Analysis of tumor microenvironment alterations in partially responsive rectal cancer patients treated with neoadjuvant chemoradiotherapy.

PURPOSE: Achieving a pathologic complete response (pCR) after neoadjuvant chemoradiotherapy (NCRT) remains a challenge for most patients with rectal cancer. Exploring the potential of combining NCRT with immunotherapy or targeted therapy for those achieving a partial response (PR) offers a promising avenue to enhance treatment efficacy. This study investigated the impact of NCRT on the tumor microenvironment in locally advanced rectal cancer (LARC) patients who exhibited a PR. METHODS: This was a retrospective, observational study. Five patients demonstrating a PR after neoadjuvant treatment for LARC were enrolled in the study. Biopsy samples before treatment and resected specimens after treatment were stained with a panel of 26 antibodies targeting various immune and tumor-related markers, each labeled with distinct metal tags. The labeled samples were then analyzed using the Hyperion imaging system. RESULTS: Heterogeneity within the tumor microenvironment was observed both before and after NCRT. Notably, tumor-associated macrophages, CD4 + T cells, CD8 + T cells, CD56 + natural killer cells, tumor-associated neutrophils, cytokeratin, and E-cadherin exhibited slight increase in abundance within the tumor microenvironment following treatment (change ratios = 0.78, 0.2, 0.27, 0.32, 0.17, 0.46, 0.32, respectively). Conversely, the number of CD14 + monocytes, CD19 + B cells, CD45 + CD4 + T cells, collagen I, α-smooth muscle actin, vimentin, and ß-catenin proteins displayed significant decreases post-treatment (change ratios = 1.73, 1.92, 1.52, 1.25, 1.52, 1.12, 2.66, respectively). Meanwhile, Foxp3 + regulatory cells demonstrated no significant change (change ratio = 0.001). CONCLUSIONS: NCRT has diverse effects on various components of the tumor microenvironment in LARC patients who achieve a PR after treatment. Leveraging combination therapies may optimize treatment outcomes in this patient population.

Busulfan for Allogeneic Hematopoietic Stem Cell Transplantation in Children with Severe Aplastic Anemia: A Retrospective Study.

INTRODUCTION: This retrospective study aimed to compare a range of conditioning regimens in children with severe aplastic anemia (SAA) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) at the Seventh Medical Center of PLA General Hospital between January 2008 and June 2017. METHODS: Patients were categorized into the Bu (Bu + Flu + Cy + ATG-F regimen) and control (Flu + Cy + ATG-F) groups, with a median follow-up time after HSCT of 3.5 (range, 3.1-6.2) and 3.7 (3.2-5.9) years in the Bu and control groups, respectively. RESULTS: No differences were observed between the two groups regarding the median time of peripheral blood neutrophil and platelet engraftment (p = 0.538 and p = 0.491); the 28-day engraftment rates of neutrophils were similar (p = 0.199), although higher for platelets with Bu (p = 0.044). Additionally, graft failure was 0% and 20.0% in the Bu and control groups, respectively (p = 0.004). In both groups, the incidence of grades III-IV (or grades II-IV) acute graft-versus-host disease (GVHD) and chronic GVHD was not significantly different (p > 0.05). Moreover, the 3-year overall survival and failure-free survival did not show significant differences (p = 0.670 and p = 0.908). DISCUSSION: In children with SAA undergoing allo-HSCT, conditioning regimen with Bu + Flu + Cy + ATG-F is capable of enhancing the myeloablation effect, promoting donor hematopoietic stem cell engraftment, and reducing the graft failure rate. Furthermore, it does not increase the incidence of complications, including GVHD.

Two new eunicellin diterpenoids from the East China Sea gorgonian Muricella sp.

A phytochemical investigation on gorgonian Muricella sp. from East China Sea resulted in the isolation of eight eunicellin diterpenoids including two new ones, muricellins A-B (1, 2). Chemical structures of these compounds were elucidated by spectroscopic techniques (1D and 2D NMR and MS) and by comparison with data reported in the literature. Anti-rheumatoid arthritis activities of 1, 3, 4, and 6 have been evaluated.

Jaspiferin G, a new isomalabaricane-type triterpenoid from the sponge Jaspis stellifera.

A new isomalabaricane-type triterpenoid, jaspiferin G (1), together with four known isomalabaricane-type triterpenoids, isogeoditin A (2), 13-(E)-isogeoditin A (3), jaspolide E (4), and 22,23-dihydrostellettin D (5), was isolated from the marine sponge Jaspis stellifera. The structures were elucidated on the basis of spectroscopic data ((1)H and (13)C NMR, (1)H(-1)H COSY, HSQC, HMBC, and ROESY) and mass spectrometry.

Involvement of periostin-sclerostin-Wnt/ß-catenin signaling pathway in the prevention of neurectomy-induced bone loss by naringin.

Periostin has an essential role in mechanotransduction in bone. Naringin, a natural flavonoid, has been evidenced for its osteoprotective role in osteoporosis, while its mechanism is far from clear. Here we show that down-regulation of periostin, and up-regulation of its downstream sclerostin and inactivation of Wnt/ß-catenin signaling were implicated in neurectomy-induced bone loss. Naringin could up-regulate periostin and prevent neurectomy-induced deterioration of BMD, trabecular microstructure and bone mechanical characteristics. In conclusion, naringin could prevent progress of disuse osteoporosis in rats, which may be mediated by increased periostin expression and subsequently inhibition of sclerostin and activation of Wnt/ß-catenin signaling pathways.

Three new polyunsaturated lipids from a Guangxi marine sponge Haliclona sp.

Three new polyunsaturated lipids, (6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraen-3-one (1), (6Z,9Z,12Z,15Z)-1-bromooctadeca-6,9,12,15-tetraen-3-one (2), and (Z)-ethyl docos-5-enoate (3), together with two known polyunsaturated lipids, 4(Z),7(Z),10(Z)-tridecatrienoic acid (4) and (6Z,9Z,12Z,15Z)-octadeca-1,6,9,12,15-pentaen-3-one (5), were isolated from the marine sponge Haliclona sp., which was collected from Guangxi, using HSCCC and HPLC methods. Chemical structures of the five compounds were elucidated by spectroscopic techniques.

Jaspiferins C-F, four new isomalabaricane-type triterpenoids from the South China Sea sponge Jaspisstellifera.

A chemical investigation of marine sponge Jaspis stellifera, collected from the South China Sea, led to the isolation of four new isomalabaricane-type triterpenoids, jaspiferins C-F (1-4). The structures of those compounds were elucidated by extensive spectroscopic methods. Jaspiferin C (1), which has the six-membered carbon ring at the side chain, was discovered for the first time from the isomalabaricane-type triterpenoids. The hypothesis of a biogenetic pathway to generate jaspiferin C (1) was depicted.

Novel polyhydroxylated steroids from the East China Sea gorgonian Echinogorgia sassapo reticulata with suppressive activity of leukotriene C4 generation and degranulation in bone marrow-derived mast cells.

The gorgonian Echinogorgia sassapo reticulata contains two new bioactive polyhydroxylated steroids, sassapols A (1), B (2), and five related known compounds (3-7). Compound 6 has been encountered for the first time in natural sources. The structures of these new compounds were defined by spectroscopic analysis. All the compounds (1-7) isolated from E. sassapo reticulata were tested for anti-inflammatory activity. Compounds 1, 3, 5, and 7 inhibited both the generation of leukotriene C4 and the degranulation reaction in mouse bone marrow-derived mast cells.

Lower peripheral blood CD4+ lymphocyte ratio is associated with severe bronchopulmonary dysplasia.

OBJECTIVE: To elucidate the characteristics of lymphocyte subsets in bronchopulmonary dysplasia (BPD) diagnosis following Jensen's criterion to understand the spectrum of lymphocytes in different degrees of BPD. STUDY DESIGN: This single-center retrospective cohort study included 120 neonates admitted to the neonatal intensive care unit between 1 July 2014 and 30 June 2021, who had undergone peripheral blood lymphocyte subpopulation detection. RESULTS: Thirty-one neonates were included in the control group, whereas 33 infants with BPD were included in the case group. In addition, we selected 56 infants with a gestational age (GA) <37 weeks without BPD who were receiving oxygen therapy. Among the three groups, the B cell and NK cell frequencies were significantly higher and the frequencies of T cells and CD4+ cells were significantly lower in the BPD group. In newborns without BPD, the distribution of T lymphocyte subsets was similar at different GAs. Comparing different degrees of BPD, the patients in the grades 2-3 BPD group had significantly lower percentages of T lymphocytes and CD4+ T cells than those in the other groups. Remarkably, the frequencies of NK cells were significantly higher in patients with grades 2-3 BPD, and the Treg cells slightly increased with BPD severity, although the differences were not significant. CONCLUSION: Healthy neonates had similar ratios of lymphocyte subsets among different GAs; although as the GAs increased, the percentage of lymphocytes increased slightly. Severe BPD was associated with lower CD4+ T cells and higher NK cells. However, whether such changes were the cause or the consequence of BPD has not been determined.

Lentivirus-mediated short hairpin RNA interference of CENPK inhibits growth of colorectal cancer cells with overexpression of Cullin 4A.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative. Immunohistochemical analysis has revealed high expression of centromere protein K (CENPK) in CRC. However, the role of CENPK in the progression of CRC is not well characterized. AIM: To evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A (CUL4A) in RKO and HCT116 cells. METHODS: Human colon cancer samples were collected and tested using a human gene expression chip. We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis. In vitro experiments verified the function of this gene. We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction (qPCR), western blot, and flow cytometry. The effect of short hairpin RNA (shRNA) virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging. To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells, we performed a series of in vitro experiments, using qPCR, western blot, MTT assay, and flow cytometry. RESULTS: We demonstrated overexpression of CENPK in human colon cancer samples. CENPK was an independent risk factor in patients with CRC. The downstream genes FBX32, CUL4A, and Yes-associated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells. Significantly delayed xenograft tumor emergence, slower growth rate, and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group. The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo. The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells, with overexpression of the CUL4A. CONCLUSION: We indicated a potential role of CENPK in promoting tumor proliferation, and it may be a novel diagnostic and prognostic biomarker for CRC.

Analysis of the Efficacy and Pharmacological Mechanisms of Action of Zhenren Yangzang Decoction on Ulcerative Colitis Using Meta-Analysis and Network Pharmacology.

OBJECTIVE: We analyzed the efficacy and pharmacological mechanisms of action of Zhen Ren Yang Zang decoction (ZRYZD) on ulcerative colitis (UC) using meta-analysis and network pharmacology. METHODS: The major databases were searched for randomized controlled trials of ZRYZD for the treatment of UC. Meta-analysis of the efficacy of ZRYZD on UC was conducted using RevMan software. Active compounds and target genes were acquired using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. UC-related genes were searched using the GeneCards database. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using RGUI. A compound-target network was constructed using Cytoscape software, and a protein-protein interaction network was constructed using the STRING database. Molecular docking simulations of the macromolecular protein targets and their corresponding ligand compounds were performed using the AutoDock tool and AutoDock Vina software. RESULTS: Meta-analysis revealed that the total effective rate and recovery rate of clinical efficacy were significantly higher in the experimental group than those of the control group. The screening identified 169 active compounds and 277 active target genes for ZRYZD. The 277 active target genes were compared with the 4,798 UC-related genes. This identified 187 active target genes of ZRYZD for UC that correlated with 138 active compounds. GO functional enrichment and KEGG pathway enrichment analyses were performed, and compound-target and protein-protein interaction networks were constructed. The key compounds and key target proteins were then selected. Finally, target protein binding with the corresponding compound was analyzed using molecular docking. CONCLUSION: Our findings demonstrate the effectiveness and safety of ZRYZD for the treatment of UC and provide insight into the underlying pharmacological mechanisms of action. Furthermore, key compounds were identified, laying the foundation for future studies on ZRYZD for the treatment of UC.

Surgical Treatment of Lateral Tibial Plateau Fractures Involving the Posterolateral Column.

OBJECTIVE: To summarize the indications and the clinical effects of a transfibular neck osteotomy approach and a combined anterolateral and posterolateral approach in the treatment of fractures of the lateral tibial plateau involving the posterolateral column. METHODS: Eleven patients with lateral tibial plateau fractures were included in the present study. The fractures were Schatzker type II or lateral platform fractures involving posterolateral column. The anterolateral combined posterolateral approach (lateral + posterolateral locking plate fixation) was applied in 7 patients and 4 patients underwent transfibular neck osteotomy (lateral + posterolateral locking plate fixation + 1/4 tubular plate edge fixation, fibular osteotomy with Kirschner wire tension band fixation, and hollow nail fixation for upper tibiofibular joint). All cases were followed up for 12-24 months, with an average follow-up of 17.5 ± 5.0 months. At the last followup, the Rasmussen radiological criteria were used to evaluate the effect of fracture reduction and fixation. The knee joint function was evaluated using the knee function evaluation criteria of the Hospital for Special Surgery (HSS). The Lachman test and the pivot-shift test were used to evaluate the anterior and posterior and rotational stability of the knee joint. The range of knee motion was recorded. RESULTS: Bone healing was achieved in all patients with fractures treated with a transfibular neck osteotomy approach and a combined anterolateral and posterolateral approach. At the last follow-up, both the Lachman test and the pivot-shift test results were negative. All patients had complete knee extension. For the combined anterolateral and posterolateral approach, the knee flexion angle was 110°-130°, with an average of 122.86° ± 7.56°. For the transfibular neck osteotomy approach, the knee flexion angle was 115°-130°, with an average of 120.00° ± 7.07°. For the patients in which the combined anterolateral and posterolateral approach was used, the Rasmussen score was 12-18 points, with an average of 16.00 ± 2.56 points. The results were excellent in 4 cases and good in 3 cases; therefore, 100% of results were excellent or good. For patients in which the transfibular neck osteotomy approach was used, the Rasmussen score was 10-18 points, with an average of 15.25 ± 3.77 points. The results were excellent in 2 cases, good in 1 case, and acceptable in 1 case; therefore, 75% of results were excellent or good. The HSS score for the combined anterolateral and posterolateral approach was 76-98 points, with an average of 88.43 ± 7.55 points. The results were excellent in 5 cases and good in 2 cases; therefore, 100% of results were excellent or good. The HSS score for the transfibular neck osteotomy approach was 74-96 points, with an average of 87.25 ± 9.43 points. The results were excellent in 3 cases and good in 1 case; therefore, 100% of results were excellent or good. There were no significant differences in operation time, surgical blood loss, fracture healing time, postoperative imaging score, and knee function evaluation between the two approaches. One patient who underwent transfibular neck osteotomy had a 3-mm step that gradually appeared, but no significant abnormalities were found in the width of the platform and the lower limb force line. One patient in whom the combined anterolateral and posterolateral approach was used showed numbness in the common peroneal nerve. No common peroneal nerve injury occurred through the transfibular neck osteotomy approach. CONCLUSIONS: The anterolateral combined posterolateral approach and the transfibular neck osteotomy approach are effective in the surgical treatment of lateral tibial plateau fractures involving the posterolateral column. However, the transfibular neck osteotomy approach is more suitable for the posterolateral plateau articular surface damaged with bone separation and displacement, deep collapse, cases involving a large range of the posterolateral column, especially fractures of the lateral tibial plateau in the upper tibiofibular syndesmosis area of the line connecting the anterior and posterior margin of the fibular head to the midpoint of the plateau.

[Effects of Dioscorea septemloba on the biomechanical and bone histomorphometric parameters of femur in the ovariectomized rats].

OBJECTIVE: To investigate the effects of the decoction of Rhizoma Dioscoreae septemlobae (RD) on the biomechanical and bone histomorphometric parameters of femur in the ovariectomized rats. METHODS: 25 female Wistar rats without pregnancy and deliver were divided into 5 groups randomly: sham (sham-operation), ovariectomy (OVX), OVX + high dosage RD (4 g/kg x d), OVX + middle dosage RD (2 g/kg x d) and OVX + low dosage RD (1 g/kg x d), n = 5 in every group. After intragastric administration 12-week period continuously, the biomechanical and the bone histomorphometric parameters of the femur of the rats in every group were determined, respectively, including percentage of trabecular bone volume (TBV%), percentage of trabecular osteoid (TOS%), mean osteoid width (MOSW), percentage of mineralizing surface (MdS%), percentage of double mineralizing surface (DMds%), percentage of DMds/MdS (%), percentage of travecular formation surface (TFS%), percentage of trabecular resorption surface (TRS%), mineral apposition rate (MAR), mineralizing lag time (MLT). RESULTS: The maximum loading, deflection, the maximum strain of the femur in the OVX group was 125.78 +/- 15.48 (N), 1.87 +/- 0.22 (mm), 9.34 +/- 1.10 (%), it was significantly lower than that in the sham group ( P< 0.05, P < 0.01), respectively. The maximum loading and maximum stress was increased in different extent in the every dose group of OVX + RD, respectively. TBV% of femur was significantily lower in the OVX group than that in the sham group (P < 0.01). The MdS%, DMds%, DMds/MdS (%), TOS%, MOSW, TRS%, MAR was significantly higher in the OVX group than that in the sham group, respectively (P < 0.01). In the RA high and middle dosage group, the TBV% of femur was significantly higher than that in the model group (P < 0.05), and the MdS%, DMds%, DMds/MdS (%), TRS%, TOS%, MOSW, MAR was significantly lower than that in the model group, respectively (P < 0.05), and MLT was decurtated slightly (P > 0.05). CONCLUSION: The decoction of RD can decline the bone turnover and the loss of bone of femur in the ovariectomized rats.

Elevated peripheral blood B lymphocytes and CD3+CD4-CD8- T lymphocytes in patients with non-small cell lung cancer: A preliminary study on peripheral immune profile.

It has been established that the tumor microenvironment (TME) has a crucial role in enabling tumors to evade from host immune responses. Previous studies demonstrated that tumor cells are not only able to reshape immune milieu at the tumor site, but also exert systemic effects, which has been demonstrated to be important for metastasis. At present, how the peripheral immune environment change in the tumor-bearing host is unclear. The present study identified a number of changes in the proportions of lymphocyte subpopulations and the levels of cytokines in patients with NSCLC, which may provide a preliminary profile of the immune environment in the peripheral blood of patients harboring a tumor. These findings expand on the present knowledge on how tumors can alter the immune system to benefit its growth and metastasis, which may provide a potential novel strategy for immunotherapy.

Overexpression of DNMT1 leads to hypermethylation of H19 promoter and inhibition of Erk signaling pathway in disuse osteoporosis.

Disuse osteoporosis (DOP) is a common complication of the lack of mechanical loading. The precise mechanism underlying DOP remains unknown, although epigenetic modifications may be a major cause. Recently, cumulative research has revealed that DNA methyltransferase (DNMT) proteins can catalyze the conversion of cytosine to 5-methylcytosine (5mC), altering the epigenetic state of DNA. Here, we report that DNMT1 expression and lncRNA-H19 methylation are upregulated in the femoral tissues of DOP rats, accompanied with inhibited Erk signaling pathway. Overexpression of DNMT1 in UMR-106 cells mimics 5mC enrichment in the H19 promoter, inhibition of Erk signaling and impairment of osteogenesis, which can be rescued by 5'-aza-deoxycytidine (5'-Aza) treatment. Moreover, local intramedullary injection of Dnmt1 siRNA (siDNMT1) in Sprague-Dawley (SD) rats abrogated disuse lncRNA-H19 (H19) downregulation, Erk signaling inhibition, histopathological changes, and bone microstructure declines in the distal femur in vivo. Therefore, our data identify for the first time a new signaling cascade in DOP: mechanical unloading causes upregulation of DNMT1 and hypermethylation of H19 promoter, which subsequently leads to downregulation of lncRNA-H19 and inhibition of the ERK signaling, suggesting a new potential therapeutic target.

[Effects of Dioscorea septemloba on bone metabolism in ovariectomized rats].

OBJECTIVE: To investigate the effects of the decoction of Rhizoma Dioscorea septemlobae (RD) on the bone metabolism in ovariectomized rats. METHOD: Thirty female, 3-month-old Wistar rats without pregnancy and deliver were randomly divided into 6 groups: sham (sham-operation), ovariectomy (OVX), OVX + diethylstilbestrol, OVX + high dose RD (4 g x kg(-1) x d(-1)), OVX + middle dose RD (2 g x kg(-1) x d(-1)) and OVX + low dose RD (1 g x kg(-1) x d(-1)) (n = 5 in every group). After 12-week period of continuous treatment, the urinary samples and blood samples were collected for the determination of serum estrodiol (E2), calcium (Ca), phosphorus (P), bone glaprotein (BGP), alkaline phosphatase (ALP), urinary calcium/creatinine (Ca/Cr), phosphorus/ creatinine (P/Cr) and deoxypyridioline/creatinine (DPD/Cr). The uteri were removed and weighed. The bone mineral density (BMD) and the biomechanical parameters of the femur of the rats in every group were determined, respectively. RESULT: The coefficient of uteri in every dose group of OVX + RD was significantly higher than that in the OVX group (P < 0.01). The concentration of serum ALP, BGP and urinary DPD/Cr, Ca/Cr in the OVX group was significantly higher than that in the sham group (P < 0.05), respectively, However, that in the every dose of OVX + RD was lower than that in the OVX group, respectively. There was no significan difference in the concentration of serum Ca, P and urinary P/Cr in every group, respectively. The bone mineral density (BMD) in the OVX group was (0.032 +/- 0.007) g x cm(-2) and was significantly lower than that in the sham group (P < 0.01). However, the value in the group of every dose OVX + RD was significantly higher than that in the OVX group (P < 0.05, P < 0.01), respectively. The maximum loading, deflection and the maximum strain of the femur in the OVX group were (125.78 +/- 15.48) N, (1.87 +/- 0.22) mm, (9.34 +/- 1.10) % and were significantly lower than those in the sham group (P < 0.05, P < 0.01), respectively. The maximum loading and maximum stress were increased in different extent in the every dose group of OVX + RD, respectively. CONCLUSION: The decoction of RD can inhibit bone absorption, decline bone turnover and improve the loss of bone in ovariectomized rats.

[Determination of flavor compounds in foxtail millet wine by gas chromatography-mass spectrometry coupled with headspace solid phase microextraction].

To comprehensively understand flavor compounds and aroma characteristics of foxtail millet wine, extraction conditions were optimized with 85 µm polyacrylate (PA), 100 µm polydimethylsiloxane (PDMS), 75 µm carboxen (CAR)/PDMS and 50/30 µm divinylbenzene (DVB)/CAR/PDMS fibers. The flavor compounds in foxtail millet wine were investigated by gas chromatography-mass spectrometry (GC-MS) coupled with headspace solid phase microextraction (HS-SPME), and the odor characteristics and intensity were analyzed by odor active values (OAVs). The samples of 8 mL were placed in headspace vials with 1.5 g NaCl, then the headspace vials were heated at 60℃ for 40 min. Using HS-SPME with different fibers, a total of 55 flavor compounds were identified from the samples, including alcohols, esters, benzene derivatives, hydrocarbons, acids, aldehydes, ketones, terpenes, phenols and heterocycle compounds. The main flavor compounds were alcohols compounds. According to their OAVs, phenylethyl alcohol, styrene, 1-methyl-naphthalene, 2-methyl-naphthalene, benzaldehyde, benzeneacetaldehyde and 2-methoxy-phenol were established to be odor-active compounds. Phenylethyl alcohol and benzeneacetaldehyde were the most prominent odor-active compounds. PA and PDMS fibers had good extraction effect for polar and nonpolar compounds, respectively. CAR/PDMS and DVB/CAR/PDMS provided a similar compounds profile for moderate polar compounds. This research comprehensively determined flavor compounds of foxtail millet wine, and provided theoretical basis for product development and quality control.

LncRNA-H19 Modulates Wnt/ß-catenin Signaling by Targeting Dkk4 in Hindlimb Unloaded Rat.

OBJECTIVE: To investigate the biological functions of long noncoding RNA-H19 (H19) in the pathogenesis of disuse osteoporosis (DOP). METHODS: Fifty-four male Sprague Dawley (SD) rats were randomly divided into three groups: baseline control (BC, 6), age-matched control (AC, 24), and hindlimb unloading (HLU, 24). The rats in the BC group were sacrificed at the beginning of the experiment, while the AC and HLU rats were sacrificed at different times (7, 14, 21 and 28 days after HLU). The DOP model was verified by micro-CT scan, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify the expression of osteogenic genes (OPG, RunX2 and OPG). Gene sequencing and bioinformatic analysis were performed to find H19 target genes and the associated signaling pathway, which were first verified on tissue samples. Further verification was performed by knocking down the H19 and related gene in rat osteoblast cell line (UMR106 cell). Then, the changes of associated signaling pathway and osteogenic function were examined to confirm the prediction of the bioinformatic analysis. RESULTS: Micro-CT scans and quantitative real-time polymerase chain reaction (qRT-PCR) tests showed progressively deteriorated trabecular bone and decreased level of osteogenic genes in the metaphysis of distal femur during HLU, indicating the successful establishment of a DOP model. According to RNA sequencing, 1351 mRNA and 464 lncRNA were abnormally expressed in response to mechanical unloading, in which the H19 decreased 2.86 fold in HLU rats. There were 1426 mRNA predicted to be the target genes of H19, and KEGG pathway analysis suggested that Wnt signaling pathway (Wnt signaling) was the top pathway responsible for these target genes. In the Wnt-associated genes targeted by H19, 11 were differentially expressed between HLU and AC rats, among which Dkk4 increased 2.44 fold in HLU rats when compared to normal controls. These results of sequencing and bioinformatic analysis were confirmed by the low expression of H19, overexpression of Dkk4 and inhibited Wnt signaling observed in DOP rats. Subsequent in vitro cell assay further demonstrated that knockdown of H19 led to upregulation of Dkk4, and inhibition of Wnt signaling and osteogenic function in UMR106 cell. These effects can be greatly reversed after application of knocking down Dkk4. CONCLUSION: Our findings demonstrated that low expression of H19, induced by mechanical unloading, leads to development of DOP through inhibition of Wnt signaling by promoting Dkk4 expression.