Efficient estimation for large-scale linkage disequilibrium patterns of the human genome.

In this study, we proposed an efficient algorithm (X-LD) for estimating linkage disequilibrium (LD) patterns for a genomic grid, which can be of inter-chromosomal scale or of small segments. Compared with conventional methods, the proposed method was significantly faster, dropped from O(nm2) to O(n2m)-n the sample size and m the number of SNPs, and consequently we were permitted to explore in depth unknown or reveal long-anticipated LD features of the human genome. Having applied the algorithm for 1000 Genome Project (1KG), we found (1) the extended LD, driven by population structure, universally existed, and the strength of inter-chromosomal LD was about 10% of their respective intra-chromosomal LD in relatively homogeneous cohorts, such as FIN, and to nearly 56% in admixed cohort, such as ASW. (2) After splitting each chromosome into upmost of more than a half million grids, we elucidated the LD of the HLA region was nearly 42 folders higher than chromosome 6 in CEU and 11.58 in ASW; on chromosome 11, we observed that the LD of its centromere was nearly 94.05 folders higher than chromosome 11 in YRI and 42.73 in ASW. (3) We uncovered the long-anticipated inversely proportional linear relationship between the length of a chromosome and the strength of chromosomal LD, and their Pearson's correlation was on average over 0.80 for 26 1KG cohorts. However, this linear norm was so far perturbed by chromosome 11 given its more completely sequenced centromere region. Uniquely chromosome 8 of ASW was found most deviated from the linear norm than any other autosomes. The proposed algorithm has been realized in C++ (called X-LD) and is available at https://github.com/gc5k/gear2, and can be applied to explore LD features in any sequenced populations.

Chemical-induced degradation of PreS2 mutant surface antigen via the induction of microautophagy.

Naturally evolved immune-escape PreS2 mutant is an oncogenic caveat of liver cirrhosis and hepatocellular carcinoma (HCC) during chronic hepatitis B virus (HBV) infection. PreS2 mutant is prevalent in above 50% of patients with HCC. In addition, intrahepatic expression of PreS2 mutant large surface antigen (PreS2-LHBS) induces endoplasmic reticulum stress, mitochondria dysfunction, cytokinesis failure, and subsequent chromosome hyperploidy. As PreS2-LHBS has no enzymatic activity, the development of PreS2-specific inhibitors can be challenging. In this study, we aim to identify inhibitors of PreS2-LHBS via the induction of protein-specific degradation. We set up a large-scale protein stability reporter platform and applied an FDA-approved drug library for the screening. We identified ABT199 as a negative modulator of PreS2-LHBS, which induced the degradation of PreS2-LHBS without affecting the general cell viability in both hepatoma and immortalized hepatocytes. Next, by affinity purification screening, we found that PreS2-LHBS interacted with HSC70, a microautophagy mediating chaperone. Simultaneously, inhibitions of lysosomal degradation or microautophagy restored the expression of PreS2-LHBS, suggesting microautophagy is involved in ABT199-induced PreS2-LHBS degradation. Notably, a 24-hr treatment of ABT199 was sufficient for the reduction of DNA damage and cytokinesis failure in PreS2-LHBS expressing hepatocytes. In addition, a persistent treatment of ABT199 for 3 weeks reversed chromosome hyperploidy in PreS2-LHBS cells and suppressed anchorage-independent growth of HBV-positive hepatoma cells. Together, this study identified ABT-199 as a negative modulator of PreS2-LHBS via mediating microautophagy. Our results indicate that long-term inhibition of PreS2-LHBS may serve as a novel strategy for the therapeutic prevention of HBV-mediated HCC.

Tumor suppressor BAP1 nuclear import is governed by transportin-1.

Subcellular localization of the deubiquitinating enzyme BAP1 is deterministic for its tumor suppressor activity. While the monoubiquitination of BAP1 by an atypical E2/E3-conjugated enzyme UBE2O and BAP1 auto-deubiquitination are known to regulate its nuclear localization, the molecular mechanism by which BAP1 is imported into the nucleus has remained elusive. Here, we demonstrated that transportin-1 (TNPO1, also known as Karyopherin ß2 or Kapß2) targets an atypical C-terminal proline-tyrosine nuclear localization signal (PY-NLS) motif of BAP1 and serves as the primary nuclear transporter of BAP1 to achieve its nuclear import. TNPO1 binding dissociates dimeric BAP1 and sequesters the monoubiquitination sites flanking the PY-NLS of BAP1 to counteract the function of UBE2O that retains BAP1 in the cytosol. Our findings shed light on how TNPO1 regulates the nuclear import, self-association, and monoubiquitination of BAP1 pertinent to oncogenesis.

Identification of Entry Inhibitors against Delta and Omicron Variants of SARS-CoV-2.

Entry inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to control the outbreak of coronavirus disease 2019 (COVID-19). This study developed a robust and straightforward assay that detected the molecular interaction between the receptor-binding domain (RBD) of viral spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor in just 10 min. A drug library of 1068 approved compounds was used to screen for SARS-CoV2 entry inhibition, and 9 active drugs were identified as specific pseudovirus entry inhibitors. A plaque reduction neutralization test using authentic SARS-CoV-2 virus in Vero E6 cells confirmed that 2 of these drugs (Etravirine and Dolutegravir) significantly inhibited the infection of SARS-CoV-2. With molecular docking, we showed that both Etravirine and Dolutegravir are preferentially bound to primary ACE2-interacting residues on the RBD domain, implying that these two drug blocks may prohibit the viral attachment of SARS-CoV-2. We compared the neutralizing activities of these entry inhibitors against different pseudoviruses carrying spike proteins from alpha, beta, gamma, and delta variants. Both Etravirine and Dolutegravir showed similar neutralizing activities against different variants, with EC50 values between 4.5 to 5.8 nM for Etravirine and 10.2 to 22.9 nM for Dolutegravir. These data implied that Etravirine and Dolutegravir may serve as general spike inhibitors against dominant viral variants of SARS-CoV-2.

Methotrexate inhibition of SARS-CoV-2 entry, infection and inflammation revealed by bioinformatics approach and a hamster model.

Background: Drug repurposing is a fast and effective way to develop drugs for an emerging disease such as COVID-19. The main challenges of effective drug repurposing are the discoveries of the right therapeutic targets and the right drugs for combating the disease. Methods: Here, we present a systematic repurposing approach, combining Homopharma and hierarchal systems biology networks (HiSBiN), to predict 327 therapeutic targets and 21,233 drug-target interactions of 1,592 FDA drugs for COVID-19. Among these multi-target drugs, eight candidates (along with pimozide and valsartan) were tested and methotrexate was identified to affect 14 therapeutic targets suppressing SARS-CoV-2 entry, viral replication, and COVID-19 pathologies. Through the use of in vitro (EC50 = 0.4 µM) and in vivo models, we show that methotrexate is able to inhibit COVID-19 via multiple mechanisms. Results: Our in vitro studies illustrate that methotrexate can suppress SARS-CoV-2 entry and replication by targeting furin and DHFR of the host, respectively. Additionally, methotrexate inhibits all four SARS-CoV-2 variants of concern. In a Syrian hamster model for COVID-19, methotrexate reduced virus replication, inflammation in the infected lungs. By analysis of transcriptomic analysis of collected samples from hamster lung, we uncovered that neutrophil infiltration and the pathways of innate immune response, adaptive immune response and thrombosis are modulated in the treated animals. Conclusions: We demonstrate that this systematic repurposing approach is potentially useful to identify pharmaceutical targets, multi-target drugs and regulated pathways for a complex disease. Our findings indicate that methotrexate is established as a promising drug against SARS-CoV-2 variants and can be used to treat lung damage and inflammation in COVID-19, warranting future evaluation in clinical trials.

Aerobic glycolysis supports hepatitis B virus protein synthesis through interaction between viral surface antigen and pyruvate kinase isoform M2.

As an intracellular pathogen, the reproduction of the hepatitis B virus (HBV) depends on the occupancy of host metabolism machinery. Here we test a hypothesis if HBV may govern intracellular biosynthesis to achieve a productive reproduction. To test this hypothesis, we set up an affinity purification screen for host factors that interact with large viral surface antigens (LHBS). This identified pyruvate kinase isoform M2 (PKM2), a key regulator of glucose metabolism, as a binding partner of viral surface antigens. We showed that the expression of viral LHBS affected oligomerization of PKM2 in hepatocytes, thereby increasing glucose consumption and lactate production, a phenomenon known as aerobic glycolysis. Reduction of PKM2 activity was also validated in several different models, including HBV-infected HepG2-NTCP-C4 cells, adenovirus mediated HBV gene transduction and transfection with a plasmid containing complete HBV genome on HuH-7 cells. We found the recovery of PKM2 activity in hepatocytes by chemical activators, TEPP-46 or DASA-58, reduced expressions of viral surface and core antigens. In addition, reduction of glycolysis by culturing in low-glucose condition or treatment with 2-deoxyglucose also decreased expressions of viral surface antigen, without affecting general host proteins. Finally, TEPP-46 largely suppressed proliferation of LHBS-positive cells on 3-dimensional agarose plates, but showed no effect on the traditional 2-dimensional cell culture. Taken together, these results indicate that HBV-induced metabolic switch may support its own translation in hepatocytes. In addition, aerobic glycolysis is likely essential for LHBS-mediated oncogenesis. Accordingly, restriction of glucose metabolism may be considered as a novel strategy to restrain viral protein synthesis and subsequent oncogenesis during chronic HBV infection.

Identifying Primate ACE2 Variants That Confer Resistance to SARS-CoV-2.

SARS-CoV-2 infects humans through the binding of viral S-protein (spike protein) to human angiotensin I converting enzyme 2 (ACE2). The structure of the ACE2-S-protein complex has been deciphered and we focused on the 27 ACE2 residues that bind to S-protein. From human sequence databases, we identified nine ACE2 variants at ACE2-S-protein binding sites. We used both experimental assays and protein structure analysis to evaluate the effect of each variant on the binding affinity of ACE2 to S-protein. We found one variant causing complete binding disruption, two and three variants, respectively, strongly and mildly reducing the binding affinity, and two variants strongly enhancing the binding affinity. We then collected the ACE2 gene sequences from 57 nonhuman primates. Among the 6 apes and 20 Old World monkeys (OWMs) studied, we found no new variants. In contrast, all 11 New World monkeys (NWMs) studied share four variants each causing a strong reduction in binding affinity, the Philippine tarsier also possesses three such variants, and 18 of the 19 prosimian species studied share one variant causing a strong reduction in binding affinity. Moreover, one OWM and three prosimian variants increased binding affinity by >50%. Based on these findings, we proposed that the common ancestor of primates was strongly resistant to and that of NWMs was completely resistant to SARS-CoV-2 and so is the Philippine tarsier, whereas apes and OWMs, like most humans, are susceptible. This study increases our understanding of the differences in susceptibility to SARS-CoV-2 infection among primates.

Stage-specific functional relationships between Tub1 and Tub2 beta-tubulins in the wheat scab fungus Fusarium graminearum.

The filamentous ascomycete Fusarium graminearum contains two ß-tubulin genes TUB1 and TUB2 that differ in functions during vegetative growth and sexual reproduction. To further characterize their functional relationship, in this study we determined the co-localization of Tub1 and Tub2 and assayed their expression levels in different mutants and roles in DON production. Tub1 co-localized with Tub2 to the same regions of microtubules in conidia, hyphae, and ascospores. Whereas deletion of TUB1 had no obvious effect on the transcription of TUB2 and two α-tubulin genes (TUB4 and TUB5), the tub2 mutant was up-regulated in TUB1 transcription. To assay their protein expression levels, polyclonal antibodies that could specifically detect four α- and ß-tubulin proteins were generated. Western blot analyses showed that the abundance of Tub1 proteins was increased in tub2 but reduced in tub4 and tub5 mutants. Interestingly, protein expression of Tub4 and Tub5 was decreased in the tub1 mutant in comparison with the wild type, despite a lack of obvious changes in their transcription. In contrast, deletion of TUB2 had no effect on translation of TUB4 and TUB5. Ectopic expression of Tub2-mCherry partially recovered the growth defect of the tub1 mutant but did not rescue its defect in sexual reproduction. Expression of Tub1-GFP in the tub2 mutant also partially rescued its defects in vegetative growth, suggesting that disturbance in the balance of α- and ß-tubulins contributes to mutant defects. The tub2 but not tub1 mutant was almost blocked in DON biosynthesis. Expression of TRI genes, toxisome formation, and DON-related cellular differentiation were significantly reduced in the tub2 mutant. Overall, our results showed that Tub1 and Tub2 share similar subcellular localization and have overlapping functions during vegetative growth but they differ in functions in DON production and ascosporogenesis in F. graminearum.

Intrahepatic hepatitis B virus large surface antigen induces hepatocyte hyperploidy via failure of cytokinesis.

Hepatitis B virus (HBV) is an aetiological factor for liver cirrhosis and hepatocellular carcinoma (HCC). Despite current antiviral therapies that successfully reduce the viral load in patients with chronic hepatitis B, persistent hepatitis B surface antigen (HBsAg) remains a risk factor for HCC. To explore whether intrahepatic viral antigens contribute directly to hepatocarcinogenesis, we monitored the mitotic progression of HBV-positive cells. Cytokinesis failure was increased in HBV-positive HepG2.2.15 and 1.3ES2 cells, as well as in HuH-7 cells transfected with a wild-type or X-deficient HBV construct, but not in cells transfected with an HBsAg-deficient construct. We show that expression of viral large surface antigen (LHBS) was sufficient to induce cytokinesis failure of immortalized hepatocytes. Premitotic defects with DNA damage and G2 /M checkpoint attenuation preceded cytokinesis in LHBS-positive cells, and ultimately resulted in hyperploidy. Inhibition of polo-like kinase-1 (Plk1) not only restored the G2 /M checkpoint in these cells, but also suppressed LHBS-mediated in vivo tumourigenesis. Finally, a positive correlation between intrahepatic LHBS expression and hepatocyte hyperploidy was detected in >70% of patients with chronic hepatitis B. We conclude that HBV LHBS provokes hyperploidy by inducing DNA damage and upregulation of Plk1; the former results in atypical chromatin structures, and the latter attenuates the function of the G2 /M DNA damage checkpoint. Our data uncover a mechanism by which genomic integrity of hepatocytes is disrupted by viral LHBS. These findings highlight the role of intrahepatic surface antigen as an oncogenic risk factor in the development of HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Target-activated streptavidin-biotin controlled binding probe.

Target-activated chemical probes are important tools in basic biological research and medical diagnosis for monitoring enzyme activities and reactive small molecules. Based on the fluorescence turn-on mechanism, they can be divided into two classes: dye-based fluorescent probes and caged-luciferin. In this paper, we introduce a new type of chemical probe in which the fluorescence turn-on is based on controlled streptavidin-biotin binding. Compared to conventional probes, the streptavidin-biotin controlled binding probe has several advantages, such as minimal background at its "OFF" state, multiple signal amplification steps, and unlimited selection of the optimal dyes for detection. To expand the scope, a new synthetic method was developed, through which a wider range of analyte recognition groups can be easily introduced to construct the binding probe. This probe design was successfully applied to image and study secreted peroxynitrite (ONOO-) at the cell surface of macrophages where information on ONOO- is difficult to obtain. As the signals are generated upon the binding of streptavidin to the biotin probe, this highly versatile design can not only be used in fluorescence detection but can also be applied in various other detection modes, such as electrochemical and enzyme-amplified luminescence detection.

Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability.

Hepatitis B virus (HBV) is a driver of hepatocellular carcinoma, and two viral products, X and large surface antigen (LHBS), are viral oncoproteins. During chronic viral infection, immune-escape mutants on the preS2 region of LHBS (preS2-LHBS) are gain-of-function mutations that are linked to preneoplastic ground glass hepatocytes (GGHs) and early disease onset of hepatocellular carcinoma. Here, we show that preS2-LHBS provoked calcium release from the endoplasmic reticulum (ER) and triggered stored-operated calcium entry (SOCE). The activation of SOCE increased ER and plasma membrane (PM) connections, which was linked by ER- resident stromal interaction molecule-1 (STIM1) protein and PM-resident calcium release- activated calcium modulator 1 (Orai1). Persistent activation of SOCE induced centrosome overduplication, aberrant multipolar division, chromosome aneuploidy, anchorage-independent growth, and xenograft tumorigenesis in hepatocytes expressing preS2- LHBS. Chemical inhibitions of SOCE machinery and silencing of STIM1 significantly reduced centrosome numbers, multipolar division, and xenograft tumorigenesis induced by preS2-LHBS. These results provide the first mechanistic link between calcium homeostasis and chromosome instability in hepatocytes carrying preS2-LHBS. Therefore, persistent activation of SOCE represents a novel pathological mechanism in HBV-mediated hepatocarcinogenesis.

PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis.

PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH(-/-) cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH(-/-) cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.

Sgo1 is a potential therapeutic target for hepatocellular carcinoma.

Shugoshin-like protein 1 (Sgo1) is an essential protein in mitosis; it protects sister chromatid cohesion and thereby ensures the fidelity of chromosome separation. We found that the expression of Sgo1 mRNA was relatively low in normal tissues, but was upregulated in 82% of hepatocellular carcinoma (HCC), and correlated with elevated alpha-fetoprotein and early disease onset of HCC. The depletion of Sgo1 reduced cell viability of hepatoma cell lines including HuH7, HepG2, Hep3B, and HepaRG. Using time-lapse microscopy, we showed that hepatoma cells were delayed and ultimately die in mitosis in the absence of Sgo1. In contrast, cell viability and mitotic progression of immortalized cells were not significantly affected. Notably, mitotic cell death induced upon Sgo1 depletion was suppressed upon inhibitions of cyclin-dependent kinase-1 and Aurora kinase-B, or the depletion of mitotic arrest deficient-2. Thus, mitotic cell death induced upon Sgo1 depletion in hepatoma cells is mediated by persistent activation of the spindle assembly checkpoint. Together, these results highlight the essential role of Sgo1 in the maintenance of a proper mitotic progression in hepatoma cells and suggest that Sgo1 is a promising oncotarget for HCC.

SH2B1 and IRSp53 proteins promote the formation of dendrites and dendritic branches.

SH2B1 is an adaptor protein known to enhance neurite outgrowth. In this study, we provide evidence suggesting that the SH2B1 level is increased during in vitro culture of hippocampal neurons, and the ß isoform (SH2B1ß) is the predominant isoform. The fact that formation of filopodia is prerequisite for neurite initiation suggests that SH2B1 may regulate filopodium formation and thus neurite initiation. To investigate whether SH2B1 may regulate filopodium formation, the effect of SH2B1 and a membrane and actin regulator, IRSp53 (insulin receptor tyrosine kinase substrate p53), is investigated. Overexpressing both SH2B1ß and IRSp53 significantly enhances filopodium formation, neurite outgrowth, and branching. Both in vivo and in vitro data show that SH2B1 interacts with IRSp53 in hippocampal neurons. This interaction depends on the N-terminal proline-rich domains of SH2B1. In addition, SH2B1 and IRSp53 co-localize at the plasma membrane, and their levels increase in the Triton X-100-insoluble fraction of developing neurons. These findings suggest that SH2B1-IRSp53 complexes promote the formation of filopodia, neurite initiation, and neuronal branching.

[Effects of promoting blood circulation to remove meridian obstruction combined with medicinal guides on related osteogenesis factors in rabbits with femoral head necrosis].

OBJECTIVE: To observe effects of blood circulation promoting compounds combined with medicinal guides on content of bone glaprotein (BGP), bone morphogenetic protein-2 (BPM-2) and expression of BMP-2 mRNA in rabbits with femoral head necrosis, and explore its mechanism. METHODS: Ninety-eight healthy Spragur-Dawley male rabbits were collected and weighted 2.2 to 2.8 kg. Eighty-four rabbits were built femoral head necrosis model by freezing left femoral head in liquid nitrogen, then randomly divided into 6 groups, 14 in each group. The 6 groups included model group,promoting blood circulation to remove meridian obstruction group,promoting blood circulation to remove meridian obstruction combined with achyranthes bidentata group,radix angelicae pubescentis, asarum group, and platycodon grandiflorum group,other 14 rabbits were sham operation group. While drug groups were administrated corresponding Chinese herb after molding,model group and shamp operation group were given saline. Recombinant human granulocyte-colony stimulating factor ( 30 microg x kg(-1) x d(-1))were injected into all rabbits for 7 days. Samples were taken on the second and fourth week,the content of BGP and BMP-2 were detected by enzyme-linked immunosorbent assay (ELSA) and radioimmunoassay (RIA), histopathological changes of left femoral head were observed by Hematoxylin and Eeosin staining (HE), and expression of BMP-2 mRNA were tested by fluorescence in situ hybridization. RESULTS: Compared with sham operation group, the rate of empty lacunae femoral head were obviously increased in model group, and the content of BGP were increased on the second week, and BMP-2 and BMP-2 mRNA were decreased on the fourth week. Compared with model group, the content of BGP, BMP-2 and BMP-2 mRNA were higher both of the second and fourth week in promoting blood circulation to remove meridian obstruction group. The rate of empty la- cunae femoral head were lower in achyranthes bidentata group, BGP, BMP-2 and BMP-2 mRNA were higher on the fourth week. The rate of empty lacunae femoral head were lower in platycodon grandiflorum group, and BGP were decreased on the second and fourth week, BMP-2 were lower on the second week ,while BMP-2 mRNA were decreased on the fourth week; the content of BMP-2 and BMP-2 mRNA were increased in radix angelicae pubescentis group on the second week; while there was no change in asarum group. CONCLUSION: Radix angelicae pubescentis can increase the content of BGP, BMP-2 and expression of BMP-2 mRNA ,which is an effective mechanism of preventing femoral head necrosis.

[Effect of different channel ushering drugs on homing of bone marrow stem cells of femoral head necrosis model rabbits].

OBJECTIVE: To observe the effect of Huogu II formula compatible with different channel ushering drugs on the homing of bone marrow stem cells of osteonecrosis of the femoral head (ONFH) induced by liquid nitrogen freezing in rabbits and discuss the mechanism for preventing and treating ONFH. METHOD: The ONFH model was established by liquid nitrogen freezing of 84 rabbits. They were randomly assigned to the model group and the Huogu II formula group and groups of Huogu II formula combining with Achyranthis Bidentatae Radix, Asari Radix et Rhizoma, Angelicae Pubescentis Radix, Platycodonis Radix. The remaining 14 rabbits were sham-operated. During the course of ONFH modeling, all of the rabbits were subcutaneously injected with recombinant human granulocyte colony-stimulating factor (rhG-CSF)(30 microg x kg(-1) x d(-1), for consecutively 7 days). Meanwhile, normal saline and decoction of the formulae were orally administrated respectively. WBC was counted in peripheral blood before and after the injection of rhG-CSF. HE stainings at the 2nd and the 4th weeks after the modeling were adopted to observe histopathological changes, vascular morphology was observed by ink perfusion, BrdU and SDF-1 were determined by immunohistochemical assay in femoral heads of the left hind leg. RESULT: Compared with the sham-operated group, the Huogu II formula group showed decrease in the ratio of empty lacuna and increase in vessel area, number of BrdU positive cells and SDF-1 level. In comparison with the model group, the Achyranthis Bidentatae Radix group displayed decreasing empty lacuna ratio and increasing vessel area at the 4th week and increasing SDF-1 at the 2nd week; the Platycodonis Radix group revealed a notably increasing empty lacuna ratio and a sharp decrease in the number of BrdU positive cells at 4th week; Asari Radix et Rhizoma and Angelicae Dubescentis Radix groups showed no remarkable change. CONCLUSION: Huogu II formula can promote the directional homing of bone marrow stem cell to the necrosis area. Channel ushering drug achyranthes can further boost above effects of Huogu II formula.

Effect of Huogu II Formula (II) with medicinal guide Radix Achyranthis Bidentatae on bone marrow stem cells directional homing to necrosis area after osteonecrosis of the femoral head in rabbit.

OBJECTIVE: To investigate the effect of Huogu II Formula (II) with medicinal guide Radix Achyranthis Bidentatae (Ach) on bone marrow stem cells (BMSCs) homing to necrosis area after osteonecrosis of the femoral head (ONFH) frozen by liquid nitrogen in rabbit as well as to explore the mechanism of prevention and treatment for ONFH. METHODS: The animal model of ONFH was established by liquid nitrogen frozen on the rabbit left hind leg. Forty-eight Japanese White rabbits were randomly assigned to sham-operated group, model group, Huogu II group, and Huogu II plus Ach group, with 12 rabbits in each. During the course of ONFH animal model establishment, all rabbits were subcutaneously injected with recombinant human granulocyte colony-stimulating factor [rhG-CSF, 30 µg/(kg·day) for continuous 7 days]. Meanwhile, normal saline and decoction of the two formulae were administrated by gavage, respectively. White blood cells (WBC) were counted in peripheral blood before and after injection of rhG-CSF. Materials were drawn on the 2nd and 4th weeks after model built; bone glutamine protein (BGP) and bone morphogenetic protein 2 (BMP2) levels in serum were tested. Histopathologic changes were observed by hematoxylin and eosin (HE) staining. BMP2 mRNA levels were detected with in situ hybridization (ISH) staining. 5-Bromo-2'-deoxyuridine (BrdU) and stromal cell derived factor 1 (SDF-1) were measured by immunohistochemical assay in femoral head of the left hind leg. RESULTS: Compared with the shamoperated group, the ratio of empty lacuna, serum BGP, and SDF-1 level in the model group increased significantly, and BMP2 in both serum and femoral head decreased significantly. However, in comparison with the model group, the empty lacuna ratio of Huogu II group and Huogu II plus Ach group decreased obviously in addition to the levels of serum BGP and BMP2, and the expressions of BMP2 mRNA, BrdU, and SDF-1 increased significantly. Above changes were particularly obvious in Huogu II plus Ach group. BGP and SDF-1 on the 2nd week and empty lacuna rate and serum BMP2 level on the 4th week in Huogu II group significantly exceeded their counterparts. On the 2nd week, only in Huogu II plus Ach group that the BrdU counting rose significantly. On the 4th week, empty lacuna rate and serum BMP2 level in Huogu II plus Ach group exceeded those in Huogu II group distinctively. CONCLUSIONS: To a certain extent, the medicinal guide Ach improves the preventive and therapeutic effects of Huogu II Formula on experimental ONFH model. The possible mechanism of this is related to its promoting effect on directional homing of BMSCs to the necrosis area.