Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

Photoactivatable CRISPR/Cas12a Sensors for Biomarkers Imaging and Point-of-Care Diagnostics.

In recent years, the CRISPR/Cas12a-based sensing strategy has shown significant potential for specific target detection due to its rapid and sensitive characteristics. However, the "always active" biosensors are often insufficient to manipulate nucleic acid sensing with high spatiotemporal control. It remains crucial to develop nucleic acid sensing devices that can be activated at the desired time and space by a remotely applied stimulus. Here, we integrated photoactivation with the CRISPR/Cas12a system for DNA and RNA detection, aiming to provide high spatiotemporal control for nucleic acid sensing. By rationally designing the target recognition sequence, this photoactivation CRISPR/Cas12a system could recognize HPV16 and survivin, respectively. We combined the lateral flow assay strip test with the CRISPR/Cas12a system to realize the visualization of nucleic acid cleavage signals, displaying potential instant test application capabilities. Additionally, we also successfully realized the temporary control of its fluorescent sensing activity for survivin by photoactivation in vivo, allowing rapid detection of target nucleic acids and avoiding the risk of contamination from premature leaks during storage. Our strategy suggests that the CRISPR/Cas12a platform can be triggered by photoactivation to sense various targets, expanding the technical toolbox for precise biological and medical analysis. This study represents a significant advancement in nucleic acid sensing and has potential applications in disease diagnosis and treatment.

Metal-Induced Energy Transfer (MIET) Imaging of Cell Surface Engineering with Multivalent DNA Nanobrushes.

The spacing between cells has a significant impact on cell-cell interactions, which are critical to the fate and function of both individual cells and multicellular organisms. However, accurately measuring the distance between cell membranes and the variations between different membranes has proven to be a challenging task. In this study, we employ metal-induced energy transfer (MIET) imaging/spectroscopy to determine and track the intermembrane distance and variations with nanometer precision. We have developed a DNA-based molecular adhesive called the DNA nanobrush, which serves as a cellular adhesive for connecting the plasma membranes of different cells. By manipulating the number of base pairs within the DNA nanobrush, we can modify various aspects of membrane-membrane interactions such as adhesive directionality, distance, and forces. We demonstrate that such nanometer-level changes can be detected with MIET imaging/spectroscopy. Moreover, we successfully employed MIET to measure distance variations between a cellular plasma membrane and a model membrane. This experiment not only showcases the effectiveness of MIET as a powerful tool for accurately quantifying membrane-membrane interactions but also validates the potential of DNA nanobrushes as cellular adhesives. This innovative method holds significant implications for advancing the study of multicellular interactions.

Exploring the role of gut microbiota in advancing personalized medicine.

Ongoing extensive research in the field of gut microbiota (GM) has highlighted the crucial role of gut-dwelling microbes in human health. These microbes possess 100 times more genes than the human genome and offer significant biochemical advantages to the host in nutrient and drug absorption, metabolism, and excretion. It is increasingly clear that GM modulates the efficacy and toxicity of drugs, especially those taken orally. In addition, intra-individual variability of GM has been shown to contribute to drug response biases for certain therapeutics. For instance, the efficacy of cyclophosphamide depends on the presence of Enterococcus hirae and Barnesiella intestinihominis in the host intestine. Conversely, the presence of inappropriate or unwanted gut bacteria can inactivate a drug. For example, dehydroxylase of Enterococcus faecalis and Eggerthella lenta A2 can metabolize L-dopa before it converts into the active form (dopamine) and crosses the blood-brain barrier to treat Parkinson's disease patients. Moreover, GM is emerging as a new player in personalized medicine, and various methods are being developed to treat diseases by remodeling patients' GM composition, such as prebiotic and probiotic interventions, microbiota transplants, and the introduction of synthetic GM. This review aims to highlight how the host's GM can improve drug efficacy and discuss how an unwanted bug can cause the inactivation of medicine.

The pig pangenome provides insights into the roles of coding structural variations in genetic diversity and adaptation.

Structural variations have emerged as an important driving force for genome evolution and phenotypic variation in various organisms, yet their contributions to genetic diversity and adaptation in domesticated animals remain largely unknown. Here we constructed a pangenome based on 250 sequenced individuals from 32 pig breeds in Eurasia and systematically characterized coding sequence presence/absence variations (PAVs) within pigs. We identified 308.3-Mb nonreference sequences and 3438 novel genes absent from the current reference genome. Gene PAV analysis showed that 16.8% of the genes in the pangene catalog undergo PAV. A number of newly identified dispensable genes showed close associations with adaptation. For instance, several novel swine leukocyte antigen (SLA) genes discovered in nonreference sequences potentially participate in immune responses to productive and respiratory syndrome virus (PRRSV) infection. We delineated previously unidentified features of the pig mobilome that contained 490,480 transposable element insertion polymorphisms (TIPs) resulting from recent mobilization of 970 TE families, and investigated their population dynamics along with influences on population differentiation and gene expression. In addition, several candidate adaptive TE insertions were detected to be co-opted into genes responsible for responses to hypoxia, skeletal development, regulation of heart contraction, and neuronal cell development, likely contributing to local adaptation of Tibetan wild boars. These findings enhance our understanding on hidden layers of the genetic diversity in pigs and provide novel insights into the role of SVs in the evolutionary adaptation of mammals.

Low-Background CRISPR/Cas12a Sensors for Versatile Live-Cell Biosensing.

The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in "on-off-on" mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the "RESET" effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer-substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 µM, and 8.3 × 10-5 U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. Moreover, we also demonstrate that these sensing systems can be easily combined with lateral flow assay (LFA), thus holding great potential in point-of-care testing, especially in poorly equipped or nonlaboratory environments.

Nanoparticle-Coupled Single-Molecule Kinetic Fingerprinting for Enzymatic Activity Detection.

The sensitive and accurate detection of biomarkers plays an important role in clinical diagnosis and drug discovery. Currently, amplification-based methods for biomarker detection are widely explored. However, the key challenges of these methods are limited reproducibility and high background noise. To overcome these limitations, we develop a robust plasmonic nanoparticle-coupled single-molecule kinetic fingerprinting (PNP-SMKF) method to achieve ultrasensitive detection of protein kinase A (PKA). Transient binding of a short fluorescent probe with the genuine target produces a distinct kinetic signature that is completely different from that of the background signal, allowing us to recognize PKA sensitively. Importantly, integrating a plasmonic nanoparticle efficiently breaks the concentration limit of the imager strand for single-molecule imaging, thus achieving a much faster imaging speed. A limit of detection (LOD) of as low as 0.0005 U/mL is readily realized. This method holds great potential as a versatile platform for enzyme detection and inhibitor screening in the future.

Safety and efficacy of topical diquafosol for the treatment of dry eye disease: An updated meta-analysis of randomized controlled trials.

Dry eye disease (DED) is a common multi-factorial disease that is characterized by tear film instability. Diquafosol tetrasodium (DQS), an ophthalmic solution, has been shown to be beneficial in the treatment of DED. The goal of this study was to provide an update on the safety and efficacy of topical 3% DQS in treating DED patients. A thorough search for all the published randomized controlled trials (RCTs) up to March 31, 2022 in CENTRAL, PubMed, Scopus, and Google Scholar databases was performed. Data were reported as standardized mean difference (SMD) with 95% confidence interval (CI). Modified Jadad scale was used for sensitivity analysis. Funnel plot and Egger's regression test assessed the publication bias. Fourteen RCTs evaluating the safety and efficacy of topical 3% DQS treatment in DED patients were included. Eight included RCTs reported data on the DED after cataract surgery. Overall findings suggest that 3% DQS treatment in DED patients was associated with significantly better improvement at 4 weeks in tear breakup time, Schirmer test, fluorescein staining scores, and Rose Bengal staining score as compared to patients treated with others eye drops including artificial tears or 01% sodium hyaluronate. However, no significant difference in ocular surface disease index was observed. Our findings suggest that 3% DQS treatment is safer and had a superior efficacy compared to artificial tears or sodium hyaluronate for treating DED in general and DED after cataract surgery.

Lysine Demethylase 1B Promotes Tear Secretion Disorder in Sjogren's Syndrome by Regulating the PAX6/CLU Axis.

The impacts of lysine demethylase 1B (KDM1B) have been probed in multiple diseases, but the effects of KDM1B on SS remained obscure. The study aimed to unravel the efficiency of KDM1B on SS progression via the paired box 6 (PAX6)/clusterin (CLU) axis. NODB10. H2b mice were selected to establish the SS model. KDM1B, Pax6, and CLU expression in SS mice was assessed. Adeno-associated viruses carrying KDM1B, Pax6, and CLU were injected into the SS mice to detect tear secretion, epithelium corneal fluorescein staining scores, and levels of specific markers of lacrimal gland epithelial cells, neurotransmitter receptors that induce secretion from the lacrimal gland, and genes encoding normal tear components. The relation among KDM1B, Pax6, and CLU was examined. The rescue experiments were conducted for verifying the interaction among KDM1B, Pax6, and CLU. KDM1B expression was elevated, while Pax6 and CLU levels were decreased in the lacrimal gland tissues of SS mouse models. KDM1B decrement and Pax6 augmentation improved tear secretion, reduced corneal fluorescein staining score, decreased levels of specific markers of lacrimal gland epithelial cells, and increased levels of neurotransmitter receptors that induce secretion from the lacrimal gland and genes encoding normal tear components. KDM1D suppressed Pax6 expression by mediating H3K4me2 demethylation. Pax6 promoted the expression of CLU at the transcriptional level by binding to the CLU promoter. Silencing of Pax6 or CLU could reverse the effects of KDM1B reduction on improving the tear secretion disorder of SS mice. Silencing KDM1B mitigates the tear secretion disorder of SS mice via modulating the Pax6/CLU axis.

The Effect of Orthokeratology Lens on the Axial Length of Globe in Children with Myopia in Asia.

The aim of this meta-analysis was to determine the effect of orthokeratology (OK) lens on the axial length of the eye globe of Asian children with myopia of low and moderate degree compared with children treated with glasses. PubMed, Embase, Web of Science, and Cochrane Library were searched to collect relative researches on the treatment of OK lens in myopia children from January 2000 to July 2021. The authors adopted the standardised mean difference (SMD) as effect size, to estimate the pooled changes of axial length in Asian myopic children. Seven articles were identified. In Asian children with myopia of low to a moderate degree, compared with glasses treatment, axial length decreased significantly in the OK Lens group (SMD = -0.84, 95%CI: -1.10 ~ -0.58), with a p-value less than 0.05. According to the follow-up time, the subgroup analysis demonstrated that, SMD = -0.68 (95%CI: -1.43 ~ 0.08, p>0.05) for six months; SMD = -1.00 (95 %CI: -1.38~-0.62, p<0.05) for 12 months; SMD=-0.71 (95%CI: -1.20~-0.21, p<0.05) for two years. In conclusion, in the early treatment, compared with glasses, children with low and moderate myopia wearing OK lenses were more effective in reducing axial elongation in Asia. Key Words: Axial length, Orthokeratology, Myopia, Meta-analysis.

A new and spontaneous animal model for ankylosing spondylitis is found in cynomolgus monkeys.

BACKGROUND: Ankylosing spondylitis is a progressive, disabling joint disease that affects millions worldwide. Given its unclear etiology, studies of ankylosing spondylitis relied heavily on drug-induced or transgenic rodent models which retain only partial clinical features. There is obviously a lack of a useful disease model to conduct comprehensive mechanistic studies. METHODS: We followed a group of cynomolgus monkeys having joint lesions reported of spinal stiffness for 2 years by conducting hematological testing, radiographic examination, family aggregation analysis, pathological analysis, and genetic testing. RESULTS: The results confirmed that these diseased animals suffered from spontaneous ankylosing spondylitis with clinical features recapitulating human ankylosing spondylitis disease progression, manifested by pathological changes and biochemical indicators similar to that of ankylosing spondylitis patients. CONCLUSION: The study offers a promising non-human primate model for spontaneous ankylosing spondylitis which may serve as an excellent substitute for its pre-clinical research.

Naringenin inhibits autophagy and epithelial-mesenchymal transition of human lens epithelial cells by regulating the Smad2/3 pathway.

Cataract is the number one cause of blindness in the world. Fibrosis of the lens is the main cause of cataract. Pathological epithelial-mesenchymal transition (EMT) plays an important role in the development of fibrotic cataract. Inhibition of EMT may be an effective treatment for fibrosis of lens epithelial cells. Naringin (NRG) is one of the major citrus flavonoids, which has many pharmacological properties, including anti-inflammatory and cardioprotective. However, the effect of NRG on cataract induced by abnormal fibrosis of LECs is not clear. Herein, we found NRG inhibited transforming growth factor ß2 (TGFß2)-induced SRA01/04 cell viability. Additionally, NRG inhibited TGFß2-induced cell migration and EMT. We further noticed that NRG inhibited autophagy and Smad2/3 phosphorylation in LECs. We therefore thought Naringenin inhibited autophagy and EMT of human LECs by regulating the Smad2/3 pathway. NRG could therefore serve as a promising drug for cataract treatment.

Expanding the application of non-invasive prenatal testing in the detection of foetal chromosomal copy number variations.

PURPOSE: The aim of this study was to assess the detection efficiency and clinical application value of non-invasive prenatal testing (NIPT) for foetal copy number variants (CNVs) in clinical samples from 39,002 prospective cases. METHODS: A total of 39,002 pregnant women who received NIPT by next-generation sequencing (NGS) with a sequencing depth of 6 M reads in our centre from January 2018 to April 2020 were enrolled. Chromosomal microarray analysis (CMA) was further used to diagnose suspected chromosomal aneuploidies and chromosomal microdeletion/microduplication for consistency assessment. RESULTS: A total of 473 pregnancies (1.213%) were positive for clinically significant foetal chromosome abnormalities by NIPT. This group comprised 99 trisomy 21 (T21, 0.254%), 30 trisomy 18 (T18, 0.077%), 25 trisomy 13 (T13, 0.064%), 155 sex chromosome aneuploidy (SCA, 0.398%), 69 rare trisomy (0.177%), and 95 microdeletion/microduplication syndrome (MMS, 0.244%) cases. Based on follow-up tests, the positive predictive values (PPVs) for the T21, T18, T13, SCA, rare trisomy, and MMS cases were calculated to be 88.89%, 53.33%, 20.00%, 40.22%, 4.88%, and 49.02%, respectively. In addition, the PPVs of CNVs of < 5 Mb, 5-10 Mb, and > 10 Mb were 54.55%, 38.46%, and 40.00%, respectively. Among the 95 cases with suspected CNVs, 25 were diagnosed as true positive and 26 cases as false positive; follow-up prenatal diagnosis by CMA was not performed for 44 cases. Moreover, among the 25 true positive cases, 10 were pathogenic, 3 were likely pathogenic, and 12 were of uncertain significance. CONCLUSION: NIPT is not only suitable for screening T21, T18, T13, and SCA but also has potential significance for CNV detection. As combined with ultrasound, extended NIPT is effective for screening MMS. However, NIPT should not be recommended for whole-chromosome aneuploidy screening.

Alternative Splicing Dynamics of the Hypothalamus-Pituitary-Ovary Axis During Pubertal Transition in Gilts.

The timing of puberty in mammals marks the point at which reproduction becomes possible. Abnormalities in the timing of puberty may exert a series of negative effects on subsequent health outcomes. Alternative splicing (AS) has not only emerged as a significant factor in the transcription of genes but it is also reported to play a role in the timing of puberty. However, to date, the changes and dynamics of AS during the onset of puberty is extremely seldom explored. In the present study, we used gilts as a research model to investigated the dynamics of AS and differentially expressed AS (DEAS) events within the hypothalamus-pituitary-ovary (HPO) axis across pre-, in-, and post-puberty. We detected 3,390, 6,098, and 9,085 DEAS events in the hypothalamus, pituitary, and ovary when compared across pre-, in-, and post-pubertal stages, respectively. Within the entire HPO axis, we also identified 22,889, 22,857, and 21,055 DEAS events in the pre-, in-, and post-pubertal stages, respectively. Further analysis revealed that the differentially spliced genes (DSGs) associated with staged DEAS events were likely to be enriched in the oxytocin signaling pathway, thyroid hormone signaling pathway, GnRH signaling pathway, and oocyte meiosis signaling pathway. The DSGs associated with DEAS events across the entire HPO axis were enriched in endocytosis signaling pathway, the MAPK signaling pathway, and the Rap1 signaling pathway. Moreover. the ASs of TAC1, TACR3, CYP19A1, ESR1, ESRRA, and FSHR were likely to regulate the functions of the certain HPO tissues during the onset of puberty. Collectively, the AS dynamics and DEAS events were comprehensively profiled in hypothalamus, pituitary, and ovary across the pre-, in-, and post-pubertal stages in pigs. These findings may enhance our knowledge of how puberty is regulated by AS and shed new light on the molecular mechanisms underlying the timing of puberty in mammals.

Identification of Circular RNAs in Hypothalamus of Gilts during the Onset of Puberty.

The disorders of puberty have shown negative outcomes on health of mammals, and the hypothalamus is thought to be the main regulator of puberty by releasing GnRH. Many studies show that the circular RNAs (circRNAs) might be implicated in the timing of puberty in mammals. However, the circRNAs in the hypothalamus of gilts have not been explored. To profile the changes and biological functions of circRNAs in the hypothalamus during the onset of puberty, RNA-seq was utilized to establish pre-, in-, and post-pubertal hypothalamic circRNAs profiles. In this study, the functions of hypothalamic circRNAs were enriched in the signaling pathway of neurotrophin, progesterone-mediated oocyte maturation, oocyte meiosis, insulin, ErbB, and mTOR, which have been highly suggested to be involved in the timing of puberty. Furthermore, 53 circRNAs were identified to be putative hypothalamus-specific expressed circRNAs, and some of them were exclusively expressed in the one of three pubertal stages. Moreover, 22 differentially expressed circRNAs were identified and chosen to construct the circRNA-miRNA-gene network. Moreover, 10 circRNAs were found to be driven by six puberty-related genes (ESR1, NF1, APP, ENPP2, ARNT, and DICER1). Subsequently, the expression changes of several circRNAs were confirmed by RT-qPCR. Collectively, the preliminary results of hypothalamic circRNAs provided useful information for the investigation of the molecular mechanism for the timing of puberty in gilts.

Identification of Specific N6-Methyladenosine RNA Demethylase FTO Inhibitors by Single-Quantum-Dot-Based FRET Nanosensors.

The fat mass and obesity-associated enzyme (FTO) can catalyze the demethylation of N6-methyladenosine (m6A) residues in mRNA, regulates the cellular level of m6A modification, and plays a critical role in human obesity and cancers. Herein, we develop a single-quantum-dot (QD)-based fluorescence resonance energy transfer (FRET) sensor for the identification of specific FTO demethylase inhibitors. The FTO-mediated demethylation of m6A can induce the cleavage of demethylated DNA to generate the biotinylated DNA fragments, which may function as capture probes to assemble the Cy5-labeled reporter probes onto the QD surface, enabling the occurrence of FRET between the QD and Cy5. The presence of inhibitors can inhibit the FTO demethylation and consequently abolish FRET between the QD and Cy5. The inhibition effect of inhibitors upon FTO demethylation can be simply evaluated by monitoring the decrease of Cy5 counts. We use this nanosensor to screen several small-molecule inhibitors and identify diacerein as a highly selective inhibitor of FTO. Diacerein can inhibit the demethylation activity of endogenous FTO in HeLa cells. Interestingly, diacerein is neither a structural mimic of 2-oxoglutarate (2-OG) nor a chelator of metal ions, and it can selectively inhibit FTO demethylation by competitively binding the m6A-containing substrate.

Correction: Development of a bidirectional isothermal amplification strategy for the sensitive detection of transcription factors in cancer cells.

Correction for 'Development of a bidirectional isothermal amplification strategy for the sensitive detection of transcription factors in cancer cells' by Yan Zhang et al., Chem. Commun., 2020, 56, 8952-8955, DOI: 10.1039/D0CC03134H.

Development of a bidirectional isothermal amplification strategy for the sensitive detection of transcription factors in cancer cells.

We developed a new strategy to sensitively detect transcription factors (TFs) based on the integration of a bidirectional isothermal exponential amplification reaction (EXPAR) with endonuclease IV (endo IV)-assisted cycle digestion of signal probes. This assay exhibits ultrahigh sensitivity with a detection limit of 1.29 × 10-14 M, and it can measure endogenous NF-κB p50 in HeLa cell extracts. Moreover, this strategy can be applied to screen TF inhibitors and detect other TFs by simply changing the TF-binding sequence.

Inhibited Maternal Bone Resorption Suppress Fetal Rat Bone Development During Pregnancy.

OBJECTIVE: To determine the relationship between maternal bone resorption and bone development in fetuses. METHODS: Female SD rats were injected with either fluorescent calcium indicator calcein alone or together with tetracycline 1 week before pregnancy, followed by fluorescence detection in fetal tibias 21 days post-treatment. Alendronate was subsequently administered to pregnant rats to inhibit maternal bone resorption, while maternal bone turnover and fetal bone development were both examined. RESULTS: The maternal fluorescent labeled calcium before pregnancy was found in the fetal tibia. This indicated that the calcium of maternal bones may be released into the maternal circulation through high bone resorption during pregnancy, thereby participating in the fetal bone development. Bone histomorphometry and serum biomarker results showed that Alendronate significantly inhibited maternal bone resorption in pregnant rats when compared to normal pregnant rats. Moreover, the body weight, bone mass, and bone length of the fetuses in the Alendronate group were significantly decreased; while no apparent abnormality in placental morphology was observed. The above results implied that when maternal bone resorption is suppressed, the development of the fetal bone shall also be suppressed. CONCLUSION: Calcium in the maternal bone is released into the maternal circulation through bone resorption during pregnancy which represents an important material source in fetal bone development. Therefore, high bone turnover during pregnancy is essential for mammalian embryonic bone development.

Preventative effects of metformin on glucocorticoid-induced osteoporosis in rats.

This study evaluated the preventative effects of metformin (Met) on glucocorticoid (GC)-induced osteoporosis in a rat model, compared with alendronate (Aln). Twenty-eight 3-month-old female Sprague-Dawley rats were randomly assigned into four groups: normal control (Ctr), methylprednisolone (MP, 13 mg/kg/day, sc, 5 days per week), MP plus Aln orally (1 mg/kg/day), and MP plus Met orally (200 mg/kg/day). After 9 weeks, serum bone metabolic biochemistry, bone densitometry and histomorphometry were performed. The GC-induced osteoporosis model was characterized by decreased osteocalcin, increased tartrate-resistant acid phosphatase-5b (TRAP-5b), and decreased bone mineral density (BMD) in the femur and fifth lumbar vertebra (L5). Histomorphometrically, MP significantly decreased trabecular bone volume, decreased bone formation and increased bone resorption in proximal metaphysis, compared with the controls. Aln and Met increased the BMDs of femur (0.305 ± 0.011 vs. 0.280 ± 0.012, P < 0.05; 0.304 ± 0.019 vs. 0.280 ± 0.012, P < 0.05) and L5 (0.399 ± 0.029 vs. 0.358 ± 0.022, P < 0.05; 0.397 ± 0.022 vs. 0.358 ± 0.022, P < 0.05), compared with the model group. Met increased osteocalcin and decreased TRAP-5b, but Aln only decreased TRAP-5b, compared with model group. In histomorphometry of tibial proximal metaphysis, Aln and Met increased trabecular bone volume (39.21 ± 2.46 vs. 30.98 ± 5.83, P < 0.05; 38.97 ± 5.56 vs. 30.98 ± 5.83, P < 0.05), while Met increased the bone formation dynamic parameters and decreased bone resorption dynamic parameters, but Aln just decreased bone resorption dynamic parameters, compared with model group significantly. These findings suggest that metformin prevents GC-induced bone loss by suppressing bone resorption and stimulating bone formation in trabecular bone. The action mode of metformin was different from alendronate, which only suppressed bone resorption.