Pyrroloquinoline quinone alleviates natural aging-related osteoporosis via a novel MCM3-Keap1-Nrf2 axis-mediated stress response and Fbn1 upregulation.

Age-related osteoporosis is associated with increased oxidative stress and cellular senescence. Pyrroloquinoline quinone (PQQ) is a water-soluble vitamin-like compound that has strong antioxidant capacity; however, the effect and underlying mechanism of PQQ on aging-related osteoporosis remain unclear. The purpose of this study was to investigate whether dietary PQQ supplementation can prevent osteoporosis caused by natural aging, and the potential mechanism underlying PQQ antioxidant activity. Here, we found that when 6-month-old or 12-month-old wild-type mice were supplemented with PQQ for 12 months or 6 months, respectively, PQQ could prevent age-related osteoporosis in mice by inhibiting osteoclastic bone resorption and stimulating osteoblastic bone formation. Mechanistically, pharmmapper screening and molecular docking studies revealed that PQQ appears to bind to MCM3 and reduces its ubiquitination-mediated degradation; stabilized MCM3 then competes with Nrf2 for binding to Keap1, thus activating Nrf2-antioxidant response element (ARE) signaling. PQQ-induced Nrf2 activation inhibited bone resorption through increasing stress response capacity and transcriptionally upregulating fibrillin-1 (Fbn1), thus reducing Rankl production in osteoblast-lineage cells and decreasing osteoclast activation; as well, bone formation was stimulated by inhibiting osteoblastic DNA damage and osteocyte senescence. Furthermore, Nrf2 knockout significantly blunted the inhibitory effects of PQQ on oxidative stress, on increased osteoclast activity and on the development of aging-related osteoporosis. This study reveals the underlying mechanism of PQQ's strong antioxidant capacity and provides evidence for PQQ as a potential agent for clinical prevention and treatment of natural aging-induced osteoporosis.

1,25-Dihydroxyvitamin D Deficiency Accelerates Aging-related Osteoarthritis via Downregulation of Sirt1 in Mice.

Emerging observational data suggest that vitamin D deficiency is associated with the onset and progression of knee osteoarthritis (OA). However, the relationship between vitamin D level and OA and the role of vitamin D supplementation in the prevention of knee OA are controversial. To address these issues, we analyzed the articular cartilage phenotype of 6- and 12-month-old wild-type and 1α(OH)ase-/- mice and found that 1,25(OH)2D deficiency accelerated the development of age-related spontaneous knee OA, including cartilage surface destruction, cartilage erosion, proteoglycan loss and cytopenia, increased OARSI score, collagen X and Mmp13 positive chondrocytes, and increased chondrocyte senescence with senescence-associated secretory phenotype (SASP). 1,25(OH)2D3 supplementation rescued all knee OA phenotypes of 1α(OH)ase-/- mice in vivo, and 1,25(OH)2D3 rescued IL-1ß-induced chondrocyte OA phenotypes in vitro, including decreased chondrocyte proliferation and cartilage matrix protein synthesis, and increased oxidative stress and cell senescence. We also demonstrated that VDR was expressed in mouse articular chondrocytes, and that VDR knockout mice exhibited knee OA phenotypes. Furthermore, we demonstrated that the down-regulation of Sirt1 in articular chondrocytes of 1α(OH)ase-/- mice was corrected by supplementing 1,25(OH)2D3 or overexpression of Sirt1 in mesenchymal stem cells (MSCs) and 1,25(OH)2D3 up-regulated Sirt1 through VDR mediated transcription. Finally, we demonstrated that overexpression of Sirt1 in MSCs rescued knee OA phenotypes in 1α(OH)ase-/- mice. Thus, we conclude that 1,25(OH)2D3, via VDR-mediated gene transcription, plays a key role in preventing the onset of aging-related knee OA in mouse models by up-regulating Sirt1, an aging-related gene that promotes articular chondrocyte proliferation and extracellular matrix protein synthesis, and inhibits senescence and SASP.

Calcareous Materials Effectively Reduce the Accumulation of Cd in Potatoes in Acidic Cadmium-Contaminated Farmland Soils in Mining Areas.

The in situ chemical immobilization method reduces the activity of heavy metals in soil by adding chemical amendments. It is widely used in farmland soil with moderate and mild heavy metal pollution due to its high efficiency and economy. However, the effects of different materials depend heavily on environmental factors such as soil texture, properties, and pollution levels. Under the influence of lead-zinc ore smelting and soil acidification, Cd is enriched and highly activated in the soils of northwestern Guizhou, China. Potato is an important economic crop in this region, and its absorption of Cd depends on the availability of Cd in the soil and the distribution of Cd within the plant. In this study, pot experiments were used to compare the effects of lime (LM), apatite (AP), calcite (CA), sepiolite (SP), bentonite (BN), and biochar (BC) on Cd accumulation in potatoes. The results showed that the application of LM (0.4%), AP (1.4%), and CA (0.4%) had a positive effect on soil pH and cations, and that they effectively reduced the availability of Cd in the soil. In contrast, the application of SP, BN, and BC had no significant effect on the soil properties and Cd availability. LM, AP, and CA treatment strongly reduced Cd accumulation in the potato tubers by controlling the total 'flux' of Cd into the potato plants. In contrast, the application of SP and BN promoted the migration of Cd from the root to the shoot, while the effect of BC varied by potato genotype. Overall, calcareous materials (LM, CA, and AP) were more applicable in the remediation of Cd-contaminated soils in the study area.

Inhibition of Nrf2 degradation alleviates age-related osteoporosis induced by 1,25-Dihydroxyvitamin D deficiency.

Previous studies have shown that 1,25(OH)2D plays an anti-osteoporosis role by an anti-aging mechanism. Oxidative stress is a key mediator of aging and bone loss; however, whether 1,25(OH)2D can exert its anti-osteoporosis effect by inhibiting oxidative stress is unclear. In this study, osteoporosis and the bone aging phenotype induced by 1,25(OH)2D deficiency in male mice were significantly rescued in vivo upon the supplementation of oltipraz, an inhibitor of Nrf2 degradation. Increased oxidative stress, cellular senescence and reduced osteogenesis of BM-MSCs from VDR knockout mice were also significantly rescued when the cells were pre-treated with oltipraz. We found that 1,25(OH)2D3 promoted Nrf2 accumulation by inhibiting its ubiquitin-proteasome degradation, thus facilitating Nrf2 activation of its transcriptional targets. Mechanistically, 1,25(OH)2D3 enhances VDR-mediated recruitment of Ezh2 and facilitation of H3K27me3 action at the promoter region of Keap1, thus transcriptionally repressing Keap1. To further validate that the Nrf2-Keap1 pathway serves as the key mediator in the anabolic effect of 1,25(OH)2D3 on bone, Nrf2-/- mice, or hBM-MSCs with shRNA-mediated Nrf2-knockdown, were treated with 1,25(OH)2D3; we found that Nrf2 knockout largely blocked the bone anabolic effect of 1,25(OH)2D3 in vivo and ex vivo, and Nrf2 knockdown in hBM-MSCs markedly blocked the role of 1,25(OH)2D3 in inhibiting oxidative stress and promoting osteogenic differentiation and bone formation. This study provides insight into the mechanism whereby 1,25(OH)2D3 postpones age-related osteoporosis via VDR-mediated activation of Nrf2-antioxidant signaling and inhibition of oxidative stress, and thus provides evidence for oltipraz as a potential reagent for clinical prevention and treatment of age-related osteoporosis.

1,25-Dihydroxyvitamin D protects against age-related osteoporosis by a novel VDR-Ezh2-p16 signal axis.

To determine whether 1,25-dihydroxyvitamin D (1,25(OH)2 D) can exert an anti-osteoporosis role through anti-aging mechanisms, we analyzed the bone phenotype of mice with 1,25(OH)2 D deficiency due to deletion of the enzyme, 25-hydroxyvitamin D 1α-hydroxylase, while on a rescue diet. 1,25(OH)2 D deficiency accelerated age-related bone loss by activating the p16/p19 senescence signaling pathway, inhibiting osteoblastic bone formation, and stimulating osteoclastic bone resorption, osteocyte senescence, and senescence-associated secretory phenotype (SASP). Supplementation of exogenous 1,25(OH)2 D3 corrected the osteoporotic phenotype caused by 1,25(OH)2 D deficiency or natural aging by inhibiting the p16/p19 pathway. The proliferation, osteogenic differentiation, and ectopic bone formation of bone marrow mesenchymal stem cells derived from mice with genetically induced deficiency of the vitamin D receptor (VDR) were significantly reduced by mechanisms including increased oxidative stress, DNA damage, and cellular senescence. We also demonstrated that p16 deletion largely rescued the osteoporotic phenotype caused by 1,25(OH)2 D3 deficiency, whereas 1,25(OH)2 D3 could up-regulate the enzyme Ezh2 via VDR-mediated transcription thereby enriching H3K27me3 and repressing p16/p19 transcription. Finally, we demonstrated that treatment with 1,25(OH)2 D3 improved the osteogenic defects of human BM-MSCs caused by repeated passages by stimulating their proliferation and inhibiting their senescence via the VDR-Ezh2-p16 axis. The results of this study therefore indicate that 1,25(OH)2 D3 plays a role in preventing age-related osteoporosis by up-regulating Ezh2 via VDR-mediated transcription, increasing H3K27me3 and repressing p16 transcription, thus promoting the proliferation and osteogenesis of BM-MSCs and inhibiting their senescence, while also stimulating osteoblastic bone formation, and inhibiting osteocyte senescence, SASP, and osteoclastic bone resorption.

A novel bacterial type II l-asparaginase and evaluation of its enzymatic acrylamide reduction in French fries.

This study reports the identification of a novel bacterial type II l-asparaginase, abASNase2, from Aquabacterium sp. A7-Y. The enzyme contains 319 amino acids and shared 35% identity with Escherichia coli type II l-asparaginase (EcAII), a commercial enzyme trademarked Elspar® that is widely used for medical applications. abASNase2 had high specific activity (458.9U/mg) toward l-asparagine, very low activity toward l-glutamine and d-glutamine and no activity toward d-asparagine. The optimal enzymatic activity conditions for abASNase2 were found to be 50mM Tris-HCl buffer (pH 9.0) at 60°C. It was very stable in the pH range of 7.0-11.0 and exhibited up to 80% relative activity after 2h below 40°C. The Km and kcat of abASNase2 were 1.8×10-3M and 241.9s-1, respectively. In addition, abASNase2's ability to remove acrylamide from fried potato strips was evaluated. Compared to untreated potato strips (acrylamide content: 0.823±0.0457mg/kg), 88.2% acrylamide was removed in the abASNase2-treated group (acrylamide content: 0.097±0.0157mg/kg). These results indicate that the novel l-asparaginase abASNase2 is a potential candidate for applications in the food processing industry.

[Effect of Acupotomy Lysis at Cervical Acupoints on Expression of Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinase 1 and Changes of Pulpiform Nucleus Ultrastructure in Rats with Degenerated Cervical Intervertebral Discs].

OBJECTIVE: To observe the effect of acupotomy lysis at acupoints around the neck on expression of matrix metalloproteinase 1 (MMP-1), MMP-2 and tissue inhibitor of metalloproteinase 1 (TIMP-1) genes and ultrastructure of pulpiform nucleus in cervical intervertebral disc degeneration (IVDD) rats, so as to explore its mechanism underlying easing IVDD. METHODS: SD rats were randomly allocated to control (n = 15), model (n = 14), Jiaji (EX-B 2, n = 13), cervico-acupoint (n = 14) and medication groups (n = 14). The cervical IVDD model was established by using static-dynamic imbalance method. For rats of the Jiaji (EX-B 2) and cervico-acupoint groups, EX-B 2-points of the cervical 2-7 segments, and peri-cervical acupoints: bilateral "Naokong" (GB 19) , "Naohu" (GV 17), "Dazhui" (GV 14), bilateral "Quyuan" (SI 13) and bilateral "Tianzong" (SI 11) were separately punctured with a needle-knife, once every 5 days for 3 times, and for rats of the medication group, Brufen Capsules (15 mg · kg(-1) · d(-1)) and Jingfukang Granule (0.5 mg · kg(-1) · d(-1)) were given by intragastric administration, once daily for 10 days. The expression levels of MMP-1, MMP-3 and TIMP-1 genes in the pulpiform nucleus of cervical intervertebral discs were detected by RT-PCR and changes of the ultrastructure of the pulpiform nucleus observed under transmission electron microscope. RESULTS: Compared to the control group, the expression levels of MMP-1 mRNA and MMP-3 mRNA of the cervical intervertebral disc tissues were significantly up-regulated in the model group (P < 0.05), and that of TIMP-1 mRNA was obviously down-regulated in the model group (P < 0.05). After the treatment, the increased expression of MMP-1 mRNA and MMP-3 mRNA and the decreased expression of TIMP-1 mRNA were reversed by acupotomy lysis and medication (P < 0.05) except TIMP-1 mRNA in the medication group (P > 0.05). No significant differences were found between the Jiaji (EX-B 2) and cervico-acupoint groups in down-regulating MMP-1 mRNA and MMP-3 mRNA expression and up-regulating TIMP-1 mRNA expression (P > 0.05). Results of electron microscope examinations showed that the ultrastructural injury changes of cells of the pulpiform nucleus were relatively milder in the Jiaji (EX-B 2) and cervico-acupoint groups, followed by the medication group in comparison with those of the model group. CONCLUSION: Acupotomy lysis at acupoints around the neck can improve the ultrastructural changes of cells of the pulpiform nucleus of cervical intervertebral discs in IVDD rats, which is possibly by regulating the expression of MMP-1, MMP-3 and TIMP-1 genes.

[Effect of Acupotome Relaxing on Expression of Type I and II Collagens of Degenerative Cervical Inter- vertebral Discs in Rats].

OBJECTIVE: To observe the effect of acupotome relaxing at cervical acupoints on type I and II collagens of degenerated cervical intervertebral discs in rats, so as to explore its potential mechanism underlying anti-degeneration of intervertebral discs. METHODS: Rats were randomly divided into control, model, Jiaji acupoints, cervical acupoints and medication groups (n = 15 in each group). The rat model of cervical intervertebral disc degeneration due to static-dynamic imbalance was made as previously specified. The Jiaji acupoints were those located along the cervical vertebra 2-7. The cervical acupoints included bilateral "Naokong"(GB 19) , "Naohu" (GV 17) , "Dazhui"(GV 14) , bilateral "Quyuan" (SI 13) and bilateral "Tianzong" (SI 11). Acupoints were treated according to the procedures of acupotome for 3 times in ten days with five days' break between every two treatment sessions. Rats of the medication group were intragastrically administered with Jing Fu Kang Granules and ibuprofen daily for ten days. Twenty days after the end of treatment, all rats were sacrificed for further examination of morphological changes of the intervertebral disc tissue. Immunoactivity of protein and mRNA expression levels of collagen type I and II of the intervertebral discs were measured by means of immunohistochemistry and RT-PCR, respectively. RESULTS: In comparison with the control group, the immunoactivity and mRNA expression levels of collagen type I and II of the intervertebral discs were significantly elevated or reduced in rats of the model group, respectively (P < 0.05). After acupotome intervention and medication, the increased and decreased expression levels of type I and II collagen proteins and genes were markedly reversed (P < 0.05). The effects of acupotome relaxing of both cervical and Jiaji acupoints were significantly superior to those of medication in down-regulating expression of type I collagen protein and mRNA, and in up-regulating that of type II collagen protein and mRNA (P < 0.05). No significant differences were found between the cervical acupoints and Jiaji acupoints groups in the above- mentioned outcomes (P > 0.05) . The degree of severity of the degenerated intervertebral discs was the worst in the model group, followed by the medication group, then the Jiaji acupoints group and cervical acupoints group, and the control group the least. CONCLUSION: Acupotome at neck acupoints can regulate the extracellular matrix of the intervertebral disc via inhibiting the transformation between type I and type II collagens, which may contribute to its effect in delaying the degenerative process of the cervical intervertebral discs.

The effects of paeoniflorin monomer of a Chinese herb on cardiac ion channels / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Because of the potential proarrhythmic effect of current antiarrhythmic drugs, it is still desirable to find safer antiarrhythmic drugs worldwide. Paeoniflorin is one of the Chinese herb monomers that have different effects on many ion channels. The present study aimed to determine the effects of paeoniflorin on cardiac ion channels.</p><p><b>METHODS</b>Whole-cell patch-clamp technique was used to record ion channel currents. L-type calcium current (I(Ca-L)), inward rectifier potassium current (I(K1)), and transient outward potassium current (I(to1)) were studied in rat ventricular myocytes and sodium current (I(Na)), slow delayed rectifier current (I(Ks)), and HERG current (I(Kr)) were investigated in transfected human embryonic kidney 293 cells.</p><p><b>RESULTS</b>One hundred µmol/L paeoniflorin reduced the peak I(Ca-L) by 40.29% at the test potential of +10 mV (from (-9.78 ± 0.52) pA/pF to (-5.84 ± 0.89) pA/pF, n = 5, P = 0.028). The steady-state activation curve was shifted to more positive potential in the presence of the drug. The half activation potentials were (-11.22 ± 0.27) mV vs. (-5.95 ± 0.84) mV (n = 5, P = 0.007), respectively. However, the steady-state inactivation and the time course of recovery from inactivation were not changed. One hundred µmol/L paeoniflorin completely inhibited the peak I(Na) and the effect was reversible. Moreover, paeoniflorin inhibited the I(K1) by 30.13% at the test potential of -100 mV (from -25.26 ± 8.21) pA/pF to (-17.65 ± 6.52) pA/pF, n = 6, P = 0.015) without effects on the reversal potential and the rectification property. By contrast, 100 µmol/L paeoniflorin had no effects on I(to1), I(Ks) or I(Kr) channels.</p><p><b>CONCLUSIONS</b>The study demonstrated that paeoniflorin blocked I(Ca-L), I(Na), and I(K1) without affecting I(to1), I(Ks), or I(Kr). The multi-channel block effect may account for its antiarrhythmic effects with less proarrhythmic potential.</p>