various flow cytometric methods for measurements cell viability, DNA cell cycle, mitochondrial potential, lysosomal activity, and intracellular content of glutathione was used. In addition, quantitative PCR, western blotting and multiplex bead array analyses were applied for evaluation of redox stress, autophagic flux, and cell signaling alterations.The results showed that realgar treatment of studied cells caused modulation of cell proliferation, induced a block in G2/M phase of the cell cycle and altered phosphorylation of IκB, Akt, ERK1/2, p38, and JNK kinases, as well as decreased mitochondrial membrane potential. Additionally, it appeared that induction of cell death by both realgar and ATO was dose-dependent, when lower (0.3 µM) dosage increased lysosomal activity and induced autophagy and higher (1.25 µM) concentration resulted in the appearance of apoptosis, while pan-caspase inhibitor attenuated more efficiently realgar- than ATO-induced cell death. Furthermore, low concentrations of ATO and realgar nanoparticles increased the content of intracellular glutathione and elevated γ-H2AX expression confirmed DNA damage preferentially at higher concentrations of both drugs used. Further analysis revealed slight differences in time-dependent phosphorylation pattern due to both realgar and ATO treatments, while significant differences were noticed between cell lines. In conclusion, realgar nanoparticles and ATO treatment induced dose-dependent activation of autophagy and apoptosis in both melanoma cell lines, when autophagy flux was determined at lower drug concentrations and the switch to apoptosis occurred at higher concentrations of both arsenic forms.

Realgar-induced apoptosis and differentiation in all-trans retinoic acid (ATRA)-sensitive NB4 and ATRA-resistant MR2 cells

Realgar has been used in Western medicine and Chinese traditional medicine since ancient times, and its promising anticancer activity has attracted much attention in recent years, especially for acute promyelocytic leukemia (APL). However, the therapeutic action of realgar treatment for APL remains to be fully elucidated. Cellular cytotoxicity, proliferation, apoptosis and differentiation were comprehensively investigated in realgar-treated cell lines derived from PML-RARα+ APL patient, including the all-trans retinoic acid (ATRA)-sensitive NB4 and ATRA-resistant MR2 cell lines. For analysis of key regulators of apoptosis and differentiation, gene expression profiles were performed in NB4 cells. Realgar was found to induce apoptosis and differentiation in both cell lines, and these effects were exerted simultaneously. Gene expression profiles indicated that genes influenced by realgar treatment were involved in the modulation of signal transduction, translation, transcription, metabolism and the immune response. Given its low toxicity, realgar is a promising alternative reagent for the therapy of APL. Our data contribute to an understanding of the underlying mechanism responsible for the therapeutic effects of realgar in the clinical treatment of APL.

Asenic accumulation following realgar administration in rats

To explore arsenic accumulation and toxicity mechanism following long-term use of realgar and provide scientific basis for safety use of realgar in clinic. The realgar which was used in the study contains 90% insoluble asenic sulfide (As2S2) and 1.696 mg x kg(-1) soluble arsenic. Two separate experiments were performed: 1) Twenty-eight fasting SD rats were orally given a single dose of realgar at the dose of 0.8 g x kg(-1) and the other four rats were given ultra-filtrated water served as control group. Blood, hearts, livers, kidneys, lungs and brains of four rats were taken out at 0.5, 1, 2, 4, 8, 16, 36 h respectively after treatment. Asenic quantity of each organ or blood sample was measured. 2) Forty SD rats were randomly divided into four groups: control group and realgar 0.02, 0.08, 0.16 g x kg(-1) groups, each group containing 5 females and 5 males. The rats were intra-gastrically treated with realgar once a day for successively 90 days, while the control group was given ultra-filtrated water. Asenic amount in blood, liver, kidney and brain of each rat was measured in fasting rats at 16 h after last dosing. Asenic amount of blood, liver, kidney, heart, lung and brain increased after single dosing of realgar at dose of 0.16 g x kg(-1), with the order from high to low blood > kidney > lung > liver > heart > brain. Asenic amount was much higher in blood than that in other organs. The feature of asenic distribution in blood following realgar administration may be the basis for its use for leukemia Ninety-day oral treatment of realgar led to significant accumulation of asenic in blood, kidney, liver and brain. The highest asenic accumulation times was found in kidney followed by liver, which was assumed to be associated with nephrotoxicity and hepatotoxicity of realgar. The highest amount of asenic was observed in blood after 90 day's administration of realgar, and the amount of asenic in organs was in the order of blood > kidney > liver > brain. Asenic can be absorbed and extensively distributed in various organs or tissesses after realgar administration in rats. Long-term use of realgar caused high asenic accumulation in various tissueses, including blood, kidney, liver, and brain. The nephrotoxicity and hepatotoxicity of realgar could be associated with the asenic accumulation in relative organs. Blood is the target of the most highest distribution and accamulation of asenic after realgar treatment, that could be associated with the efficacy of realgar on the treatment of leakemia.

Realgar-mediated growth inhibition on HaCaT human keratinocytes is associated with induction of apoptosis

Traditional Chinese medicine has long been used to treat a variety of ailments including skin diseases. Our previous study has revealed the ethanolic extract of realgar, a common ingredient used in psoriasis treatment in Chinese medicine, to possess potent anti-proliferative action on cultured HaCaT cells of human keratinocyte origin. In the present study, the mechanisms of action of the observed growth inhibitory action of realgar were investigated. Several bioassay methods were employed to elucidate whether cellular apoptosis is involved in the realgar-induced growth inhibition of the skin cells. Morphologically, nuclear condensation and DNA fragmentation were observed when HaCaT cells were exposed to the realgar extract. DNA fragmentation induced by the treatment of realgar was also evident as detected by gel electrophoresis and the TUNEL method. Cell cycle analysis by propidium iodide (PI) staining demonstrated the appearance of sub-G1 peak and cell cycle arrest at the G1 phase upon realgar treatment. Quantitative analysis by annexin V-PI staining revealed that the realgar-induced apoptotic event was dose-dependent. Furthermore, realgar was able to activate caspase-3 expression when examined by Western blot analysis. Our experimental data unambiguously confirm that induction of cellular apoptosis is mainly responsible for the observed growth inhibition brought about by realgar on the HaCaT keratinocytes, and this finding helps place the traditional use of this mineral for psoriasis treatment on a scientific footing.

Research on Realgar Inducing Apoptosis of Leukemia

Objective To study the influence of realgar on apoptosis and Bcl-2 expression of leukemia.Methods Realgar treatment group and adriamycin(ADR)treatment group were established,and the indexes involved with OD value of cell strain,morphologic alteration of apoptosis,and positive expressive rate of Bcl-2 were observed in the two groups.Results Compared with ADR treatment group,OD value and Bcl-s positive expressive rate were significantly decreased(P＜0.05)and apoptotic rate was significantly increased in realgar group(P＜0.05).Conclusion Realgar has excellent effect to induce apoptosis of leukemia. Key words: Realgar ; Apoptosis ;

High Expression of bcl-xL in K562 Cells and Its Role in the Low Sensitivity of K562 to Realgar-Induced Apoptosis

Arsenic compounds (As₂O₃orAs₄S₄) have been used successfully for the treatment of acute promyelocytic leukemia (APL) for quite a long time. It has been noticed that the sensitivity to apoptosis induced by As₂O₃varies among various leukemia cells. It was reported by several groups that As₂O₃ could induce apoptosis in APL-derived NB4 cells at concentrations of 0.5–1 µmol/l, whereas in other leukemia cells like K562, As₂O₃ has no effects at the same concentration. K562 cells undergo apoptosis only when the concentration of As₂O₃is greater than 2 µmol/l. Another arsenic compound, realgar (As₄S₄), a traditional Chinese mineral medicine, has been used to treat APL effectively and demonstrated to have lower toxicity than As₂O₃. It would be interesting to know whether NB4 and K562 cells will show different sensitivity to realgar as well and if there is a difference, what is the cellular mechanism of it. In our present study, K562 cells were much less sensitive than NB4 cells to apoptosis induced by realgar. We confirm that the expression of bcl-x_L is significantly higher in K562 cells than that in NB4 cells and is not downregulated upon realgar treatment. K562 cells become sensitive to realgar at clinically acceptable concentrations when bcl-x_L expression level is downregulated by transfecting bcl-x_L antisense RNA vector into the cells. Our results suggest that the increased bcl-x_L expression in K562 cells contributes to its insensitivity to realgar-induced apoptosis.

Modulation of gene expression networks underlying Realgar-Induced differentiation of acute promyelocytic leukemia cells

To elucidate the molecular mechanism of the differentiation of acute promyelocytic leukemia (APL) cell line NB4 induced by realgar. The response of NB4 cell to realgar was explored with a cDNA microarray representing 1003 different human genes. The analysis of gene expression profiles indicated that 8 genes were up-regulated and 33 genes were down-regulated 48 hrs after realgar treatment. Among the 8 up-regulated genes, 2 genes were involved in ubiquitin proteasome degradation pathway. Some genes related to RNA processing, protein synthesis and signal transduction were down-regulated. The ubiquitin-proteasome degradation pathway may play an important role in the degradatipn of PML/ RAR a fusion protein and the differentiation of NB4 cells.