Nanopore analysis of salvianolic acids in herbal medicines.

Natural herbs, which contain pharmacologically active compounds, have been used historically as medicines. Conventionally, the analysis of chemical components in herbal medicines requires time-consuming sample separation and state-of-the-art analytical instruments. Nanopore, a versatile single molecule sensor, might be suitable to identify bioactive compounds in natural herbs. Here, a phenylboronic acid appended Mycobacterium smegmatis porin A (MspA) nanopore is used as a sensor for herbal medicines. A variety of bioactive compounds based on salvianolic acids, including caffeic acid, protocatechuic acid, protocatechualdehyde, salvianic acid A, rosmarinic acid, lithospermic acid, salvianolic acid A and salvianolic acid B are identified. Using a custom machine learning algorithm, analyte identification is performed with an accuracy of 99.0%. This sensing principle is further used with natural herbs such as Salvia miltiorrhiza, Rosemary and Prunella vulgaris. No complex sample separation or purification is required and the sensing device is highly portable.

Unambiguous discrimination of all 20 proteinogenic amino acids and their modifications by nanopore.

Natural proteins are composed of 20 proteinogenic amino acids and their post-translational modifications (PTMs). However, due to the lack of a suitable nanopore sensor that can simultaneously discriminate between all 20 amino acids and their PTMs, direct sequencing of protein with nanopores has not yet been realized. Here, we present an engineered hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a sole Ni2+ modification. It enables full discrimination of all 20 proteinogenic amino acids and 4 representative modified amino acids, Nω,N'ω-dimethyl-arginine (Me-R), O-acetyl-threonine (Ac-T), N4-(ß-N-acetyl-D-glucosaminyl)-asparagine (GlcNAc-N) and O-phosphoserine (P-S). Assisted by machine learning, an accuracy of 98.6% was achieved. Amino acid supplement tablets and peptidase-digested amino acids from peptides were also analyzed using this strategy. This capacity for simultaneous discrimination of all 20 proteinogenic amino acids and their PTMs suggests the potential to achieve protein sequencing using this nanopore-based strategy.

Simultaneous Identification of Major Thyroid Hormones by a Nickel Immobilized Biological Nanopore.

Thyroid hormones (THs) are a variety of iodine-containing hormones that demonstrate critical physiological impacts on cellular activities. The assessment of thyroid function and the diagnosis of thyroid disorders require accurate measurement of TH levels. However, largely due to their structural similarities, the simultaneous discrimination of different THs is challenging. Nanopores, single-molecule sensors with a high resolution, are suitable for this task. In this paper, a hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore containing a single nickel ion immobilized to the pore constriction has enabled simultaneous identification of five representative THs including l-thyroxine (T4), 3,3',5-triiodo-l-thyronine (T3), 3,3',5'-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2) and 3,3'-diiodo-l-thyronine (3,3'-T2). To automate event classification and avoid human bias, a machine learning algorithm was also developed, reporting an accuracy of 99.0%. This sensing strategy is also applied in the analysis of TH in a real human serum environment, suggesting its potential use in a clinical diagnosis.

Nanopore Signatures of Nucleoside Drugs.

Nucleoside drugs, which are analogues of natural nucleosides, have been widely applied in the clinical treatment of viral infections and cancers. The development of nucleoside drugs, repurposing of existing drugs, and combined use of multiple drug types have made the rapid sensing of nucleoside drugs urgently needed. Nanopores are emerging single-molecule sensors that have high resolution to resolve even minor structural differences between chemical compounds. Here, an engineered Mycobacterium smegmatis porin A hetero-octamer was used to perform general nucleoside drug analysis. Ten nucleoside drugs were simultaneously detected and fully discriminated. An accuracy of >99.9% was consequently reported. This sensing capacity was further demonstrated in direct nanopore analysis of ribavirin buccal tablets, confirming its sensing reliability against complex samples and environments. No sample separation is needed, however, significantly minimizing the complexity of the measurement. This technique may inspire nanopore applications in pharmaceutical production and pharmacokinetics measurements.

Nanopore Discrimination of Nucleotide Sugars.

Nucleotide sugars, the glycosyl donors in the biosynthesis of carbohydrates, are critical ingredients in the growth and development of all living organisms. A variety of nucleotide sugars simultaneously exist in biological samples. They, however, have only minor structural differences, which make them extremely difficult to discriminate. In this work, a phenylboronic acid (PBA)-modified Mycobacterium smegmatis porin A (MspA) hetero-octamer was applied to sense nucleotide sugars. Five representative nucleotide sugars, including guanosine diphosphate mannose (GDP-Man), adenosine diphosphate glucose (ADP-Glc), uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), uridine diphosphate glucose (UDP-Glc), and uridine diphosphate glucoronic acid (UDP-GlcA), were successfully distinguished. A custom machine learning algorithm was also employed to automatically identify events, reporting a general accuracy of 99.4%. This sensing strategy provides a rapid, direct, and accurate method for identifying different nucleotide sugars. However, single-molecule identification of nucleotide sugars has never been previously reported, to the best of our knowledge.

Single-Molecule Interconversion between Chiral Configurations of Boronate Esters Observed in a Nanoreactor.

Isomers of some chemical compounds may be dynamically interconvertible. Due to a lack of sensing methods with a sufficient resolution, however, direct monitoring of such processes can be difficult. Engineered Mycobacterium smegmatis porin A (MspA) nanopores can be applied as nanoreactors so that chemical reactions can be directly monitored. Here, an MspA modified with a phenylboronic acid (PBA) adapter was prepared and was used to observe dynamic interconversion between chiral configurations of boronate esters, which appears as telegraphic switching on top of nanopore events. The mechanism of this behavior was further confirmed by trials with different halogenated catechols, dopamine, adenosine, 1,2-propanediol, and (2R,3R)-2,3-butanediol, and its generality has been demonstrated. These results suggest that an engineered MspA possesses an exceptional resolution in its monitoring of chemical reaction processes and may inspire the future design of nanopore small-molecule sensors.

Discrimination of Ribonucleoside Mono-, Di-, and Triphosphates Using an Engineered Nanopore.

Ribonucleotides, which widely exist in all living organisms and are essential to both physiological and pathological processes, can naturally appear as ribonucleoside mono-, di-, and triphosphates. Natural ribonucleotides can also dynamically switch between different phosphorylated forms, posing a great challenge for sensing. A specially engineered nanopore sensor is promising for full discrimination of all canonical ribonucleoside mono-, di-, and triphosphates. However, such a demonstration has never been reported, due to the lack of a suitable nanopore sensor that has a sufficient resolution. In this work, we utilized a phenylboronic acid (PBA) modified Mycobacterium smegmatis porin A (MspA) hetero-octamer for ribonucleotide sensing. Twelve types of ribonucleotides, including mono-, di-, and triphosphates of cytidine (CMP, CDP, CTP), uridine (UMP, UDP, UTP), adenosine (AMP, ADP, ATP), and guanosine (GMP, GDP, GTP) were simultaneously discriminated. A machine-learning algorithm was also developed, which achieved a general accuracy of 99.9% for ribonucleotide sensing. This strategy was also further applied to identify ribonucleotide components in ATP tablets and injections. This sensing strategy provides a direct, accurate, easy, and rapid solution to characterize ribonucleotide components in different phosphorylated forms.

p300 arrests intervertebral disc degeneration by regulating the FOXO3/Sirt1/Wnt/ß-catenin axis.

The transcription factor p300 is reportedly involved in age-associated human diseases, including intervertebral disc degeneration (IDD). In this study, we investigate the potential role and pathophysiological mechanism of p300 in IDD. Clinical tissue samples were collected from patients with lumbar disc herniation (LDH), in which the expression of p300, forkhead box O3 (FOXO3), and sirtuin 1 (Sirt1) was determined. Nucleus pulposus cells (NPCs) isolated from clinical degenerative intervertebral disc (IVD) tissues were introduced with oe-p300, oe-FOXO3, Wnt/ß-catenin agonist 1, C646 (p300/CBP inhibitor), or si-p300 to explore the functional role of p300 in IDD and to characterize the relationship between p300 and the FOXO3/Sirt1/Wnt/ß-catenin pathway. Also, we established a rat IDD model by inducing needle puncture injuries in the caudal IVDs for further verification of p300 functional role. We found that p300 was downregulated in the clinical tissues and NPCs of IDD. Overexpression of p300 promoted the proliferation and autophagy of NPCs while inhibiting cell apoptosis, which was associated with FOXO3 upregulation. p300 could increase the expression of FOXO3 by binding to the Sirt1 promoter, and thus, contributed to inactivation of the Wnt/ß-catenin pathway. In vivo results further displayed that p300 slowed down the progression of IDD by disrupting the Wnt/ß-catenin pathway through the FOXO3/Sirt1 axis. Taken together, we suggest that p300 can act to suppress IDD via a FOXO3-dependent mechanism, highlighting a potential novel target for treatment of IDD.

Discrimination between Different DNA Lesions by Monitoring Single-Molecule Polymerase Stalling Kinetics during Nanopore Sequencing.

O6-Carboxymethylguanosine (O6-CMG), O6-methylguanosine (O6-MeG), and abasic site (AP site) are DNA lesions induced by alkylating agents. Identification of these lesions in DNA may aid in understanding their relevance to carcinogenesis and may be used for diagnosis. Nanopore sequencing (NPS) may directly report nucleotide modifications solely from the nanopore readout. However, the conventional NPS strategy still suffers from interferences from neighboring sequences. Instead, by observation of the enzymatic stalling kinetics caused by the O6-CMG, O6-MeG, or AP site, discrimination between different DNA lesions is directly achieved. This strategy is not interfered with by the sequence context around the lesion. The lesion, which retards the movement of the DNA through the pore, efficiently prohibits misreading of the DNA lesion. These results suggest a new strategy in the identification of DNA lesions or DNA modifications. It also provides a high-resolution biophysical tool to investigate enzymatic kinetics caused by DNA lesions and the corresponding enzymes.

A Nanopore Based Molnupiravir Sensor.

Nucleoside analogues are reagents that resemble the structure of natural nucleosides and are widely applied in antiviral and anticancer therapy. Molnupiravir, a recently reported nucleoside analogue drug, has shown its inhibitory effect against SARS-CoV-2. Rapid tracing of molnupiravir and its metabolites is important in the evaluation of its pharmacology effect, but direct sensing of molnupiravir as a single molecule has not been reported to date. Here, we demonstrate a nanopore-based sensor with which direct sensing of molnupiravir and its two major metabolites ß-d-N4-hydroxycytidine and its triphosphate can be achieved simultaneously. In conjunction with a custom machine learning algorithm, an accuracy of 92% was achieved. This sensing strategy may be useful in the current pandemic and is in principle suitable for other nucleoside analogue drugs.

Non-binary Encoded Nucleic Acid Barcodes Directly Readable by a Nanopore.

A large collection of unique molecular barcodes is useful in the simultaneous sensing or screening of molecular analytes. Though the sequence of DNA has been widely applied to encode for molecular barcodes, decoding of these barcodes is normally assisted by sequencing. We here demonstrate a barcode system based solely on self-assembly of synthetic nucleic acids and direct nanopore decoding. Each molecular barcode is composed of "n" distinct information nodes in a non-binary manner and can be sequentially scanned and decoded by a Mycobacterium smegmatis porin A (MspA) nanopore. Nanopore events containing step-shaped features were consistently reported. 14 unique information nodes were developed which in principle could encode for 14n unique molecular barcodes in a barcode containing "n" information nodes. These barcode probes were adapted to detect different antibody proteins or cancer-related microRNAs, suggesting their immediate application in a wide variety of sensing applications.

[Comparative study on effectiveness of percutaneous endoscopic and Wiltse-approach transforaminal lumbar interbody fusion in the treatment of lumbar spondylolisthesis].

OBJECTIVE: To compare the effectiveness of percutaneous endoscopic transforaminal lumbar interbody fusion (PE-TLIF) and Wiltse-approach TLIF (W-TLIF) in the treatment of lumbar spondylolisthesis. METHODS: The clinical data of 47 patients with lumbar spondylolisthesis who met the selection criteria between July 2018 and June 2019 were retrospectively analyzed, in which 21 patients were treated with PE-TLIF (PE-TLIF group) and 26 patients were treated with W-TLIF (W-TLIF group). There was no significant difference between the two groups in age, gender, disease duration, level of spondylolisthesis vertebrae, spondylolisthesis degree, spondylolisthesis type, and preoperative visual analogue scale (VAS) score of low back pain and leg pain, lumbar Japanese Orthopaedic Association (JOA) score, and the disc height (DH), segmental lordosis (SL), and Taillard index (TI) of the operated vertebrae ( P>0.05). The operation time, intraoperative blood loss, postoperative drainage, postoperative bedridden time, and complications were compared between the two groups. The VAS score and JOA score were used to evaluate the improvement of pain and function. At last follow-up, DH, SL, and TI of operated vertebrae were measured by X-ray films, and lumbar CT was performed to evaluate the interbody fusion. RESULTS: Compared with W-TLIF group, the operation time in PE-TLIF group was significantly longer, but the intraoperative blood loss and postoperative drainage were significantly less, and the postoperative bedridden time was significantly shorter ( P<0.05). There were 2 cases of transient lower limb radiating pain in PE-TLIF group and 1 case of superficial incision infection in W-TLIF group. There was no significant difference in the incidence of complications (9.5% vs. 3.8%) between the two groups ( χ 2=0.037, P=0.848). The patients in both groups were followed up 12-24 months, with an average of 17.3 months in PE-TLIF group and 17.7 months in W-TLIF group. The VAS scores of low back pain and leg pain, and the JOA scores of the two groups significantly improved at each time point after operation when compared with those before operation ( P<0.05). Compared with W-TLIF group, the VAS scores of low back pain in PE-TLIF group significantly lower at 3 days and 3 months after operation ( P<0.05), and the JOA score of PE-TLIF group was significantly higher at 3 months after operation ( P<0.05), and there was no significant difference in each score at any other time point between the two groups ( P>0.05). At last follow-up, the DH, SL, and TI of operated vertebrae of the two groups significantly improved when compared with those before operation ( P<0.05), and there was no significant difference in the differences of each parameter between the two groups ( P>0.05). According to Suk's standard, the fusion rates of PE-TLIF group and W-TLIF group were 90.5% (19/21) and 92.3% (24/26), respectively, with no significant difference ( χ 2=0.000, P=1.000). At last follow-up, there was no case of Cage sunk into the adjacent vertebral body, or dislodgement of Cage anteriorly or posteriorly in both groups. CONCLUSION: PE-TLIF and W-TLIF are both effective in the treatment of grade Ⅰ and Ⅱ lumbar spondylolisthesis. Although the operation time is prolonged, PE-TLIF has less intraoperative blood loss and postoperative drainage, shorter postoperative bedridden time, and can get more obvious short-term improvement of low back pain and function.

Nanopore Sequencing Accurately Identifies the Cisplatin Adduct on DNA.

Cisplatin, which selectively binds to N7 atoms of purines to inhibit normal replication and transcription, is a widely applied chemotherapeutic drug in the treatment of cancer. Though direct identification of cisplatin lesions on DNA is of great significance, existing sequencing methods have issues such as complications of preamplification or enrichment-induced false-positive reports. Direct identification of cisplatin lesions by nanopore sequencing (NPS) is in principle feasible. However, relevant investigations have never been reported. By constructing model sequences (83 nucleotides in length) containing a sole cisplatin lesion, identification of corresponding lesions by NPS is achieved with <10 ng of input sequencing library. Moreover, characteristic high-frequency noises caused by cisplatin lesions are consistently observed during NPS, clearly identifiable in corresponding high-pass filtered traces. This feature is, however, never observed in any other combinations of natural DNA bases and could be taken as a reference to identify cisplatin lesions on DNA. Further investigations demonstrate that cisplatin stalls the replication of phi29 DNA polymerase, which appears as a ∼5 pA level fluctuation in the single-molecule resolution. These results have confirmed the feasibility of NPS to identify cisplatin lesions at the genomic level and may provide new insights into understanding the molecular mechanism of platinum-based drugs.

Single Molecule Ratcheting Motion of Peptides in a Mycobacterium smegmatis Porin A (MspA) Nanopore.

Diverse functions of proteins, including synthesis, catalysis, and signaling, result from their highly variable amino acid sequences. The technology allowing for direct analysis of protein sequences, however, is still unsatisfactory. Recent developments of nanopore sequencing of DNA or RNA have motivated attempts to realize nanopore sequencing of peptides in a similar manner. The core challenge has been to achieve a controlled ratcheting motion of the target peptide, which is currently restricted to a limited choice of compatible enzymes. By constructing peptide-oligonucleotide conjugates (POCs) and measurements with nanopore-induced phase-shift sequencing (NIPSS), direct observation of the ratcheting motion of peptides has been successfully achieved. The generated events show a clear sequence dependence on the peptide that is being tested. The method is compatible with peptides with either a conjugated N- or C-terminus. The demonstrated results suggest a proof of concept of nanopore sequencing of peptide and can be useful for peptide fingerprinting.

Metallic Oxide-Induced Self-Assembly of Block Copolymers to Form Polymeric Hybrid Micelles with Tunable Stability for Tumor Microenvironment-Responsive Drug Delivery.

Since block copolymers are able to self-assemble into various polymeric architectures, it is intriguing to explore a unique self-assembly strategy for polymers. Two different metallic oxides [manganese dioxide (MnO2) and zinc oxide (ZnO)] are displayed herein to demonstrate this self-assembly mechanism of polymers. In situ generation of metallic oxides induces self-assembly of block copolymers to form polymeric hybrid micelles with tunable stability in aqueous solutions. These final ZnO-cross-linked polymeric micelles exhibited a high drug loading capacity of 0.41 mg mg-1 toward doxorubicin (DOX), whereas DOX-loaded ZnO-cross-linked polymeric micelles could be broken down into Zn2+ and polymer scraps, which facilitated drug release in tumor microenvironments. Both in vitro and in vivo investigations showed that the drug-loaded ZnO-cross-linked polymeric micelles effectively suppressed tumor growth. Accordingly, the present study demonstrates a novel strategy of polymer self-assembly for fabricating polymeric architectures that can potentially provide insight for developing other polymeric architectures.

Clinical therapeutic effects of combined methotrexate and other chemotherapeutic agents in treating children and young patients with osteosarcoma: A protocol for systematic review and meta-analysis.

BACKGROUND: Osteosarcoma is one of the most common primary bone tumour in children and young patients, and the third most common among adults. Its main treatment option is currently based on neoadjuvant or adjuvant chemoradiotherapy along with the lesion's surgical resection. The current study's primary aim is to examine the clinical therapeutic impacts of combined methotrexate, along with other chemotherapeutic agents to treat children and young adults suffering from osteosarcoma. METHODS: We will perform a comprehensive literature search in English database (PubMed, EMBASE, Cochran Library CINAHL, and PsycINFO) and Chinese database (Chinese National Knowledge Infrastructure, VIP information database, Chinese Biomedical Database, and WanFang Database) with no language restriction from their inception to the search date. Additionally, two independent authors will screen the works of literature obtained from these databases, obtain information, and examine the risks of data included for the studies' bias. Furthermore, we intend to employ the Q statistics as well as I2 statistics to calculate heterogeneity among each study's analysis. Accordingly, we will utilize the funnel plots and Egger test to assess the possibility of publication bias where relevant. RESULTS: The current study aims to provide significant information regarding the clinical therapeutic impacts of combines methotrexate along with other chemotherapeutic agents to treat children and young adults suffering from osteosarcoma. CONCLUSIONS: The present study will generate compelling evidence of combined methotrexate as well as other chemotherapeutic agents for osteosarcoma among children and young adults. Also, it will provide clinical practice suggestions. ETHICS AND DISSEMINATION: The study is founded upon published data. Therefore, there is no requirement for ethics approval. OSF REGISTRATION NUMBER: March 26, 2021.osf.io/a23rc. (https://osf.io/a23rc/).

[Comparison of the effectiveness of oblique lumbar interbody fusion and posterior lumbar interbody fusion for treatment of Cage dislodgement after lumbar surgery].

OBJECTIVE: To compare the clinical and radiological effectiveness of oblique lumbar interbody fusion (OLIF) and posterior lumbar interbody fusion (PLIF) in the treatment of Cage dislodgement after lumbar surgery. METHODS: The clinical data of 40 patients who underwent revision surgery due to Cage dislodgement after lumbar surgery betweem April 2013 and March 2017 were retrospectively analyzed. Among them, 18 patients underwent OLIF (OLIF group) and 22 patients underwent PLIF (PLIF group) for revision. There was no significant difference between the two groups in age, gender, body mass index, intervals between primary surgery and revision surgery, number of primary fused levels, disc spaces of Cage dislodgement, and visual analogue scale (VAS) scores of low back pain and leg pain, Oswestry disability index (ODI), the segmental lordosis (SL) and disc height (DH) of the disc space of Cage dislodgement, and the lumbar lordosis (LL) before revision ( P>0.05). The operation time, intraoperative blood loss, hospital stay, and complications of the two groups were recorded and compared. The VAS scores of low back pain and leg pain were evaluated at 3 days, 3, 6, and 12 months after operation, and the ODI scores were evaluated at 3, 6, and 12 months after operation. The SL and DH of the disc space of Cage dislodgement and LL were measured at 12 months after operation and compared with those before operation. CT examination was performed at 12 months after operation, and the fusion of the disc space implanted with new Cage was judged by Bridwell grading standard. RESULTS: The intraoperative blood loss in the OLIF group was significantly less than that in the PLIF group ( t=-12.425, P=0.000); there was no significant difference between the two groups in the operation time and hospital stay ( P>0.05). Both groups were followed up 12-30 months, with an average of 18 months. In the OLIF group, 2 patients (11.1%) had thigh numbness and 1 patient (5.6%) had hip flexor weakness after operation; 2 patients (9.1%) in the PLIF group had intraoperative dural sac tear. The other patients' incisions healed by first intention without early postoperative complications. There was no significant difference in the incidence of complications between the two groups ( χ 2=0.519, P=0.642). The VAS scores of low back pain and leg pain, and the ODI score of the two groups at each time point after operation were significantly improved when compared with those before operation ( P<0.05); there was no significant difference between the two groups at each time point after operation ( P>0.05). At 12 months after operation, SL, LL, and DH in the two groups were significantly increased when compared with preoperative ones ( P<0.05); SL and DH in the OLIF group were significantly improved when compared with those in the PLIF group ( P<0.05), and there was no significant difference in LL between the two groups ( P>0.05). CT examination at 12 months after operation showed that all the operated disc spaces achieved bony fusion. According to the Bridwell grading standard, 12 cases were grade Ⅰ and 6 cases were grade Ⅱ in the OLIF group, and 13 cases were grade Ⅰ and 9 cases were grade Ⅱ in the PLIF group; there was no significant difference between the two groups ( Z=-0.486, P=0.627). During follow-up, neither re-displacement or sinking of Cage, nor loosening or fracture of internal fixation occurred. CONCLUSION: OLIF and PLIF can achieve similar effectiveness in the treatment of Cage dislodgement after lumbar surgery. OLIF can further reduce intraoperative blood loss and restore the SL and DH of the disc space of Cage dislodgement better.

Direct microRNA Sequencing Using Nanopore-Induced Phase-Shift Sequencing.

MicroRNAs (miRNAs) are a class of short non-coding RNAs that function in RNA silencing and post-transcriptional gene regulation. However, direct characterization of miRNA is challenging due to its unique properties such as its low abundance, sequence similarities, and short length. Although urgently needed, single molecule sequencing of miRNA has never been demonstrated, to the best of our knowledge. Nanopore-induced phase-shift sequencing (NIPSS), which is a variant form of nanopore sequencing, could directly sequence any short analytes including miRNA. In practice, NIPSS clearly discriminates between different identities, isoforms, and epigenetic variants of model miRNA sequences. This work thus demonstrates direct sequencing of miRNA, which serves as a complement to existing miRNA sensing routines by the introduction of the single molecule resolution. Future engineering of this technique may assist miRNA-based early stage diagnosis or inspire novel cancer therapeutics.

Effect of Ginsenoside Rg1 on the intervertebral disc degeneration rats and the degenerative pulposus cells and its mechanism.

OBJECTIVE: To explore the effect of ginsenoside Rg1 on intervertebral disc degeneration (IVDD) in vivo and in vitro and its mechanism. METHODS: 60 rats were underwent surgery to construct rat models of IVDD and divided in the sham group, model group and gradient G-Rg1 groups (10 mg/kg/d, 20 mg/kg/d and 40 mg/kg/d).The change of histology was observed by HE staining, the water content and the expression of ß-catenin in IVD were detected. Rat nucleus pulposus cells(NPCs) were isolated from IVDD rats and divided in D-NPCs group, and gradient G-Rg1 groups(20 µg/ml, 50 µg/ml and 100 µg/ml).The cell proliferation activity, cell apoptosis rate,the expression of proteins related to ECM and Wnt/ß-catenin were detected respectively, Finally the agonist of Wnt/ß-catenin pathway LiCl was used for reversed experiments. RESULTS: In vivo, G-Rg1 treatment could improve the structural disorganization, low water content, NPCs number and aggrecan and collagenⅡ expression in IVD and down-regulate the expression of ß-catenin. In vitro NPCs, G-Rg1 treatment could improve the low cell proliferation, high apoptosis rate and low expression of aggrecan and collagenⅡ in degenerative NPCs in a dose-dependent manner.G-Rg1 treatment could down-regulate the expression of proteins related to ß-catenin signal and LiCl could reverse the increase of cell proliferation and ECM synthesis, decrease of apoptosis of degenerative NPCs induced by G-Rg1. CONCLUSION: G-Rg1 could promote ECM synthesis of degenerative NPCs and inhibiting its apoptosis, improve the IVDD via inhibiting the Wnt/ß-catenin pathway.