Plastrum testudinis Ameliorates Oxidative Stress in Nucleus Pulposus Cells via Downregulating the TNF-α Signaling Pathway.

BackgroundPlastrum testudinis (PT), a widely used traditional Chinese medicine, exerts protective effects against bone diseases such as intervertebral disc degeneration (IDD). Despite its effectiveness, the molecular mechanisms underlying the effects of PT on IDD remain unclear. Methods In this study, we used a comprehensive strategy combining bioinformatic analysis with experimental verification to investigate the possible molecular mechanisms of PT against IDD. We retrieved targets for PT and IDD, and then used their overlapped targets for protein-protein interaction (PPI) analysis. In addition, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to investigate the anti-IDD mechanisms of PT. Moreover, in vivo and in vitro experiment validations including hematoxylin-eosin (HE) and safranine O-green staining, senescence-associated ß-galactosidase (SA-ß-gal) assay, cell immunofluorescence staining, intracellular ROS measurement and Western blot analysis were performed to verify bioinformatics findings. Results We identified 342 and 872 PT- and IDD-related targets (32 overlapping targets). GO enrichment analysis yielded 450 terms related to oxidative stress and inflammatory response regulation. KEGG analysis identified 48 signaling pathways, 10 of which were significant; the TNF-α signaling pathway had the highest p-value, and prostaglandin G/H synthase 2 (PTGS2), endothelin-1 (EDN1), TNF-α, JUN and FOS were enriched in this pathway. Histopathological results and safranin O/green staining demonstrated that PT attenuated IDD, and SA-ß-gal assay showed that PT ameliorated nucleus pulposus cell (NPC) senescence. An ROS probe was adopted to confirm the protective effect of PT against oxidative stress. Western blot analyses confirmed that PT downregulated the protein expression of PTGS2, EDN1, TNF-α, JUN and FOS in the TNF-α signaling pathway as well as cellular senescence marker p16, proinflammatory cytokine interleukin-6 (IL6), while PT upregulated the expression of NPC-specific markers including COL2A1 and ACAN in a concentration-dependent manner. Conclusions To the best of our knowledge, this study is the first to report that PT alleviates IDD by downregulating the protein expression of PTGS2, EDN1, TNF-α, JUN and FOS in the TNF-α signaling pathway and upregulating that of COL2A1 and ACAN, thus suppressing inflammatory responses and oxidative stress in NPCs.

miR-330-5p in Small Extracellular Vesicles Derived From Plastrum testudinis-Preconditioned Bone Mesenchymal Stem Cells Attenuates Osteogenesis by Modulating Wnt/ß-Catenin Signaling.

The bone microenvironment is crucial for the growth and development of different types of osteocytes. Small extracellular vesicles (sEVs) secreted by bone mesenchymal stem cells are delivered to target cells where their contents regulate biological functions. Here, we evaluated the osteogenic effects and mechanism of sEVs derived from Plastrum testudinis-preconditioned bone mesenchymal stem cells (PT-sEV). The osteogenic effects of PT-sEV were evaluated by the differentiation of osteoblasts and the alternation of bone quality and quantity in ovariectomized rats. The specific mechanism was explored by high-throughput sequencing and verified by transfection with the corresponding miRNA mimic and inhibitor. RNA-sequence identified a unique enrichment of a set of miRNAs in PT-sEV compared with sEVs derived from untreated BMSCs. Overexpression or inhibition in vitro indicated that the osteogenic inducing potential of sEVs was mainly attributable to miR-330-5p, one of the most dramatically downregulated miRNAs in the PT-sEV fraction. Dual luciferase reporter assays showed that miR-330-5p negatively regulated osteogenesis by directly binding to the 3' untranslated region of Tnc. Additional experiments showed that Tnc regulated Wnt/ß-catenin signaling, and rescue experiment showed that miR-330-5p could restore ß-catenin expression; additionally, animal experiments indicated that Wnt signaling was inactivated in the ovariectomized rats. These data demonstrated the regenerative potential of PT-sEV, which induced osteogenic differentiation of pre-osteoblasts, leading to bone formation. This process was achieved by delivering miR-330-5p, which regulated Tnc to control Wnt/ß-catenin signaling.

Protective effect of plastrum testudinis extract on dopaminergic neurons in a Parkinson's disease model through DNMT1 nuclear translocation and SNCA's methylation.

The pathological characteristics of Parkinson's disease (PD) include dopaminergic neuron damage, specifically disorders caused by dopamine synthesis, in vivo. Plastrum testudinis extract (PTE) and its bioactive ingredient ethyl stearate (PubChem CID: 8122) were reported to be correlated with tyrosine hydroxylase (TH), which is a biomarker of dopaminergic neurons. This suggests that PTE and its small-molecule active ingredient ethyl stearate have potential for development as a therapeutic drug for PD. In this study, we treated 6-hydroxydopamine (6-OHDA)-induced model rats and PC12 cells with PTE. The mechanism of action of PTE and ethyl stearate was investigated by western blotting, bisulfite sequencing PCR (BSP), real-time PCR, immunofluorescence and siRNA transfection. PTE effectively upregulated the TH expression and downregulated the alpha-synuclein expression in both the substantia nigra and the striatum of the midbrain in a PD model rat. The PC12 cell model showed that both PTE and its active monomer ethyl stearate significantly promoted TH expression and blocked alpha-synuclein, agreeing with the in vivo results. BSP showed that PTE and ethyl stearate increased the methylation level of the Snca intron 1 region. These findings suggest that some of the protective effects of PTE on dopaminergic neurons are mediated by ethyl stearate. The mechanism of ethyl stearate may involve disrupting the abnormal aggregation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) with alpha-synuclein by releasing DNMT1, upregulating Snca intron 1 CpG island methylation, and ultimately, reducing the expression of alpha-synuclein.

Plastrum testudinis extract suppresses osteoclast differentiation via the NF-κB signaling pathway and ameliorates senile osteoporosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Plastrum testudinis (PT) is a kind of single traditional Chinese medicine that can tonify kidney and strengthen bone. Plastrum testudinis extract (PTE) has been approved to promote the osteogenic differentiation of bone marrow-derived mesenchymal stem cells in vitro. However, the mechanism by which PTE reduces osteoclast differentiation has not yet been reported. AIM OF THE STUDY: To explore the potential of PTE as a therapeutic treatment for bone loss caused by senile osteoporosis (SOP). MATERIALS AND METHODS: We evaluated whether PTE could inhibit RANKL-induced osteoclast differentiation both in vitro and in vivo, and investigated PTE-induced phenotypes of human peripheral blood monocytes. RESULTS: We found that PTE inhibited osteoclast differentiation and bone resorption in vitro in a concentration-dependent manner and that PTE treatment is most effective during the early stages of osteoclastogenesis. Moreover, we found that PTE could block the NF-κB signaling pathway in vitro, leading to the down-regulation of osteoclast-specific genes including C-FOS and NFATC1. The results from our in vivo mouse study suggest that PTE treatment suppresses osteoclast formation and mitigates bone loss caused by SOP. Notably, we also found that PTE inhibited RANKL-induced osteoclast differentiation in human peripheral blood monocytes. CONCLUSION: Our results suggest that PTE treatment suppresses osteoclastogenesis and ameliorates bone loss caused by SOP by selectively blocking the nuclear translocation of NF-κB/p50.

Network pharmacology integrated with experimental validation reveals the regulatory mechanism of plastrum testudinis in treating senile osteoporosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Plastrum testudinis (PT) has been used in traditional Chinese medicine to treat bone diseases such as senile osteoporosis (SOP) for thousands of years. However, the underlying mechanisms remain largely unknown. AIM OF THE STUDY: This study aims to investigate the possible molecular mechanism of PT in the treatment of SOP using an integrated strategy of network pharmacology and experimental validation. MATERIALS AND METHODS: The compounds of PT and its targets were identified through the BATMAN-TCM database. The SOP-related targets were retrieved from the GeneCards database. Protein-protein interaction information was obtained by inputting the intersection targets into the STRING database. Cytoscape software was used to construct a protein-protein interaction network and a PT-compound-target-SOP network. Using Cytoscape and R software, we conducted GO function and KEGG pathway enrichment analyses. We also conducted in vivo and in vitro experiments to verify the network pharmacology findings. RESULTS: In total, 6 active compounds and 342 targets of PT were screened, of which 57 common targets were related to SOP. The GO biological process enrichment analysis identified 880 entries, mainly relating to the regulation of hormone response, the cell apoptotic process, the apoptotic signaling pathway, NF-kappaB transcription factor activity, fatty acid transportation, osteoclast differentiation, macrophage activation, and inflammatory response. The KEGG pathway enrichment analysis identified 52 entries, including 14 related signaling pathways, which mainly involved the TNF, MAPK, IL-17, AGE-RAGE, estrogen, relaxin, and other signaling pathways. Our in vivo experiments confirmed that PT alleviates SOP, while the in vitro experiments demonstrated that PT exerts a suppressive effect on osteoclast differentiation and bone resorption in a concentration-dependent manner. Furthermore, we observed that PT downregulates the expression of osteoclast-specific genes, including C-FOS, TNF, and BDNF, in the MAPK signaling pathway. CONCLUSION: Through network pharmacology and experimental validation, this study is the first to report that PT downregulates the expression of osteoclast-specific genes, including C-FOS, TNF, and BDNF, in the MAPK signaling pathway, thus exerting a suppressive effect on osteoclast differentiation and bone resorption, which may be the molecular mechanism for PT treatment of SOP.

Identification of Plastrum Testudinis used in traditional medicine with DNA mini-barcodes

ABSTRACT The carapace of the tortoise Chinemys reevesii is an ingredient of "Guijia", a traditional Chinese medicine. However, C. reevesii is difficult to raise in aquaculture and is rare in the wild. Counterfeit tablets are made from carapaces of other species. In addition to C. reevesii, other species including Mauremys sinensis, Indotestudo elongate and Trachemys scripta have been used in Plastrum Testudinis as well. After processing, these carapaces are difficult to identify on the basis of morphological characteristics, which impedes law enforcement. Our study used DNA barcoding technology to identify C. reevesii and its substitutes. We extracted concentrated genomic DNA for PCR amplification. Based on the analysis of 61 full-length COI sequences, we designed four pairs of mini-barcode primers: Tu-A, Tu-B, Tu-C and Tu-D. The Tu-B primers sequenced genomic DNA with a success rate of 76.47%, and the Tu-D primers sequenced genomic DNA with a success rate of 88.24%. The identification efficiency of these two mini-barcodes was 70.59% and 64.71%, and the overall identification efficiency was approximately 76.47%. Similarly, a set of mini barcode systems was generated, which may provide an effective and low-cost method for the identification of authentic tortoise shells.

Vit D promotes proliferation and differentiation of mesenchymal stem cells through regulates extracts of plastrum testudinis / 局解手术学杂志

Objective To explore the effect and the mechanism of vitamin D(Vit D) promotes proliferation and differentiation of mesenchymal stem cells (MSCs) through regulates extracts of plastrum testudinis (PTE).Methods Established the PGL3-Id1 promoter and transfered rat MSCs.PTE combined with 10-6,10-7,10-8mol/L Vit D respectively were acted on the transfected MSCs for 36 hours.The level of Id1 promoter were detected by luciferase activity measurement.1,3,30,100 pg/mL PTE combined with Vit D of 10-7 mol/L were acted on MSCs for 36 hours,3 days and 7 days,and the VDR expression were detected by RT-PCR test.Results PTE promoted the expression of Id1 in MSCs,the expression of Id1 was inhibited when PTE combined with Vit D (P ＜ 0.01),and it was significantly different among different dosis of Vit D(P ＜0.01).The expression of VDR was inhibited in different degree when PTE combined with Vit D for 36 hours,3 days and 7 days.PTE combed with large dose of Vit D for 36 hours had significant effect of inhibition,and the difference was statistically significant (P ＜ 0.05).The inhibiting effect was more obvious when PTE combined with large dose of Vit D for 3 days and 7 days.When different doses of PTE combined with Vit D for a same duration,the difference of VDR expression was statistically significant (P ＜ 0.05).Meanwhile,when same doses of PTE combined with Vit D for different durations,the difference of VDR expression at 7 days and 36 hours was statistically significant (P ＜ 0.05).Conclusion The proliferation of MSCs which promoted by PTE was inhibited by Vit D,and the nuclear receptor VDR may be one of the targets of drug action for PTE regulating proliferation and differentiation of MSCs.

Extracts from plastrum testudinis reverse glucocorticoid-induced spinal osteoporosis of rats via targeting osteoblastic and osteoclastic markers.

Extracts from plastrum testudinis (PTE), an important traditional Chinese medicine, have been demonstrated promotion of osteoblastic function in vitro. This study aims to investigate the protective effect of PTE on glucocorticoid-induced osteoporosis(GIOP) in vivo and analyze therapeutic targets of PTE on GIOP. SD rats were randomly assigned to two experiments: preventive and therapeutic experiments, in which rats respectively received oral PTE at the same time of glucocorticoid injection or after glucocorticoid injection inducing osteoporosis. BMD, microarchitecture, biomechanics, bone metabolism markers and histomorphology were evaluated. mRNA and protein expression of OPG, Runx2, CTSK and MMP9 were examined.Results showed bone quality and bone quantity were significantly elevated by PTE. Histomorphometry showed thicker and denser bone trabecularsand more osteoblasts and less osteoclasts in group of PTE intervention. The mRNA expression of OPG was significantly upregulated whereas expression of CTSK was significantly downregulatedin different groups of PTE intervention. Stronger immunostaining for Runx2 and weaker immunostaining for CTSK were observed in groups of PTE intervention. This demonstrated that PTE may reverse GIOP in prevention and management via targeting OPG, Runx2 and CTSK in mRNA and protein levels.

Changes of microRNA Expression During Neuronal Differentiation of Neural Stem Cells Induced by Plastrum Testudinis Extract / 广州中医药大学学报

Objective To explore the changes of microRNA expression during the neuronal differentiation of neural stem cells (NSCs) induced by Plastrum testudinis extract (PTE) . Methods NSCs were isolated from the hippocampus of 14-day SD rat fetus and were obtained after primary culture. The abilities of NSCs differenting into neurons were identified by immunofluorescent staining. NSCs were randomly divided into three groups, blank control group, and low- and high-dose PTE groups ( 3, 30 μg/mL PTE) . After the cells were incubated with PTE for 7 days, total RNA was isolated and then the expression of miR-124 and miR-9 was observed by real-time fluorescence quantitative polymerase chain reaction ( qRT-PCR) . Results Compared with the blank control group, the expression of miR-124 and miR-9 remained unchanged in low-dose PTE group, but was significantly increased in high-dose PTE group ( P<0.05). Conclusion PTE promotes NSCs differentiating into neurons, which might be associated with the up-regulation of miR-124 and miR-9.

Improvement in Soxhlet Extraction with Ore and Shell Hard Chinese Materia Medica / 中国药房

OBJECTIVE:To discuss the improvement in Soxhlet extraction with ore and shell hard Chinese meteria madica taking Plastrum Testudinis as example.METHODS:On the basis of normal Soxhlet extracter,larger Soxhlet extracter on Plastrum Testudinis was experimented using saturated salt-water-bath,quadruplicity cover circle and timer.Large Soxhlet extracter was compared with normal Soxhlet extracter and stirring-reflux method by HPLC analysis,yield and clearance rate of DPPH.RESULTS:There were no significant difference between normal and larger Soxhlet extracters,but the stirring-reflux method was inferior to these two methods.CONCLUSION:The improved larger Soxhlet extraction is a thorough,high-efficient,convenient,economical and healthy extracting method for ore and shell hard Chinese materia medica.

In-Vitro Induction of Mesenchymal Stem Cells Differentiation intoNeurons in Adult Rats by Serum ContainingCarapax et Plastrum Testudinis / 广州中医药大学学报

[Objective] To observe the effect of serum containing Carapax et Plastrum Testudinis (CPT) in inducing the differentiation of mesenchymal stem cells (MSC) of adult rats into neurons. [Methods] The fifth generation of MSC were induced by the serum containing CPT (sero-CPT) and the expressions of neurofilament (NF) protein and glial fibrillary acidic protein (GFAP), the marks of neuron-like cells were detected by immunohistochemical method. [Results] The expression of NF protein was positive after the stimulation of sero-CPT and the expression reached the peak 12 hours after induction. [Conclusion] Sero-CPT can induce the differentiation of MSC into neurons in vitro.

Effect of Plastrum Testudinis on Three Subtypes of NOS Expression in Rats with Focal Cerebral Ischemia / 中药新药与临床药理

Objective: To explore the effect of Plastrum Testudinis on the three subtypes of NOS (nNOS,iNOS,eNOS) expression in rats with focal cerebral ischemia. Methods:Rat model of focal cerebral ischemia was set up by inserting nylon thread into the internal carotid artery . The expressions of nNOS, iNOS and eNOS was detected by immunohistochemical method to observe the effects of Plastrum Testudinis. Results: Expressions of nNOS and iNOS in cerebral cortex and caudate-putamen of Plastrum Testudinis treatment group were significantly lower than those of model control group (P

Effect of Plastrum Testudinis on Neural Stem Cell After Focal Cerebral Ischemia / 广州中医药大学学报

Objective To investigate the effect of Plastrum Testudinis on neural stem cell after focal cerebral ischemia.Methods Rat models of focal cerebral ischemia were established by inserting nylon thread into the anterior cerebral artery.Immunohistochemical method was used to detect the expression of the neuroepithelial stem cell protein(Nestin),neurologic deficit and histological features were also observed.Results Plastrum Testudinis can decrease the neurologic dificit score and promote the expression of Nestin.Conclusion Plastrum Testudinis can alleviate the neurological symptoms and promote the proliferation of neural stem cell after focal cerebral ischemia.

Effect of Extract of Carapax et Plastrum Testudinis on Nuclear Receptor in the Proliferation of Rat Mesenchymal Stem Cell in Vitro / 广州中医药大学学报

[Objective] To observe the effect of extract of Carapax et Plastrum Testudinis (ECPT) on nuclear receptor in the proliferation of rat mesenchymal stem cell (MSC) in vitro. [Methods] The rat MSC dissociated from bone marrow by density gradient method were cultured and identified by marking of bromodeoxyuridine (Brdu) and staining of CD44. Then different doses of ECPT (3333, 333.3 and 33.33 ?g/mL) were respectively added into in-vitro cultured MSC for 12, 24, 72 and 120 hours. The expression of retinoic acid receptor-?(RAR?), vitamin D receptor (VDR), estrogen receptor (ER), glucocorticoid receptor (GR), thyroid hormone receptor-?(TR?) and peroxisome proliferator-activated receptor ?(PPAR?) in MSC was detected by immunohistochemistry and immunofluorescence methods. [Results] The number of RAR?-and VDR-positive cells in ECPT groups was higher than that in the control group (P

Effects of Plastrum Testudinis on Os teogenesis of Cultured Rat Mesenchymal Stem Cells / 中药新药与临床药理

Objective To investigate the effects of kidney-tonifying Plastrum Testudinis on o steogenesis of cultured rat mesenchymal stem cells(MSCs ).Methods MSCs were isolated from adult rats wi th density gradient separation meth od.Osteogenesis of MSCs in the culture a dded with different concentrations of serum containing Plastrum Testudinis was evalu-ated by detecting bone glaprotein(BGP)level and observing the morphologic al features of MSCs and by alkaline ph os-phatase(ALP)and Von Kossa immunohistochemical m ethods.Results Morphological examination showed t hat the MSCs attachment formed soon after seedin g ,and grew into colonies with the appearance of fibroblastic cells.Seru m containing Plastrum Testudinis promoted the expression of alkaline phosphatase(ALP)in MSCs ,and increased BGP level and the number of calcified deposition in dose -dependant manner,the difference being significant in comparison wi th the control groups(P

Influences of Ultra-fine Crushing Method on Dissolution Rate and Decocting Rate of Water-Soluble Protein in Plastrum Testudinis / 中药新药与临床药理

Objective To compare the effects of two crushing methods on dissolution rate and decocting rate of water-soluble protein in Plastrum Testudinis.Methods The dissolution rate of water-soluble protein was compared in Plastrum Testudinis processed by the traditional crushing method(passing 100-eyes screen,fine powder)and ultra-fine crushing method(passing 300-eye screen,ultra-fine powder);besides,orthogonal design was applied to study the process technical condition of decocting rate in Plastrum Testudinis.Results Water-soluble protein in fine powder of Plastrum Testudinis was hardly detected while the dissolution rate of water-soluble protein in ultra-fine powder was 51.2 %in 20 minutes and up to 70.5 %at 2 hours.Three influencing factors of fineness,decocting frequencies and decocting time were measured with orthogonal test.The results of variance analysis showed that fineness significantly influenced the decocting rate(P .05).Conclusion The ultra-fine powder technique is propitious to the increase of dissolution rate and decocting rate of water-soluble protein in Plastrum Testudinis.