Therapeutic importance and diagnostic function of circRNAs in urological cancers: from metastasis to drug resistance.

Circular RNAs (circRNAs) are a member of non-coding RNAs with no ability in encoding proteins and their aberrant dysregulation is observed in cancers. Their closed-loop structure has increased their stability, and they are reliable biomarkers for cancer diagnosis. Urological cancers have been responsible for high mortality and morbidity worldwide, and developing new strategies in their treatment, especially based on gene therapy, is of importance since these malignant diseases do not respond to conventional therapies. In the current review, three important aims are followed. At the first step, the role of circRNAs in increasing or decreasing the progression of urological cancers is discussed, and the double-edged sword function of them is also highlighted. At the second step, the interaction of circRNAs with molecular targets responsible for urological cancer progression is discussed, and their impact on molecular processes such as apoptosis, autophagy, EMT, and MMPs is highlighted. Finally, the use of circRNAs as biomarkers in the diagnosis and prognosis of urological cancer patients is discussed to translate current findings in the clinic for better treatment of patients. Furthermore, since circRNAs can be transferred to tumor via exosomes and the interactions in tumor microenvironment provided by exosomes such as between macrophages and cancer cells is of importance in cancer progression, a separate section has been devoted to the role of exosomal circRNAs in urological tumors.

Genome-wide CNV analysis reveals candidate genes associated with milk production traits in water buffalo (Bubalus bubalis).

Buffaloes are vital contributors to the global dairy industry. Understanding the genetic basis of milk production traits in buffalo populations is essential for breeding programs and improving productivity. In this study, we conducted whole-genome resequencing on 387 buffalo genomes from 29 diverse Asian breeds, including 132 river buffaloes, 129 swamp buffaloes, and 126 crossbred buffaloes. We identified 36,548 copy number variant (CNVs) spanning 133.29 Mb of the buffalo genome, resulting in 2,100 copy number variant regions (CNVRs), with 1,993 shared CNVRs being found within the studied buffalo types. Analyzing CNVRs highlighted distinct genetic differentiation between river and swamp buffalo subspecies, verified by evolutionary tree and principal component analyses. Admixture analysis grouped buffaloes into river and swamp categories, with crossbred buffaloes displaying mixed ancestry. To identify candidate genes associated with milk production traits, we employed 3 approaches. First, we used Vst-based population differentiation, revealing 11 genes within CNVRs that exhibited significant divergence between different buffalo breeds, including genes linked to milk production traits. Second, expression quantitative loci (eQTL) analysis revealed differential expression of CNVR-driven genes (DECGs) associated with milk production traits. Notably, known milk production-related genes were among these DECGs, validating their relevance. Last, a genome-wide association study (GWAS) identified 3 CNVRs significantly linked to peak milk yield. Our study provides comprehensive genomic insights into buffalo populations and identifies candidate genes associated with milk production traits. These findings facilitate genetic breeding programs aimed at increasing milk yield and improving quality in this economically important livestock species.

Structural diversity, hypothetical biosynthesis, chemical synthesis, and biological activity of Ganoderma meroterpenoids.

Covering: 2018 to 2022Meroterpenoids found in fungal species of the genus Ganoderma and known as Ganoderma meroterpenoids (GMs) are substances composed of a 1,2,4-trisubstituted benzene and a polyunsaturated side chain. These substances have attracted the attention of chemists and pharmacologists due to their diverse structures and significant bioactivity. In this review, we present the structures and possible biosynthesis of representative GMs newly found from 2018 to 2022, as well as chemical synthesis and biological activity of some interesting GMs. We propose for the first time a plausible biosynthetic pathway for GMs, which will certainly motivate further research on the biosynthetic pathway in Ganoderma species, as well as on chemical synthesis of GMs as important bioactive compounds for the purpose of drug development.

1H-Indazoles derivatives targeting PI3K/AKT/mTOR pathway: Synthesis, anti-tumor effect and molecular mechanism.

The PI3K/AKT/mTOR signaling pathway is one of the most common abnormal activation pathways in tumor cells, and has associated with multiple functions such as tumor cell growth, proliferation, migration, invasion, and tumor angiogenesis. Here, a series of 3-amino-1H-indazole derivatives were synthesized, and their antiproliferative activities against HT-29, MCF-7, A-549, HepG2 and HGC-27 cells were evaluated. Among them, W24 exhibited the broad-spectrum antiproliferative activity against four cancer cells with IC50 values of 0.43-3.88 µM. Mechanism studies revealed that W24 inhibited proliferation by affecting the DNA synthesis, induced G2/M cell cycle arrest and apoptosis by regulating Cyclin B1, BAD and Bcl-xL, meanwhile induced the change of intracellular ROS and mitochondrial membrane potential in HGC-27 cells. Moreover, W24 inhibited the migration and invasion of HGC-27 cells by decreasing EMT pathway related proteins and reducing the mRNA expression levels of Snail, Slug and HIF-1α. Furthermore, W24 displayed low tissue toxicity profile and good pharmacokinetic properties in vivo. Therefore, 3-amino-1H-indazole derivatives might serve as a new scaffold for the development of PI3K/AKT/mTOR inhibitor and anti-gastric cancer reagent.

Dispersive liquid-liquid microextraction based on a supramolecular solvent followed by high-performance liquid chromatographic analysis of lignans in Forsythiae Fructus.

A supramolecular solvent-based dispersive liquid-liquid microextraction was proposed for the extraction and determination of lignans in Forsythiae Fructus combined with high-performance liquid chromatography. The supramolecular solvent, consisting of tetrabutylammonium bromide and n-hexanol, was mixed with the sample solution to extract the analytes by a vortex. After accomplishing the extraction, the extraction phase was separated by centrifugation and collected for high-performance liquid chromatography analysis. In this work, the important extraction variables such as the type and amount of extraction solvent, pH and salt amount in the sample phase, and extraction time were optimized. The synthesis of supramolecular solvent was studied and its microstructure was characterized by transmission electron microscopy. Under the optimal conditions, the analytes' enrichment factors were between 6 and 170 for the proposed procedure. Satisfactory linear ranges (r ≥ 0.99), detection limits (0.025-0.4 ng/ml), precisions (< 9.2%), and accuracies (recoveries: 96.5%-104.8%) were obtained. The method has been successfully applied to the preconcentration of lignans in Forsythiae Fructus with simple and rapid operation, low cost, and environmental friendliness.

Application of instantaneous nebulization dispersive liquid-phase microextraction combined with HPLC for the determination of chalcone and isoflavone in traditional Chinese medicines.

A simple and rapid instantaneous nebulization dispersive liquid-phase microextraction method was developed, and combined with high-performance liquid chromatography for determination of the contents of seven analytes in traditional Chinese medicines. In this study, using the sprinkler device to achieve instantaneous synchronous dispersion and extraction, only one spray can rapidly achieve the concentration and enrichment of seven kinds of chalcone and isoflavones. The key factors affecting the extraction efficiency were optimized including the type and volume of extractant, the pH and salt concentration of the sample phase, and the number of dispersion. Under the optimal conditions, the enrichment factor of the target analytes ranged from 103.1 to 180.9, with good linearity and correlation coefficients above 0.9970. The limits of detection ranged from 0.02 to 0.15 ng/mL, with good accuracy (recoveries 91.1 to 108.9%) and precision (relative standard deviations 1.5-7.1%). This method has short extraction time (2 s), low organic solvent consumption and high enrichment effect, so it has a wide application prospects.

Anterior bone loss: A common phenomenon which should be considered as bone remodeling process existed not only in patients underwent cervical disk replacement but also those with anterior cervical diskectomy and fusion.

OBJECTIVE: Anterior bone loss (ABL) was considered as a non-progressive process secondary only to motion-preserving implant and has been noticed recently in cervical disk replacement (CDR) let alone patients with anterior cervical diskectomy and fusion (ACDF). Our purpose is to reveal this unnoticed phenomenon in ACDF and further explore its clinical and radiological outcomes. METHODS: A total of 77 patients underwent ACDF with a minimum follow-up of at least one year were retrospectively reviewed. The average follow-up time was 22.51 ± 16.31 months. There were 50 patients in group A with ABL, while there were 27 patients in group B without ABL. ABL was measured and classified into four grades according to Kieser's methods. Clinical evaluation, radiological parameters and fusion rate were recorded. RESULTS: The incidence of bone ABL was 64.9% of Zero-P and 55.2% of endplates. The incidence of upper and lower endplates was 61% and 49%, respectively, and such difference was not significant. Mild ABL occurred in 22%, moderate ABL in 38% and severe ABL of 40% patients underwent ACDF with ABL. ABL would not affect both clinical outcomes and fusion rate. However, ABL would result in a higher incidence of subsidence. CONCLUSION: ABL should be considered as a common phenomenon that both CDR and ACDF owned a non-progressive process which confined in one year. ABL would result in a higher incidence of subsidence. Luckily, this phenomenon does not have an effect on postoperative clinical and fusion rate.

Nine New Pregnane Glycosides from the Cultivated Medicinal Plant Marsdenia tenacissima.

The ethnobotanical plant Marsdenia tenacissima has been used for hundreds of years for Dai people in Yunnan Province, China. Previously, chemical investigations on this plant have revealed that pregnane glycosides were the main biological constituents. Nine new pregnane glycosides, marsdeosides A-I (1-9), were isolated from cultivated dried stems of the medicinal plant Marsdenia tenacissima in this study. The structures were analyzed by extensive spectroscopic analysis, including 1D, 2D NMR, HRESIMS, and IR spectroscopic analysis. The absolute configurations of the sugar moieties were identified by comparing the Rf values and specific optical rotations with those of the commercially available standard samples and the data reported in the literature. Marsdeosides A (1) featured an unusual 8,14-seco-pregnane skeleton. Compounds 1, 8, and 9 showed activity against nitric oxide production in lipopolysaccharide-activated macrophage RAW264.7, with IC50 values of 37.5, 38.8, and 42.8 µM (L-NMMA was used as a positive control, IC50 39.3 µM), respectively. This study puts the knowledge of the chemical profile of the botanical plant M. tenacissima one step forward and, thereby, promotes the sustainable utilization of the resources of traditional folk medicinal plants.

[Anti-glioma mechanism of pterostilbene by regulating apoptosis and GSDME-mediated pyroptosis pathways: a study based on network pharmacology and experimental research].

This study aimed to explore the anti-glioma effect of natural compound pterostilbene(PTE) through regulating pyroptosis and apoptosis pathways, and to analyze the possible anti-glioma pathways and targets of PTE by network pharmacology and molecular docking. In this study, the action targets of PTE and the glioma targets were obtained by network pharmacology to construct a target network and a protein-protein interaction(PPI) network to predict the possible action targets of PTE against glioma. Molecular docking was performed on the core targets by AutoDock and the action pathways of PTE against glioma were predicted by enrichment analysis. In addition, the effect of PTE on the viability of U87MG and GL261 glioma cells was detected by CCK-8 assay. Clone formation assay and cell scratching assay were used to explore the effect of different concentrations of PTE on the proliferation and migration, respectively of glioma cells. Hoechst staining was used to observe PTE-induced apoptosis in glioma cells. The changes in mitochondrial membrane potential were detected by JC-1 staining. The pyroptosis-inducing effect of PTE on glioma cells was observed by inverted microscopy and lactate dehydrogenase(LDH) assay. Hoechst 33342/PI dual staining assay was performed to detect the integrity of glioma cell membranes. The expressions of pyroptosis and apoptosis-related proteins in glioma cells after PTE induction were determined by Western blot. In this study, 37 anti-glioma targets of PTE were obtained, and enrichment analysis suggested that PTE exerted anti-glioma effects through various signaling pathways including cancer pathway, proteoglycan in cancer, PI3K/AKT pathway, and apoptosis regulatory pathway. Molecular docking revealed that PTE had good binding activity with the main targets. Compared with the control group, PTE significantly reduced the viability as well as the proliferation, migration and adhesion abilities of U87MG and GL261 cells; it induced the apoptosis of the two glioma cells and the decrease of mitochondrial membrane potential in U87MG cells, and the effects increased with the increase of drug concentration. Compared with the conditions in the control group, glioma cells in the PTE group had increased pyroptosis-specific appearance and gradually increased LDH release; the number of PI positive cells was significantly elevated with the increase of PTE concentration as revealed by Hoechst 33342/PI staining; the expression levels of apoptosis-related factors cleaved PARP1 and B-cell lymphoma-2(Bcl-2) associated X(BAX) in the PTE group were markedly up-regulated, while the expression level of Bcl-2 was markedly down-regulated; the activation levels of pyroptosis-related proteins cleaved caspase-3 and gasdermin E-N(GSDME-N) had a remarkable rise in the PTE group, while no significant changes were found in the activation levels of gasdermin D-N(GSDMD-N) and cleaved caspase-1. In summary, PTE plays an anti-glioma role by inhibiting cell viability, proliferation, and migration and activating the caspase-3/GSDME-mediated pyroptosis pathway and mitochondrial apoptosis pathway.

Novel Insight into the Potential Role of Acylglycerophosphate Acyltransferases Family Members on Triacylglycerols Synthesis in Buffalo.

Acylglycerophosphate acyltransferases (AGPATs) are the rate-limiting enzymes for the de novo pathway of triacylglycerols (TAG) synthesis. Although AGPATs have been extensively explored by evolution, expression and functional studies, little is known on functional characterization of how many members of the AGPAT family are involved in TAG synthesis and their impact on the cell proliferation and apoptosis. Here, 13 AGPAT genes in buffalo were identified, of which 12 AGPAT gene pairs were orthologous between buffalo and cattle. Comparative transcriptomic analysis and real-time quantitative reverse transcription PCR (qRT-PCR) further showed that both AGPAT1 and AGPAT6 were highly expressed in milk samples of buffalo and cattle during lactation. Knockdown of AGPAT1 or AGPAT6 significantly decreased the TAG content of buffalo mammary epithelial cells (BuMECs) and bovine mammary epithelial cells (BoMECs) by regulating lipogenic gene expression (p < 0.05). Knockdown of AGPAT1 or AGPAT6 inhibited proliferation and apoptosis of BuMECs through the expression of marker genes associated with the proliferation and apoptosis (p < 0.05). Our data confirmed that both AGPAT1 and AGPAT6 could regulate TAG synthesis and growth of mammary epithelial cells in buffalo. These findings will have important implications for understanding the role of the AGPAT gene in buffalo milk performance.

[Cross-Sectional Study of Nutritional Status and Dietary Nutrient Intake in Children with Duchenne Muscular Dystrophy (DMD) in China].

Objective: To investigate the dietary nutrient intake and the nutritional status of children with Duchenne muscular dystrophy (DMD), and to explore the correlation between them, so as to provide theoretical basis for the formulation of proper nutritional treatment for children with DMD. Methods: A total of 223 children aged 2 to 14 years who came to West China Second University Hospital, Sichuan University from July 2017 to April 2021, and who were diagnosed with DMD by genetic testing were enrolled as the subjects of the study. Dietary assessment was conducted with a 3-day 24-hour dietary recall, and serum vitamin D level was measured by chemiluminescence method. Results: Only 33.2% of the children with DMD were found to be of normal nutritional status. The incidences of stunted growth, underweight, overweight and obesity were 13.5%, 14.4%, 14.3% and 8.1%, respectively. Among the children with DMD, those with serum vitamin D deficiency and insufficiency accounted for 9.0% and 89.7%, respectively. According to the dietary recall of the children with MDM, the daily energy ratio of carbohydrate, protein and fat were (47.40±6.64)%, (14.46±2.22)%, and (38.17±5.30)%, respectively. The daily intake of dietary calcium and vitamin D were (433.32±164.39) mg per day and (155.73±89.30) IU per day, respectively. The ratio of daily protein intake to the estimated average requirement for protein ( P=0.003) and ratio of daily energy intake to the estimated energy requirement ( P=0.007) were lower in children with stunted growth than those of DMD children of normal nutritional status. Conclusion: The dietary structure of children with DMD is obviously not suited to their condition and nutritional deficiency coexists with overnutrition among them. Further research needs to be done for developing appropriate nutritional guidance programs and standardized nutritional management measures for children with DMD.

Synthesis and bioevaluation of N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amines as tubulin polymerization inhibitors with anti-angiogenic effects.

A new series of N-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-amine derivatives as tubulin polymerization inhibitors were synthesized, and evaluated for the anti-proliferative activities. A structure-activity relationship study revealed that the free amino moiety of 1H-pyrazolo[3,4-b]pyridin-3-amine played an essential role in the anti-proliferative activities. Especially, compound 15c displayed the strongest anti-proliferation against MCF-7 cells with IC50 value of 0.067 ± 0.003 µM, and high selectivity over the normal human embryonic lung WI-38 cells with IC50 value of 23.41 ± 1.53 µM. Further mechanistic studies revealed that 15c showed strong anti-tubulin polymerization activity, changed the morphology of tubulin, and arrested the cell cycle at the G2/M transition in MCF-7 cells. Molecular docking analysis suggested that 15c well occupied the colchicine-binding pocket of tubulin. Additionally, 15c demonstrated anti-angiogenic activities with blocking the migration, invasion and tube formation, disrupting the newly formed tube, and regulating both MMP-9 and TIMP-1 in HUVEC cells. In summary, our results highlight that compound 15c is a potential antitumor compound that are worthy of further development.

FPR2-based anti-inflammatory and anti-lipogenesis activities of novel meroterpenoid dimers from Ganoderma.

Four pairs of novel meroterpenoid dimers, (±)-applandimeric acids A-D (1-4) with an unprecedented spiro[furo[3,2-b]benzofuran-3,2'-indene] core were isolated from the fruiting bodies of Ganoderma applanatum. Their planar structures were unambiguously determined via extensive spectroscopic analysis. Their relative and absolute configurations were confirmed through calculated internuclear distance, coupling constant, 13C NMR with DP4 + analysis and electronic circular dichroism (ECD). Furthermore, the molecular docking-based method was used to evaluate their interaction with formyl peptide receptor 2 (FPR2) associated with inflammation. Interestingly, (±)-applandimeric acid D (4) can bond with FPR2 by some key hydrogen bonds. Furthermore, an in vitro bioassay verified that 4 can inhibit the expression of FPR2 with IC50 value of 7.93 µM. In addition, compared to the positive control LiCl (20 mM), 4 showed comparable anti-lipogenesis activity at the concentration of 20 µM. Meanwhile, 4 can suppress the protein levels of peroxisome proliferators-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding protein-ß (C/EBP-ß), adipocyte fatty acid-binding protein 4 (FABP4), and fatty acid synthase (FAS) through activating AMP-activated protein kinase (AMPK) signaling pathway. Thus, our findings indicate that compound 4 could be a lead compound to treat obesity and obesity-related diseases by inhibiting lipid accumulation in adipocyte and alleviating inflammation.

Functional triterpenoids from medicinal fungi Ganoderma applanatum: A continuous search for antiadipogenic agents.

Previously, we have demonstrated the antiadipogenic benefits of Ganoderma triterpenoids (GTs), which indicated GTs have potential therapeutic implications for obesity. In this study, the EtOAc extract of Ganoderma applanatum was further phytochemically investigated for searching new antiadipogenic agents, which led to the isolation of a total of 15 highly oxygenated lanostane triterpenoids, including 9 new compounds (1-9) and 6 known analogues (10-15). Structurally, ganodapplanoic acids A and B (1, 2) are two rearranged 6/6/5/6-fused lanostane-type triterpenoids with an unusual C-13/C-15 oxygen bridge moiety. In addition, the EtOAc extract (GAE) and isolates (1-4,6-15) were assayed for their antiadipogenic effects in 3T3-L1 adipocytes. The results revealed that compound 9 effectively repressed adipogenesis through down-regulating the expression of major proteins (PPARγ, CEBPß and FAS) involving differentiation and adipogenesis in 3T3-L1 adipocytes. Thus, the present study further demonstrated the antiadipogenic potential of GTs and provided a possible perspective for obesity treatment.

Lepipyrrolins A-B, two new dimeric pyrrole 2-carbaldehyde alkaloids from the tubers of Lepidium meyenii.

Nine new pyrrole alkaloids, including two undescribed dimeric pyrrole 2­carbaldehyde alkaloids, lepipyrrolins A-B (1-2), seven pyrrole-alkaloid derivatives, macapyrrolins D-J (3-9), along with three known ones (10-12) were isolated from the rhizomes of Lepidium meyenii. Their structures and absolute configurations were demonstrated by extensive spectroscopic data (1D, 2D NMR, HRESIMS), and calculated electronic circular dichroism (ECD) experiment. Compounds 1, 3-12 were tested for their nitric oxide inhibitory effects. Furthermore, compound 1 was evaluated for its cytotoxic activity against five human tumor cell lines (HL-60, SMMC-7221, A549, MCF-7, and SW480) in vitro, and displayed selective cytotoxicity against SMMC-7721 with IC50 value of 16.78 ± 0.49 µM.

Time- and dose-dependent correlations between patient-controlled epidural analgesia and intrapartum maternal fever.

BACKGROUND: To investigate the relationship between intrapartum maternal fever and the duration and dosage of patient-controlled epidural analgesia (PCEA). METHODS: This observational study included 159 pregnant women who voluntarily accepted PCEA. During labor, patients with body temperature ≥ 38 °C were classified into the Fever group, (n = 42), and those with body temperature < 38 °C were classified into the No-fever group (n = 117). The outcome measures included the duration of PCEA, number of PCEA, and total PCEA amount. Body temperature and parturient variables, including interpartum fever status and the duration of any fever were monitored. RESULTS: The total PCEA duration and total PCEA amount in the Fever group were significantly higher than the corresponding values in the No-fever group (both, p < 0.05). The duration of fever was weakly correlated with the duration of PCEA (R2 = 0.08) and the total PCEA amount (R2 = 0.05) (both, p < 0.05). The total and effective PCEA were higher in the Fever group than in the No-fever group (both, p < 0.05). The total PCEA duration and total PCEA amount were positively correlated with the incidence of fever (both, p < 0.05). The diagnostic cutoff value for fever was 383 min, with a sensitivity of 78.6% and specificity of 57.3%. The mean temperature-time curves showed that parturients who developed fever had a steeper rise in temperature. CONCLUSIONS: This study showed that there were weak time- and dose-dependent correlations between PCEA and maternal fever during delivery. A total PCEA duration exceeding 6.3 h was associated with an increase in the duration of maternal intrapartum fever.

Pyrrolizidine alkaloids from the seeds of Scleropyrum wallichianum.

Two new pyrrolizidine alkaloids, sclerwalins A and B (1 and 2), and one known 9-O-E-hydroxysenecioylretronecine (3) were first isolated from the seeds of Scleropyrum wallichianum. Their chemical structures were elucidated by extensive 1 D NMR and 2 D NMR (HSQC, HMBC, COSY, and ROESY), MS and IR spectra. Cytotoxicities of all isolates were evaluated against five human tumor cell lines (HL-60, A-549, SMMC-7721, MCF-7 and SW480).[Formula: see text].

Morphological Changes and Component Characterization of Coffee Silverskin.

Nuclear magnetic resonance (NMR) spectroscopy was used for the qualitative and quantitative analysis of aqueous extracts of unroasted and roasted coffee silverskin (CS). Twenty compounds were identified from 1D and 2D NMR spectra, including caffeine, chlorogenic acid (CGA), trigonelline, fructose, glucose, sucrose, etc. For the first time, the presence of trigonelline was detected in CS. Results of the quantitative analysis showed that the total amount of the main components after roasting was reduced by 45.6% compared with values before roasting. Sugars in the water extracts were the main components in CS, and fructose was the most abundant sugar, its relative content accounting for 38.7% and 38.4% in unroasted and roasted CS, respectively. Moreover, 1D NMR combined with 2D NMR technology shows application prospects in the rapid, non-destructive detection of CS. In addition, it was observed by optical microscopy and scanning electron microscopy (SEM) that the morphology of CS changed obviously before and after roasting.

[Expression of Ras-Associated Protein 1 in the Vascular Endothelium of Bone Tissue with Non-Traumatic Osteonecrosis of Femoral Head].

Objective: To investigate the difference in the expression of Ras-associated protein 1 (Rap1) in necrotic and healthy areas of non-traumatic osteonecrosis of femoral head (NONFH) patients. Methods: Femoral head tissue samples from 30 cases of NONFH and 30 cases of traumatic osteonecrosis of the femoral head (TONFH) were collected after hip replacement surgery, respectively. No significant difference of Association Research Circulation Osseous (ARCO) staging was found between the NONFH and the TONFH groups ( Z=-0.769, P=0.442). In the NONFH group, 8 patients were ARCO stage IIIb, 10 were stage IV, and 12 were stage V, while in the TONFH ground, 11 patients were ARCO stage IIIb, 9 were stage IV, and 10 were stage V. There were 19 males and 11 females in the NONFH group, with an average age of 49.6 yr. (26-69 yr.), and 16 males and 14 females in the TONFH group, with an average age of 54.2 yr. (37-68 yr.). There was no significant difference in gender or age between the two groups ( P>0.05). Specimens were collected from different bone areas, including those from the necrotic areas (area A) and the healthy areas (area B) of the NONFH group, and those from the healthy areas (area B') of the TONFH group, i.e., the control group. Western blot and quantitative real-time reverse transcription PCR (qRT-PCR) were used to analyze the different expression of Rap1, vascular endothelial growth factor (VEGF) protein, phosphoinositide 3-kinase (PI3K), and Akt protein and their corresponding mRNA in the three areas of bone tissue. HE staining and immunohistochemisty staining were done in order to observe the morphological changes of each area. Results: Western blot results indicated that there was no statistical difference in the relative expression of Rap1, VEGF, PI3K, and Akt proteins ( P>0.05). The relative expressions of Rap1, VEGF, PI3K, and Akt proteins in the area A were lower than those in the area B and the difference was statistically significant ( P<0.05). qRT-PCR results showed that the relative expressions of Rap1, VEGF, PI3 K and Akt mRNA in area A were lower than those of area B, and a statistical difference was found ( P<0.05). The relative expression of the mRNA of Rap1, VEGF , PI3 K and Akt in area B and area B' were not significantly different ( P>0.05). HE staining and immunohistochemisty staining showed that chondrocytes decreased in the necrotic area (area A) of NONFH, chondrocytes nucleus disappeared, subchondral bone trabeculae were broken, bone trabeculae thickened, and empty bone lacunae appeared. Granulation tissues composed of new capillaries and fibrous cells have proliferated and crawled around the necrotic area. Positive expressions of the Rap1, VEGF, PI3K and Akt proteins in area A were weaker than those of the normal area. In addition, there were positive expressions of Rap1, PI3K and Akt on the trabecular bone of both area A and area B at similar intensity of expression. There were strong positive expressions of Rap1, VEGF, PI3K and Akt on the intima of arterioles and venules, and on the peripheral stromal cell membrane, but the positive expression in area A was significantly lower than that in area B. However, the positive expression positions and intensity of all indicators were similar in area B and area B'. Conclusion: The necrosis in NONFH may be related to vascular endothelial damages caused by the inhibition of the Rap1-PI3K/Akt signaling pathways and the subsequent decline in the protein expression.

Meroapplanins A-E: Five Meroterpenoids with a 2,3,4,5-Tetrahydropyridine Motif from Ganoderma applanatum.

Meroapplanins A-E (1-5) with a 2,3,4,5-tetrahydropyridine fragment were isolated from the fruiting bodies of Ganoderma applanatum. Their structures were elucidated by comprehensive spectroscopic analyses. Their absolute configurations were established based on the X-ray diffraction, electronic circular dichroism (ECD), and calculated nuclear magnetic resonance (NMR) with DP4+ analysis. A plausible biosynthetic pathway for 1-5 was proposed. Furthermore, compounds 1-5, together with optically pure compounds 1a-4a and 1b-4b, were evaluated for their protective effects in PC12 cells damaged by H2O2. The results showed that 3b had protective activity with a cell viability of 82.58 ± 1.31%, compared with the model group (cell viability: 65.27 ± 1.48%).