Synthesis of the Isodityrosine Moiety of Seongsanamide A-D and Its Derivatives.

The concise and highly convergent synthesis of the isodityrosine unit of seongsanamide A-D and its derivatives bearing a diaryl ether moiety is described. In this work, the synthetic strategy features palladium-catalyzed C(sp3)-H functionalization and a Cu/ligand-catalyzed coupling reaction. We report a practical protocol for the palladium-catalyzed mono-arylation of ß-methyl C(sp3)-H of an alanine derivative bearing a 2-thiomethylaniline auxiliary. The reaction is compatible with a variety of functional groups, providing practical access to numerous ß-aryl-α-amino acids; these acids can be converted into various tyrosine and dihydroxyphenylalanine (DOPA) derivatives. Then, a CuI/N,N-dimethylglycine-catalyzed arylation of the already synthesized DOPA derivatives with aryl iodides is described for the synthesis of isodityrosine derivatives.

Network pharmacological analysis of corosolic acid reveals P4HA2 inhibits hepatocellular carcinoma progression.

BACKGROUND: Corosolic acid is a pentacyclic triterpene acid with hypoglycemic, anti-inflammatory, and anti-cancer effects. However, its potential targets in hepatocellular carcinoma (HCC) are unknown, hindering clinical utilization. METHODS: Differentially expressed proteins of the Bel-7404 cell line were identified with tandem mass tag analysis and differentially expressed genes (DEGs) of an HCC TCGA dataset using bioinformatics. Gene functions and pathways were inferred using the DAVID database. Online databases were used to establish P4HA2 expression in HCC (GEPIA2) and its relationship with patient survival (UALCAN and The Human Protein Atlas), the association between P4HA2 expression and immune cell infiltration (TIMER2), and DNA methylation of the P4HA2 gene (MethSurv). Cell proliferation, cell cycle, and cell death were assessed with PI and SYTOX-Green staining, CCK-8, and colony formation assays. Protein expression levels were detected by Western blotting. RESULTS: A total of 44 differentially expressed proteins and 4498 DEGs were identified. Four genes whose proteins were also found in the differential protein profile but with opposing expressions were selected as candidate targets. The candidate gene prolyl 4-hydroxylase subunit alpha 2 (P4HA2) was recognized as the only potential target due to its high expression in public datasets, association with poor patient survival, and relation to immune cell infiltration in HCC tissues. Moreover, the DNA methylation status in 4 CpG islands of the P4HA2 gene correlated with a poor prognosis. Furthermore, corosolic acid treatment inhibited the proliferation of HCC cell lines Bel-7404 and HepG2 in a dose-dependent manner, caused G2/M phase cell cycle arrest, and promoted cell death. In addition, the treatment reduced P4HA2 protein levels. CONCLUSION: Our results indicate that P4HA2 is a potential target of corosolic acid. Thus, they contribute to understanding molecular changes in HCC after corosolic acid treatment and facilitate finding new treatment regimens.

Increased Reactive Oxygen Species and Distinct Oxidative Damage in Resveratrol-suppressed Glioblastoma Cells.

Background and Aim: Glioblastoma multiforme (GBM) is a highly aggressive brain malignancy that lacks reliable treatments. Resveratrol possesses anti-cancer effects, but its activity against glioblastoma cells is variable for unknown reasons. One mechanism through which anti-cancer drugs exert their effects is oxidative damage caused by increased reactive oxygen species (ROS) production. Thus, the present study examined the relationship between oxidative stress and sensitivity to resveratrol in glioblastoma cells. Methods: Two GBM cell lines (U251 and LN428) were exposed to 100 µM resveratrol for 48 h, and proliferation and apoptosis were assessed. ROS generation was evaluated using 2'-7'-dichlorodihydrofluorescein diacetate-based flow cytometry and fluorescent microscopy. Immunocytochemical staining and western blotting were conducted at regular intervals to profile the expression patterns of superoxide dismutase-2 (SOD2), catalase, caspase-9, caspase-3, and sulfotransferases (SULTs) in untreated and resveratrol-treated GBM cells. Results: Resveratrol-treated U251 cells, but not resveratrol-treated LN428 cells, exhibited remarkable growth arrest and extensive apoptosis accompanied by elevated intracellular ROS levels and attenuated SOD2 and catalase expression. Mitochondrial impairment and more distinct increases in the expression of activated caspase-9 and caspase-3 were detected in U251 cells following resveratrol treatment. The levels of resveratrol metabolic enzymes (SULT1A1 and SULT1C2) were lower in U251 cells than in LN428 cells. Conclusions: Resveratrol increased ROS generation and induced oxidation-related cellular lesions in U251 cells by activating an ROS-related mitochondrial signal pathway. The levels of SULTs and ROS may indicate the therapeutic outcomes of resveratrol treatment in GBM.

Synthesis and Cytotoxic Activity of Novel C-23-Modified Asiatic Acid Derivatives.

We selectively oxidized the C-23 hydroxyl group in an asiatic acid (AA) derivative and then, for the first time with AA, modification of the C-23 carboxyl group was conducted to synthesize a series of new AA derivatives. The evaluation of their cytotoxic activities against two human cancer cell lines (SKOV-3 and HCT116) using the MTT assay in vitro revealed a distinctive structure activity relationship (SAR) associated with the intramolecular hydrogen bonding of the amide moiety at C-23. According to the established SAR, the cytotoxic activities of four promising compounds were then evaluated against MCF-7, A549, A2780, HepG2 and HL-60 cancer cell lines. Compound 10 had the best cytotoxic activity among all tested derivatives in the HL-60 cell line, giving IC50 = 0.47 µM, while showing no cytotoxic effect against human normal cells (HUVEC).

STAT3 signaling statuses determine the fate of resveratrol-treated anaplastic thyroid cancer cells.

BACKGROUNDS: Anaplastic thyroid cancer/ATC is highly lethal malignancy without reliable chemotherapeutic drug. Resveratrol possesses anti-ATC activities but encounters resistance in some cases due to certain unknown reason(s). OBJECTIVE: Because signal transducer and activator of transcription/STAT3 signaling is critical for ATC cell survival and the main molecular target of resveratrol, its roles in determining the fates of resveratrol-treated ATC cells were investigated here. METHODS: Human THJ-11T, THJ-16 and THJ-21T ATC cell lines were treated by 100 µM resveratrol and their growth, statuses of STAT3 signaling and STAT3-related gene expression were examined. The relevance of STAT3 activation with resveratrol resistance was elucidated using STAT selective inhibitor AG490. Leukemia inhibitory factor/LIF expression and phosphorylated-STAT3/p-STAT3 nuclear translocation in ATC tissues were immunohistochemically analyzed. RESULTS: Resveratrol inhibited proliferation, p-STAT3 nuclear translocation as well as LIF and STAT3 expression of THJ-16T and THJ-21T but not THJ-21T cells which showed LIF upregulation and more frequent p-STAT3 nuclear translocation. AG490 significantly prevent p-STAT3 nuclear translocation, and reversed the resveratrol tolerance of THJ-11T cells. Immonohistochemical staining revealed 14.3% (4/28) of LIF and 3.6% (1/28) of p-STAT3 detection in noncancerous ATC-surrounding tissues, which increased to 89.5% (17/19) and 52.6% (10/19) respectively among ATC specimens. The correlative analysis indicated the relevance of LIF expression and STAT3 activation (r= 0.825; P< 0.01). CONCLUSIONS: The status of STAT3 activation and LIF expression are closely correlated with the therapeutic effect of resveratrol on ATCs. Frequent LIF upregulation and STAT3 activation are the unfavorable factors of ATCs and the potential targets of anti-ATC therapy.

Antiepileptic geissoschizine methyl ether is an inhibitor of multiple neuronal channels.

Geissoschizine methyl ether (GM) is an indole alkaloid isolated from Uncaria rhynchophyll (UR) that has been used for the treatment of epilepsy in traditional Chinese medicine. An early study in a glutamate-induced mouse seizure model demonstrated that GM was one of the active ingredients of UR. In this study, electrophysiological technique was used to explore the mechanism underlying the antiepileptic activity of GM. We first showed that GM (1-30 µmol/L) dose-dependently suppressed the spontaneous firing and prolonged the action potential duration in cultured mouse and rat hippocampal neurons. Given the pivotal roles of ion channels in regulating neuronal excitability, we then examined the effects of GM on both voltage-gated and ligand-gated channels in rat hippocampal neurons. We found that GM is an inhibitor of multiple neuronal channels: GM potently inhibited the voltage-gated sodium (NaV), calcium (CaV), and delayed rectifier potassium (IK) currents, and the ligand-gated nicotinic acetylcholine (nACh) currents with IC50 values in the range of 1.3-13.3 µmol/L. In contrast, GM had little effect on the voltage-gated transient outward potassium currents (IA) and four types of ligand-gated channels (γ-amino butyric acid (GABA), N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainite (AMPA/KA receptors)). The in vivo antiepileptic activity of GM was validated in two electricity-induced seizure models. In the maximal electroshock (MES)-induced mouse seizure model, oral administration of GM (50-100 mg/kg) dose-dependently suppressed generalized tonic-clonic seizures. In 6-Hz-induced mouse seizure model, oral administration of GM (100 mg/kg) reduced treatment-resistant seizures. Thus, we conclude that GM is a promising antiepileptic candidate that inhibits multiple neuronal channels.

Dendrobium candidum aqueous extract attenuates isoproterenol-induced cardiac hypertrophy through the ERK signalling pathway.

CONTEXT: The pharmacological functions of Dendrobium candidum Wall. ex Lindl. (Orchidaceae) in cardiac hypertrophy remains unclear. OBJECTIVE: To evaluate whether D. candidum aqueous extract (DCAE) can attenuate experimental cardiac hypertrophy. MATERIALS AND METHODS: Cardiac hypertrophy in SD rats was induced by subcutaneously injection of isoproterenol (2 mg/kg), once a day for ten days. Rats were gavaged with DCAE (0.13 and 0.78 g/kg) daily for one month. At the end of treatment, measurement of left ventricular systolic pressure (LVSP), heart-to-body weight ratio (HW/BW), left ventricular/tibia length (LV/TL), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) levels, haematoxylin-eosin staining, and Masson's trichrome staining were conducted. In cultured H9c2 cells, DCAE (2 mg/mL) and U0126 (10 µM) were added 2 h before the isoproterenol (10 µM) stimulus. Phalloidin staining was used to evaluate cellular hypertrophy. The mRNA expression of ANP and BNP was measured by qRT-PCR. The expression of p-ERK was determined by immunoblotting. RESULTS: DCAE treatment significantly reduced the following indicators in vivo: (1) the LVSP (16%); (2) HW/BW (13%); (3) LV/TL (6%); (4) ANP (39%); (5) BNP (32%). In cultured H9c2 cells, phalloidin staining showed that DCAE relieved cellular hypertrophy (53% reduction). Furthermore, immunoblotting showed that DCAE can significantly inhibit p-ERK protein expression in vivo and in vitro (39% and 27% reduction, respectively). DISCUSSION AND CONCLUSIONS: DCAE prevents cardiac hypertrophy via ERK signalling pathway and has the potential for treatment of cardiac hypertrophy.

Gender Differences in Arrhythmias: Focused on Atrial Fibrillation.

There are significant differences in clinical presentation and treatment of atrial fibrillation (AF) between women and men. The primary goal of AF management is to restore sinus rhythm and to prevent various complications, including stroke and heart failure. In many areas of AF, such as prevalence, clinical manifestations, morbidity, risk factors, pathophysiology, treatment strategies, and complications, gender-specific variability is observed and needs to be further addressed by large-scale population researches or randomized clinical trials, which help to promote the customization of AF treatment programs, hence to maximize the success rate of AF therapy in both sexes. This review highlights our current understanding of these gender differences in AF and how these differences affect treatment decisions on AF.

HMGB1/TLR4 promotes hypoxic pulmonary hypertension via suppressing BMPR2 signaling.

High mobility group box 1 (HMGB1), a critical nonclassical inflammatory cytokine, has been found up-regulated in patients with idiopathic pulmonary arterial hypertension (IPAH), but its role in vascular remodeling of pulmonary hypertension (PH) is still unknown. In present study, we demonstrated that the plasma level of inflammatory cytokine including HMGB1, interleukin 1ß (IL-1ß), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) were elevated in hypoxia-induced pulmonary hypertension rats model. Moreover, expressions of HMGB1 and Toll like receptor-4 (TLR4) in pulmonary arteries were obviously up-regulated accompanied with down-regulation of bone morphogenetic protein receptor 2 (BMPR2) signaling, characterized by decline of phosphorylated Smad1/5/8 (p-Smsd1/5/8) and inhibitor of differention 1 (Id1) expression. In cultured primary pulmonary arterial smooth muscle cells (PASMCs), we found that HMGB1 incubation significantly promoted proliferation and migration of PASMCs, down-regulated p-Smsd1/5/8 and Id1 expression, which can be abrogated by HMGB1 inhibitors saquinavir, glycyrrhizn and TLR4 inhibitors TAK-242. Furthermore, saquinavir, glycyrrhizn and TAK-242 treatment significantly attenuated the development of PH in rats by recovering homodynamic parameters, pulmonary vascular remodeling and BMPR2 signaling pathway. In summary, our results suggest that HMGB1/TLR4 signaling promotes hypoxia-induced pulmonary hypertension via suppressing BMPR2 signaling.

Different structures of berberine and five other protoberberine alkaloids that affect P-glycoprotein-mediated efflux capacity.

Berberine, berberrubine, thalifendine, demethyleneberberine, jatrorrhizine, and columbamine are six natural protoberberine alkaloid (PA) compounds that display extensive pharmacological properties and share the same protoberberine molecular skeleton with only slight substitution differences. The oral delivery of most PAs is hindered by their poor bioavailability, which is largely caused by P-glycoprotein (P-gp)-mediated drug efflux. Meanwhile, P-gp undergoes large-scale conformational changes (from an inward-facing to an outward-facing state) when transporting substrates, and these changes might strongly affect the P-gp-binding specificity. To confirm whether these six compounds are substrates of P-gp, to investigate the differences in efflux capacity caused by their trivial structural differences and to reveal the key to increasing their binding affinity to P-gp, we conducted a series of in vivo, in vitro, and in silico assays. Here, we first confirmed that all six compounds were substrates of P-gp by comparing the drug concentrations in wild-type and P-gp-knockout mice in vivo. The efflux capacity (net efflux) ranked as berberrubine > berberine > columbamine ~ jatrorrhizine > thalifendine > demethyleneberberine based on in vitro transport studies in Caco-2 monolayers. Using molecular dynamics simulation and molecular docking techniques, we determined the transport pathways of the six compounds and their binding affinities to P-gp. The results suggested that at the early binding stage, different hydrophobic and electrostatic interactions collectively differentiate the binding affinities of the compounds to P-gp, whereas electrostatic interactions are the main determinant at the late release stage. In addition to hydrophobic interactions, hydrogen bonds play an important role in discriminating the binding affinities.

Correlation of Reactive Oxygen Species Levels with Resveratrol Sensitivities of Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid carcinoma (ATC) is the most lethal thyroid malignancy without a reliable therapeutic agent. Resveratrol possesses cancer-suppressive effects, while its effect(s) on ATC cells remains unknown. Because oxidative damage caused by increased reactive oxygen species (ROS) is one of the therapeutic effects of anticancer drugs and oxidative stress-caused mitochondria swelling is observed in resveratrol-treated cancer cells, the oxidative statuses and their relevance with resveratrol sensitivities are elucidated using THJ-16T and THJ-11T ATC cells established from two human anaplastic thyroid carcinoma cases. The results revealed that resveratrol-treated THJ-16T rather than THJ-11T cells showed remarkable growth arrest and extensive apoptosis accompanied with the elevated ROS generation and the attenuated superoxide dismutase 2 (SOD2) and catalase (CAT) levels. Mitochondrial impairment and the enhanced caspase-9/caspase-3 activation are found only in resveratrol-sensitive THJ-16T cells. Treatment with the antioxidant N-acetylcysteine (NAC) partly attenuated resveratrol-induced ROS generation and apoptosis of THJ-16T cells. The levels of resveratrol metabolic enzymes (SULT1A1 and SULT1C2) in THJ-16T cells were lower than those in THJ-11T cells and therefore reversely related with resveratrol sensitivities of ATC cells. Our findings demonstrate the ability of resveratrol to increase ROS generation and oxidative-related cellular lesions in resveratrol-sensitive THJ-16T cells presumably through activating the ROS-mitochondrial signal pathway. The levels of SULTs and ROS may reflect the response manners of ATC cells to resveratrol.

[Analysis of Regularity of Acupoint Selection in Acupuncture Treatment of Urticaria Based on Data Mining Technology].

OBJECTIVE: To analyze the regularity of acupoint selection, main acupoints and theoretical basis in acupuncture treatment of urticaria. METHODS: Papers collected from the time of establishment of each database to September of 2017 were retrieved from databases CNKI, CBM, VIP and WF by using keywords of "acupuncture" "moxibustion" "blood-letting therapy" "autohemotherapy" "cupping" "acupoint catgut embedding" "auricular points" "acupoint injection" "fire-needle" (or red-hot needle), "dermal needle" "needle-embedding" "urticaria" in both Chinese and English. The collected papers were brought into analysis according to the inclusion and exclusion criteria, from which the prescriptions for acupuncture treatment of urticaria were subjected into descriptive statistical analysis, association rule analysis, and cluster analysis by using Access 2010, Clementine 18.0 and Stata software. RESULTS: Outcomes of analysis indicated that the treatment methods of urticaria with acupuncture and moxibustion, with different emphases, may be classified into eight categories. For treating the exterior syndrome of urticaria, acupoints of the Bladder Meridian, Governor Vessel and Conception Vessel were often employed to harmonize Ying and Wei and to dispel the pathogenic wind, while for treating the interior syndrome, acupoints of the Large Intestine Meridian, Spleen Meridian, and Stomach Meridian were usually used to invigorate the spleen to dispel dampness and to regulate blood circulation. The top five frequently used acupoints were Quchi (LI 11), Xuehai (SP 10), Zusanli (ST 36), Sanyinjiao (SP 6) and Geshu (BL 17). It was crucial to make use of the specific acupoints with adequate meridian-qi, such as He-Sea points, Back-Shu points, and Yuan-Primary points. There were some fixed forms in the combination of acupoints, including LI 11, SP 10, Dazhui (GV 14) and auricular Lung, Shenmen, Fengxi, Adrenal gland, which had the highest confidence coefficient for the meridian points and ear acu-points, respectively. The outcomes of cluster analysis about the acupoint prescriptions showed that 12 acupoint groups as the SP 6-Hegu (LI 4)-LI 11-SP 10-ST 36, etc. were frequently used. CONCLUSION: The regularity of acupuncture treatment of urticaria can be discovered using data mining technology, resulting in an in-depth understanding and having a solid theoretical basis.

Metabolism, pharmacokinetics, and hepatic disposition of xanthones and saponins on Zhimu treatments for exploratively interpreting the discrepancy between the herbal safety and timosaponin A3-induced hepatotoxicity.

Timosaponin A3, a saponin in Zhimu, elicited hepatotoxicity via oxidative stress. However, the clinical medication of Zhimu has been historically regarded as safe, probably associated with the antioxidants it contains. However, the related information on the in vivo levels of timosaponin A3 and antioxidants remained unclear on Zhimu treatments. Therefore, a combination of the in vitro metabolism, including microbiota-mediated and liver-mediated metabolism, and in vivo pharmacokinetics and hepatic disposition, was conducted for three xanthones (neomangiferin, mangiferin, and norathyriol) and three saponins (timosaponin B2, timosaponin B3, and timosaponin A3) on Zhimu treatments. Consequently, following oral administration of Zhimu decoction to rats, those saponins and xanthones were all observed in the plasma with severe liver first-pass effect, where mangiferin was of the maximum exposure. Despite the ignorable content in the herb, timosaponin A3 elicited sizable hepatic exposure as the microbiota-mediated metabolite of saponins in Zhimu. The similar phenomenon also occurred to norathyriol, the microbiota-mediated metabolite of xanthones. However, the major prototypes in Zhimu were of limited hepatic exposure. We deduced the hepatic collection of norathyriol, maximum circulating levels of mangiferin, and timosaponin B2 and mangiferin interaction may directly or indirectly contribute to the whole anti-oxidation of Zhimu, and then resisted the timosaponin A3-induced hepatotoxicity. Thus, our study exploratively interpreted the discrepancy between herbal safety and timosaponin A3-induced hepatotoxicity. However, given the considerable levels and slow eliminated rate of timosaponin A3 in the liver, more attention should be paid to the safety on the continuous clinical medication of Zhimu in the future.

Resveratrol Suppresses the Growth and Enhances Retinoic Acid Sensitivity of Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is a highly lethal undifferentiated malignancy without reliable therapies. Retinoic acid (RA) has been employed to promote redifferentiation of thyroid cancers by increasing their I131 uptake and radio-sensitivity, but its effect(s) on ATCs has not yet been ascertained. Likewise, resveratrol induces cancer redifferentiation but, also in this case, its effects on ATCs remain unknown. These issues have been addresses in the current study using three human ATC cell lines (THJ-11T, THJ-16T, and THJ-21T) through multiple experimental approaches. The results reveal that RA exerts a small inhibitory effect on these cell lines. In comparison with normally cultured cells, the total cell number in resveratrol-treated THJ-16T and THJ-21T cultures significantly decreased (p < 0.05), and this effect was accompanied by reduced Cyclin D1 immuno-labeling, increased apoptotic fractions, and distinct caspase-3 activation. Resveratrol failed to inhibit growth but enhanced RA sensitivity of THJ-11T cells, suppressed peroxisome proliferator-activated receptor-ß/δ (PPAR-ß/δ), and upregulated cellular retinoic acid-binding protein 2 (CRABP2) and retinoic acid receptor beta (RAR-ß) expression. Increased thyroglobulin (Tg) and E-cadherin levels and appearance of membranous E-cadherin were evidenced in resveratrol-treated THJ-11T cells. Our results demonstrate for the first time: (1) the therapeutic value of resveratrol by itself or in combination with RA in the management of ATCs, (2) the capacity of resveratrol to overcome RA resistance in ATC cells by reprogramming CRABP2/RAR- and fatty acid-binding protein 5 (FABP5)/PPAR-ß/δ-mediated RA signaling, and (3) the redifferentiating potential of resveratrol in ATC cells.

Malignant transformation of nasal chondromesenchymal hamartoma in adult: a case report and review of the literature.

Nasal chondromesenchymal hamartoma (NCMH) is an extremely rare benign tumor arising in the sinonasal tract, predominantly involving infants and children. To date, only 27 cases are reported in the international literature and there have been no reported cases of malignant transformation. We present a 40-year-old female patient with nasal obstruction and bloody rhinorrhea. Computed tomography (CT) of the nose and paranasal sinuses confirmed a heterogeneous polypoid soft-tissue mass filling the nasal cavity and extending into the maxillary and ethmoid sinus. The patient underwent a complete radical resection. Histological and immunohistochemical analyses showed a portion of the mass was consistent with typical NCMH. However, some areas of mass exhibited cytological atypia, marked mitotic activity and foci of necrosis. The atypical mesenchymal spindle cells were immunoreactive for vimentin, CD99 and smooth muscle actin (SMA) diffusely. The cartilaginous cells were immunopositive for S-100 protein. Ki-67 index was high in atypical areas, accounting for 50%. A rapid mass recurrence was observed at the original site only 3 months after surgery. The final diagnosis of NCMH with malignant transformation was made. To our knowledge, this is the first report of malignant transformation occurring in an adult with NCMH. Although NCMH commonly develops in the neonate or young infants and exhibits benign histological appearance and favorable prognosis, there is a possibility of malignant transformation in adult patients. Thoroughly histological inspections are suggested to be necessary to accurately diagnose this tumor when it is encountered in adults.

Bioinformatics analysis of rabbit haemorrhagic disease virus genome.

BACKGROUND: Rabbit haemorrhagic disease virus (RHDV), as the pathogeny of Rabbit haemorrhagic disease, can cause a highly infectious and often fatal disease only affecting wild and domestic rabbits. Recent researches revealed that it, as one number of the Caliciviridae, has some specialties in its genome, its reproduction and so on. RESULTS: In this report, we firstly analyzed its genome and two open reading frameworks (ORFs) from this aspect of codon usage bias. Our researches indicated that mutation pressure rather than natural is the most important determinant in RHDV with high codon bias, and the codon usage bias is nearly contrary between ORF1 and ORF2, which is maybe one of factors regulating the expression of VP60 (encoding by ORF1) and VP10 (encoding by ORF2). Furthermore, negative selective constraints on the RHDV whole genome implied that VP10 played an important role in RHDV lifecycle. CONCLUSIONS: We conjectured that VP10 might be beneficial for the replication, release or both of virus by inducing infected cell apoptosis initiate by RHDV. According to the results of the principal component analysis for ORF2 of RSCU, we firstly separated 30 RHDV into two genotypes, and the ENC values indicated ORF1 and ORF2 were independent among the evolution of RHDV.