Research on different compound combinations of Realgar-Indigo naturalis formula to reverse acute promyelocytic leukemia arsenic resistance by regulating autophagy through mTOR pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: In China, the Chinese patent drug Realgar-Indigo naturalis Formula (RIF) is utilized for the therapy of acute promyelocytic leukemia (APL). Comprising four traditional Chinese herb-Realgar, Indigo naturalis, Salvia miltiorrhiza, and Pseudostellaria heterophylla-it notably includes tetra-arsenic tetra-sulfide, indirubin, tanshinone IIa, and total saponins of Radix Pseudostellariae as its primary active components. Due to its arsenic content, RIF distinctly contributes to the therapy for APL. However, the challenge of arsenic resistance in APL patients complicates the clinical use of arsenic agents. Interestingly, RIF demonstrates a high remission rate in APL patients, suggesting that its efficacy is not significantly compromised by arsenic resistance. Yet, the current state of research on RIF's ability to reverse arsenic resistance remains unclear. AIM OF THE STUDY: To investigate the mechanism of different combinations of the compound of RIF in reversing arsenic resistance in APL. MATERIALS AND METHODS: The present study utilized the arsenic-resistant HL60-PMLA216V-RARα cell line to investigate the effects of various RIF compounds, namely tetra-arsenic tetra-sulfide (A), indirubin (I), tanshinone IIa (T), and total saponins of Radix Pseudostellariae (S). The assessment of cell viability, observation of cell morphology, and evaluation of cell apoptosis were performed. Furthermore, the mitochondrial membrane potential, changes in the levels of PMLA216V-RARα, apoptosis-related factors, and the PI3K/AKT/mTOR pathway were examined, along with autophagy in all experimental groups. Meanwhile, we observed the changes about autophagy after blocking the PI3K or mTOR pathway. RESULTS: Tanshinone IIa, indirubin and total saponins of Radix Pseudostellariae could enhance the effect of tetra-arsenic tetra-sulfide down-regulating PMLA216V-RARα, and the mechanism was suggested to be related to inhibiting mTOR pathway to activate autophagy. CONCLUSIONS: We illustrated that the synergistic effect of different compound combinations of RIF can regulate autophagy through the mTOR pathway, enhance cell apoptosis, and degrade arsenic-resistant PMLA216V-RARα.

Dissection of scientific compatibility of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia from the perspective of toxicology.

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar-Indigo naturalis formula (RIF), a first-line drug for the treatment of acute promyelocytic leukemia (APL),is also a TCM formula entirely designed based on TCM theories. There have been studies that explain the scientific connotation of the compatibility of RIF from the perspective of pharmacodynamics. However, as one of the arsenic-containing preparations, the safety of realgar is widely concerned, and there has not been systematic studies to explain the scientific connotation of RIF from the perspective of toxicology. AIM OF THIS STUDY: Dissection of scientific compatibility of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia from the perspective of toxicology. MATERIALS AND METHODS: We used normal mice and an APL model to explore (i) the effects of different components on intestinal permeability, (ii) the changes in intestinal flora, and (iii) toxic effects. At the same time, a bionic extraction method was used to study the effects of different components on the dissolution of soluble arsenic in realgar under the acidic environment in the stomach and the alkaline environment in the intestinal tract. RESULTS: Salvia miltiorrhiza Bunge can repair the intestinal mucosal barrier, maintain the homeostasis of intestinal flora, intervene in the dissolution process of realgar, reverse the increase in intestinal permeability and the disturbance of intestinal flora caused by realgar, and reduce toxicity. CONCLUSION: From the perspective of toxicology, we propose new insights into the definition of the roles of each component in the RIF formula, namely realgar is the monarch, Indigo naturalis is the minister, Salvia miltiorrhiza Bungeis the assistant.

Realgar-Induced Neurotoxicity: Crosstalk Between the Autophagic Flux and the p62-NRF2 Feedback Loop Mediates p62 Accumulation to Promote Apoptosis.

Realgar is a traditional Chinese medicine that contains arsenic. It has been reported that the abuse of medicine-containing realgar has potential central nervous system (CNS) toxicity, but the toxicity mechanism has not been elucidated. In this study, we established an in vivo realgar exposure model and selected the end product of realgar metabolism, DMA, to treat SH-SY5Y cells in vitro. Many assays, including behavioral, analytical chemistry, and molecular biology, were used to elucidate the roles of the autophagic flux and the p62-NRF2 feedback loop in realgar-induced neurotoxicity. The results showed that arsenic could accumulate in the brain, causing cognitive impairment and anxiety-like behavior. Realgar impairs the ultrastructure of neurons, promotes apoptosis, perturbs autophagic flux homeostasis, amplifies the p62-NRF2 feedback loop, and leads to p62 accumulation. Further analysis showed that realgar promotes the formation of the Beclin1-Vps34 complex by activating JNK/c-Jun to induce autophagy and recruit p62. Meanwhile, realgar inhibits the activities of CTSB and CTSD and changes the acidity of lysosomes, leading to the inhibition of p62 degradation and p62 accumulation. Moreover, the amplified p62-NRF2 feedback loop is involved in the accumulation of p62. Its accumulation promotes neuronal apoptosis by upregulating the expression levels of Bax and cleaved caspase-9, resulting in neurotoxicity. Taken together, these data suggest that realgar can perturb the crosstalk between the autophagic flux and the p62-NRF2 feedback loop to mediate p62 accumulation, promote apoptosis, and induce neurotoxicity. Realgar promotes p62 accumulation to produce neurotoxicity by perturbing the autophagic flux and p62-NRF2 feedback loop crosstalk.

Crosstalk between autophagy and the Keap1-Nrf2-ARE pathway regulates realgar-induced neurotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar, the main component of which is As2S2 or As4S4 (≥90%), is a traditional Chinese natural medicine that has been used to treat carbuncles, furuncles, snake and insect bites, abdominal pain caused by parasitic worms, and epilepsy in China for many years. Because realgar contains arsenic, chronic or excessive use of single-flavor realgar and realgar-containing Chinese patent medicine can lead to drug-induced arsenic poisoning, but the exact mechanism underlying its toxicity to the central nervous system is unclear. AIM OF THE STUDY: The aim of this study was to clarify the mechanism of realgar-induced neurotoxicity and to investigate the effects of realgar on autophagy and the Keap1-Nrf2-ARE pathway. MATERIALS AND METHODS: We used rats treated with the autophagy inhibitor 3-methyladenine (3-MA) or adeno-associated virus (AAV2/9-r-shRNA-Sqstm1, sh-p62) to investigate realgar-induced neurotoxicity and explore the specific relationship between autophagy and the Keap1-Nrf2-ARE pathway (the Nrf2 pathway) in the cerebral cortex. Molecular docking analysis was used to assess the interactions among the Nrf2, p62 and Keap1 proteins. RESULTS: Our results showed that arsenic from realgar accumulated in the brain and blood to cause neuronal and synaptic damage, decrease exploratory behavior and spontaneous movement, and impair memory ability in rats. The mechanism may have involved realgar-mediated autophagy impairment and continuous activation of the Nrf2 pathway via the LC3-p62-Keap1-Nrf2 axis. However, because this activation of the Nrf2 pathway was not sufficient to counteract oxidative damage, apoptosis was aggravated in the cerebral cortex. CONCLUSIONS: This study revealed that autophagy, the Nrf2 pathway, and apoptosis are involved in realgar-induced central nervous system toxicity and identified p62 as the hub of the LC3-p62-Keap1-Nrf2 axis in the regulation of autophagy, the Nrf2 pathway, and apoptosis.

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.

Target RNA expression omics approach to reveal the liver detoxification effect induced by Chinese medicine prescription Niu Huang Jie Du against realgar overexposure to mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Niu Huang Jie Du prescription (NHJD) is a traditional Chinese medicine (TCM) widely used in patients suffering from excessive inner fire toxin (Huo Du Nei Sheng) syndrome, such as sore throat, gingival swelling, and pain, mouth and tongue sores, etc. This formula contains realgar (As4S4) which is one of the 28 toxic medicinal materials promulgated by the Chinese Ministry of Health. Many studies reported its toxicity on the liver and kidney, and the detoxification effect of NHJD. However, its detoxification mechanism is still unclear. AIM OF THE STUDY: To clarify the detoxification mechanism of NHJD to realgar, this study evaluated the detoxification effect of NHJD on realgar exposure in mice, and analyzed differences in mRNA expression profiles in liver tissues and associated functional predictions. MATERIAL AND METHODS: ICR mice were administered with NHJD, realgar, and CMC-Na as blank control for 12 weeks, respectively. Liver injury was evaluated by histopathologic examination and liver mRNA gene were sequenced by Illumina. Differentially expressed gene, functionally enrichment and protein association network analysis were conducted. RESULTS: 43 genes were screened out, among which 15 genes in the realgar group were decreased, but the extent of the decline has been restored in the NHJD group. The remaining 28 genes have exactly the opposite trends. Functional module analysis revealed that those detoxification function-related genes were primarily for positive regulation of glutathione metabolism, P450 on the metabolism of exogenous compounds, oxidative stress and immune-related, etc. CONCLUSIONS: The results indicated that realgar mainly causes liver damage by changing the common enzymes of drug metabolism, especially the expression of genes related to CYPs, GSTs family, oxidative stress, and complement immunity, while the TCM prescription NHJD has a regulatory effect on the abnormal expression of corresponding genes. Our results will provide some clues for the detoxification mechanism of arsenic-containing TCM prescriptions.

Acid Water-ground Nano-realgar Is Superior to Crude Realgar in Promoting Apoptosis of MCF-7 Breast Cancer Cells.

OBJECTIVE: Realgar is a traditional mineral Chinese medicine with antitumor effects, but it has high toxicity and low efficacy in its crude form. The purpose of this study was to optimize realgar to increase its efficacy and therapeutic potential. METHODS: Crude realgar (CR) was mechanically ground to obtain nano-realgar (NR), and then nano-realgar processed products (NRPPs) were obtained using three different traditional Chinese medicine processing methods: grinding in water, acid water, and alkali water, respectively. RESULTS: By analyzing the size distribution of nanoparticles and the content of arsenic trioxide (As2O3; ATO), we found that acid water-ground NRPPs had the characteristics of high purity and low toxicity. The effects of CR, NR, and NRPPs on proliferation, cell cycle, and apoptosis of MCF-7 cells were detected, and the ability of NRPPs to induce apoptosis in MCF-7 cells was analyzed. The results showed that the average particle size of acid water-ground NRPPs was 137.7 nm, and the content of ATO was 2.83 mg/g. Acid water-ground NRPPs showed better effects on inhibiting proliferation, cell cycle, and apoptosis of MCF-7 cells than CR and NR. Western blot assays further confirmed that acid water-ground NRPPs upregulated the protein expression of TP53, Bax, cytochrome c, caspase-9, and caspase-3 in MCF-7 cells (P<0.05) and inhibited the expression of Bcl-2 (P<0.05). CONCLUSION: These results suggest that acid water-ground NRPPs can induce apoptosis of MCF-7 cells through regulating mitochondrial-mediated apoptosis, providing evidence for the clinical application of realgar.

An Experimental Study Reveals the Protective Effect of Autophagy against Realgar-Induced Liver Injury via Suppressing ROS-Mediated NLRP3 Inflammasome Pathway.

Realgar, a poisonous traditional Chinese medicine, has been shown to cause liver injury when used for long periods or overdoses. However, the underlying molecular mechanisms and therapeutic targets have not been fully elucidated. The aim of this study is to explore the role of autophagy in sub-chronic realgar exposure-induced liver injury. Here, the liver injury model was established by continuously administrating mice with 1.35 g/kg realgar for 8 weeks. 3-methyladenine (3-MA) and rapamycin (RAPA) were used to regulate autophagy. The results showed that realgar induced abnormal changes in liver function, pathological morphology, expression of inflammatory cytokines, and upregulated NLRP3 inflammasome pathway in mouse livers. RAPA treatment (an inducer of autophagy) significantly improved realgar-induced liver injury and NLRP3 inflammasome activation, while 3-MA (an inhibitor of autophagy) aggravated the realgar-induced liver injury and NLRP3 inflammasome activation. Furthermore, we found that realgar-induced NLRP3 inflammasome activation in mouse livers is mediated by ROS. RAPA eliminates excessive ROS, inhibits NF-κB nuclear translocation and down-regulates the TXNIP/NLRP3 axis, consequently suppressing ROS-mediated NLRP3 inflammasome activation, which may be the underlying mechanism of the protective effect of autophagy on realgar-induced liver injury. In conclusion, the results of this study suggest that autophagy alleviates realgar-induced liver injury by inhibiting ROS-mediated NLRP3 inflammasome activation. Autophagy may represent a therapeutic target in modulating realgar-induced liver injury.

Realgar facilitates the Nrf2-Keap1-p62 positive feedback signaling axis via MAPKs and AKT to interfere with autophagy-induced apoptosis and oxidative stress in the hippocampus.

Realgar, as a commonly used traditional Chinese medicine, exerts both pharmacological and biological effects. However, the mechanism by which it causes nervous system injury remains unclear. This study aimed to elucidate the specific mechanism underlying the hippocampal neurotoxicity caused by realgar. Nrf2 is an important receptor of exogenous toxic substances and oxidative stress. We utilized a p38-specific inhibitor (SB20358), ERK1/2-specific inhibitor (PD98059), JNK-specific inhibitor (SP600125) and AKT-specific inhibitor (LY249002) to establish the corresponding animal models and explore how realgar activates Nrf2. We established an Nrf2-shRNA gene silencing model in rats and an autophagy-specific inhibitor treatment model to further explore realgar-induced neurotoxicity and the role of Nrf2 in realgar-induced damage to the hippocampus. The results showed that realgar passed through the blood-brain barrier and accumulated in brain tissue to induce central nervous system toxicity. The specific mechanism was that realgar activated MAPKs and AKT signaling molecules to activate the Nrf2-Keap1-p62 positive feedback signaling axis, induced abnormal autophagy initiation and degradation, and promoted oxidative damage and apoptosis in neurons. Effective measures should be taken to prevent and control the arsenic poisoning caused by realgar in the early stage, and this study provides a theoretical and practical basis for the rational use of drugs in the clinic.

Oxidative stress induced by realgar in neurons: p38 MAPK and ERK1/2 perturb autophagy and induce the p62-Keap1-Nrf2 feedback loop to activate the Nrf2 signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Due to the modernization of traditional Chinese medicine (TCM) and the influence of traditional medication habits (TCM has no toxicity or side effects), arsenic poisoning incidents caused by the abuse of realgar and realgar-containing Chinese patent medicines have occurred occasionally. However, the potential mechanism of central nervous system toxicity of realgar remains unclear. AIM OF THE STUDY: This study aimed to clarify the specific mechanism of realgar-induced neurotoxicity. MATERIALS AND METHODS: In this study, the roles of ERK1/2 and p38 MAPK in realgar-induced neuronal autophagy and overactivation of the nuclear factor erythroid-derived factor 2-related factor (Nrf2) signalling pathways was investigated in vivo and in vitro. RESULTS: The arsenic in realgar passed through the blood-brain barrier and accumulated in the brain, resulting in damage to neurons, synapses and myelin sheaths in the cerebral cortex and a decrease in the total antioxidant capacity. The specific mechanism is that the excessive activation of Nrf2 is regulated by the upstream signalling molecules ERK1/2 and p38MAPK. At the same time, p38 MAPK and ERK1/2 interfere with autophagy, thereby promoting autophagy initiation but causing subsequent dysfunctional autophagic degradation and inducing the p62-Keap1-Nrf2 feedback loop to promote Nrf2 signalling pathway activation and nerve cell apoptosis. CONCLUSIONS: This study confirmed the role of the signalling molecules p38 MAPK and ERK1/2 in perturbing autophagy and inducing the p62-Keap1-Nrf2 feedback loop to activate the Nrf2 signalling pathway in realgar-induced neurotoxicity.

Realgar (α-As4S4) Treats Myelodysplastic Syndromes through Reducing DNA Hypermethylation.

DNA hypermethylation is an epigenetic modification that plays a critical role in the oncogenesis of myelodysplastic syndromes (MDS). Aberrant DNA methylation represses the transcription of promotors of tumor suppressor genes, inducing gene silencing. Realgar (α-As4S4) is a traditional medicine used for the treatment of various diseases in the ancient time. Realgar was reported to have efficacy for acute promyelocytic leukemia (APL). It has been demonstrated that realgar could efficiently reduce DNA hypermethylation of MDS. This review discusses the mechanisms of realgar on inhibiting DNA hypermethylation of MDS, as well as the species and metabolisms of arsenic in vivo.

Realgar (α-As4S4) Treats Myelodysplastic Syndromes through Reducing DNA Hypermethylation / 中国结合医学杂志

DNA hypermethylation is an epigenetic modification that plays a critical role in the oncogenesis of myelodysplastic syndromes (MDS). Aberrant DNA methylation represses the transcription of promotors of tumor suppressor genes, inducing gene silencing. Realgar (α-As4S4) is a traditional medicine used for the treatment of various diseases in the ancient time. Realgar was reported to have efficacy for acute promyelocytic leukemia (APL). It has been demonstrated that realgar could efficiently reduce DNA hypermethylation of MDS. This review discusses the mechanisms of realgar on inhibiting DNA hypermethylation of MDS, as well as the species and metabolisms of arsenic in vivo.

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

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar 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 STUDY: In 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 METHODS: Mice 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. RESULTS: Sub-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. CONCLUSION: Sub-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.

Arsenic concentration in peripheral blood is correlated with efficacy of a Traditional Chinese Medicine regimen containing realgar for the treatment of myelodysplastic syndrome.

OBJECTIVE: To explore the relationship between the efficacy of realgar for the treatment of myelodysplastic syndromes with multilineage dysplasia (MDS-MLD) and arsenic concentration in the peripheral blood of patients. METHODS: In this prospective study, a total of 50 MDS-MLD patients were treated with traditional Chinese drugs containing realgar for 3 months in Xiyuan Hospital from March 2018 to January 2019. Routine blood examination as well as liver and kidney function were monitored before and after treatment. The concentration of arsenic in the peripheral blood was measured using an atomic fluorescence spectrometer after treatment. The correlation between clinical effect and arsenic concentration was analyzed by Spearman's method. RESULTS: The treatment response rate was 54%. Two patients (4% ) achieved complete remission, 50% (25 of 50) showed hematologic improvement, and 23 patients had stable disease (23% ). No disease progression was observed. Arsenic concentration in the peripheral blood ranged from 14.60 to 85.96 µg/L. Clinical efficacy was positively correlated with arsenic concentration (P < 0.05). The incidence of mild adverse reactions was 16%. CONCLUSION: A relatively high concentration of arsenic in the peripheral blood may improve the clinical efficacy of realgar in MDS-MLD patients.

Proteomics analysis in the kidney of mice following oral feeding Realgar.

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar, a famous traditional Chinese mineral medicine, has been toxic to the renal system. However, the underlying mechanism of Realgar nephrotoxicity is still unclear. AIM OF THE STUDY: This study aimed to investigate the potential mechanism of Realgar-induced nephrotoxicity by using a label-free quantitative proteomic method. MATERIALS AND METHODS: 36 mice were randomly divided into four groups: Control group, 0.5-, 1.0, and 2.0 g/kg Realgar group. After one week, serum biochemical parameters and renal histopathological examination were performed. Label-free quantitative proteomics was used to identify differentially expressed proteins which were subsequently analyzed with bioinformatics methods. Western blot was utilized to verify the six representative protein expressions. RESULTS: The results showed that 2.0 g/kg Realgar significantly increased blood urea nitrogen and induced the formation of tube cast of renal tubules, while the lower-dose of 0.5 g/kg and 1.0 g/kg Realgar showed no changes. Label-free proteomic analysis identified 3138 proteins, and 272 of those proteins were screened for significant changes in a dose-dependent manner. Functional enrichment analysis suggested that these proteins could affect the apoptotic process and oxidative stress. Representative proteins in the 2.0 g/kg Realgar group, including Cat, Bad, Cycs, Nqo1, Podxl, and Hmox1, were verified by western blot. CONCLUSIONS: The results in this study suggest that apoptosis and oxidative stress might be related to the Realgar-induced nephrotoxicity in mice. Moreover, the strategy of proteomics could contribute to the understanding of the mechanisms of nephrotoxicity in mice exposed to Realgar.

Clinical Response to Traditional Chinese Herbs Containing Realgar (As2S2) is Related to DNA Methylation Patterns in Bone Marrow DNA from Patients with Myelodysplastic Syndrome with Multilineage Dysplasia.

PURPOSE: DNA methylation is known to play an important role in myelodysplastic syndrome (MDS). We previously showed that Chinese herbs (CHs) containing realgar (As2S2) were effective at treating MDS with multilineage dysplasia (MDS-MLD). We tested whether the response to CH treatment was related to changes in DNA methylation in MDS-MLD. PATIENTS AND METHODS: First, the Illumina methylation 850K array BeadChip assay was used to assess the pretreatment methylation status in bone marrow cells from eight MDS-MLD patients and 3 healthy donors. The eight MDS-MLD patients were then treated with CHs for six months, the arsenic concentration was measured following treatment. The patients were subsequently divided into "effective" and "ineffective" treatment response groups and the DNA methylation patterns of the two groups were compared. Finally, the BeadChip data were validated by pyrosequencing. RESULTS: Five of the eight MDS-MLD patients showed hematological improvement (effective-treatment group), while three showed disease progression (ineffective-treatment group) (positive response rate: 62.5%). The arsenic concentrations in the patients ranged from 26.60 to 64.16 µg/L (median 48.4 µg/L) and were not significantly different between the two groups (p = 0.27). Compared with the healthy controls, three genes were hypomethylated and 110 were hypermethylated in the ineffective-treatment group. However, in the group showing hematological improvement, 102 genes were markedly hypomethylated and 87 hypermethylated. The effective-treatment group had a higher proportion of hypomethylated sites than the ineffective-treatment group (53.9% vs 2.6%, respectively; chi-square test) (p < 0.0001). Two hypermethylated and two hypomethylated genes were selected for validation by pyrosequencing (all p < 0.05). CONCLUSION: MDS-MLD patients may present different DNA methylation subtypes. CHs containing realgar may be effective for treating MDS-MLD patients with the hypomethylation subtype.

Arsenic-Related Health Risk Assessment of Realgar-Containing NiuHuangJieDu Tablets in Healthy Volunteers Po Administration.

Realgar, an arsenic-containing traditional Chinese medicine of As2S2, has significant therapeutic effects for hundreds of years. NiuHuangJieDu tablets (NHJDT) is one of the most commonly prescribed realgar-containing preparations for the treatment of sore throat, swelling, and aching of gums. However, realgar-containing TCMs raise great safety concerns due to the adverse effects reported by arsenic poisoning. In this study, the arsenic-related health risk assessment of NHJDT was conducted in healthy volunteers after single and multiple doses oral administration. Blood, plasma, and urine samples were collected after dosing at predetermined time points or periods. Simple, rapid, and sensitive methods were established for the quantification of total arsenic and arsenic speciation in biological samples. The total arsenic and arsenic speciation were determined by hydride generation-atomic fluorescence spectrometry (HG-AFS) and high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS), respectively. No significant fluctuation of total arsenic was observed in human blood, and no traces of arsenic speciation were found in human plasma. Dimethylarsenic acid was detected as the predominated arsenic species in human urine after dosing. Therapeutic dose administration of NHJDT was relatively safe in single dose for the limited blood arsenic exposure, but long-term medication may still pose health risks due to the accumulation of arsenics in blood and its extremely slow excretion rate. Therefore, arsenic exposure should be carefully monitored during realgar-containing TCM medication, especially for long-term regimen. The results obtained in this study will provide scientific references for the clinical application of realgar and its-containing TCMs.

Realgar increases defenses against infection by Enterococcus faecalis in Caenorhabditis elegans.

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar 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 STUDY: This 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 METHODS: Infection 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. RESULTS: Realgar 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. CONCLUSION: Realgar 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.

[Preliminary study of Realgar and arsenic trioxide on gut microbiota of mice].

The aim of this paper was to observe the effect of Realgar and arsenic trioxide on gut microbiota. The mice were divided into low-dose Realgar group(RL), medium-dose Realgar group(RM), high-dose Realgar group(RH), and arsenic trioxide group(ATO), in which ATO and RL groups had the same trivalent arsenic content. Realgar and arsenic trioxide toxicity models were established after intragastric administration for 1 week, and mice feces were collected 1 h after intragastric administration on day 8. The effects of Realgar on gut microbiota of mice were observed through bacterial 16 S rRNA gene sequences. The results showed that Lactobacillus was decreased in all groups, while Ruminococcus and Adlercreutzia were increased. The RL group and ATO group were consistent in the genera of Prevotella, Ruminococcus, and Adlercreutzia but different in the genera of Lactobacillus and Bacteroides. Therefore, the effects of Realgar and arsenic trioxide with the same amount of trivalent arsenic on gut microbiota were similar, but differences were still present. Protective bacteria such as Lactobacillus were reduced after Realgar administration, causing inflammation. At low doses, the number of anti-inflammatory bacteria, such as Ruminococcus, Adlercreutzia and Parabacteroides increased, which can offset the slight inflammation caused by the imbalance of bacterial flora. At high doses, the flora was disturbed and the number of Proteobacteria was increased, with aggravated intestinal inflammation, causing edema and other inflammatory reactions. Based on this, authors believe that the gastrointestinal reactions after clinical use of Realgar may be related to flora disorder. Realgar should be used at a small dose in combination with other drugs to reduce intestinal inflammation.

Liver metabonomics study on the protective effect of glycyrrhetinic acid against realgar-induced liver injury.

Glycyrrhetinic acid (GA) is the bioactive ingredient in Glycyrrhizae Radix et Rhizoma. Our previous study has reported that GA has protective effect on realgar-induced hepatotoxicity. However, the details of the hepatoprotective mechanisms of GA on realgar-induced liver injury remain to be elucidated. In the study, mice were divided into control, GA-control, realgar, and co-treated groups. Their liver tissues were used for metabonomics study by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method. The results illustrate that GA significantly ameliorate the liver injury and metabolic perturbations caused by realgar. Some metabolites, such as phenylalanine, pyroglutamic acid (PGA), proline, carnitine, nicotinamide, choline, lysophosphatidylcholine (LPC) 16 : 0 and LPC 18 : 2 were found responsible for the hepatoprotective effect of GA. These metabolites are associated with the methylation metabolism of arsenic, cell membrane structure, energy metabolism and oxidative stress. From the results of this study, we infer that the potential hepatoprotective mechanism of GA on realgar-induced liver injury may be associated with reducing arsenic accumulation and its methylation metabolism in the liver, promoting the conjugation of arsenic and GSH to play detoxification effect, and ameliorating the liver metabolic perturbations caused by realgar.