Mitochondrial biogenesis disorder and oxidative damage promote refractory apical periodontitis in rat and human.

AIM: To elucidate whether mitochondrial biogenesis disorder and damage from oxidative stress promote refractory apical periodontitis (RAP) in rat and human. METHODOLOGY: Twenty Enterococcus faecalis-induced RAPs were established in the maxillary first molars of male Wistar rats. Concurrently, 12 periapical lesion specimens from patients presenting with RAP were obtained by apicoectomy. Radiographic examination and histologic analysis were conducted to evaluate periapical bone tissue destruction and morphological changes. The expression of key regulators of mitochondrial biogenesis, PGC-1α and Nrf2, were detected by immunohistochemistry and double immunofluorescence staining, Western blot and real-time PCR were also assayed. Mitochondrial ROS (mtROS) was identified by MitoSOX staining. Mitochondrial function was detected by the quantification of ATP production, mitochondrial DNA (mtDNA) copy number and activities of mitochondrial respiratory chain complexes. Furthermore, mitochondrial oxidative stress was evaluated by the determination of 3-nitrotyrosine (3-NT), 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-deoxyguanosine (8-OHdG) expression levels, as well as malondialdehyde (MDA) expression and antioxidant capacity. Student's t-test was performed to determine significance between the groups; p < .05 was considered significant. RESULTS: In the maxilla, significantly more bone resorption, greater number of periapical apoptotic cells and Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were observed in the RAP group compared with the control group (p < .01). PGC-1α and Nrf2 were significantly reduced in rat and human RAP lesions compared to the control group (p < .01) at both the mRNA and protein levels. Double immunofluorescence analysis of PGC-1α or Nrf2 with TOMM20 also indicated that mitochondrial biogenesis was impaired in RAP group (p < .01). Additionally, mitochondrial dysfunction was observed in RAP group, as reflected by increased mtROS, decreased ATP production, reduced mtDNA copy number and complexes of the mitochondrial respiratory chain. Finally, the expression levels of mitochondrial oxidative stress markers, 3-NT, 4-HNE and 8-OHdG, were significantly increased in the RAP group (p < .01). Consistent with this, systemic oxidative damage was also present in the progression of RAP, including increased MDA expression and decreased antioxidant activity (p < .01). CONCLUSIONS: Mitochondrial biogenesis disorder and damage from oxidative stress contribute to the development of RAP.

The role of oxidative stress biomarkers in the development of peri-implant disease: A systematic review and meta-analysis.

OBJECTIVES: To analyze the role of oxidative stress (OS) biomarkers in peri­implant diseases using a systematic review and meta-analysis approach. DATE: The review incorporated cross-sectional studies, randomized controlled trials, and case-control trials to evaluate the differences in OS biomarkers of peri­implant disease. SOURCES: A comprehensive literature search was conducted in electronic databases such as PubMed, Scopus, Embase, Web of Science, and CNKI, and no restrictions were applied during the search process. STUDY SELECTION: A total of 452 studies were identified, of which 18 were eligible for inclusion. Risk of bias and sensitivity analysis were assessed using Egger's test and funnel plots. RESULTS: We found that the levels of glutathione peroxidase (GSH-Px) in the peri­implant sulcus fluid (PISF) of patients with peri­implant diseases were significantly reduced (SMD = -1.40; 95 % CI = 1.70, -1.11; p < 0.001), while the levels of total myeloperoxidase (MPO) and malondialdehyde (MDA) were significantly increased (SMD = 0.46; 95 % CI = 0.12, 0.80; p = 0.008; SMD = 0.28; 95 % CI = 0.01, 0.56; p = 0.043). However, there were no significant differences of MPO concentration (SMD = 0.38; 95 % CI = -0.39, 1.15; p = 0.331) and superoxide dismutase (SOD)(SMD = -0.43; 95 % CI = -1.94, 1.07; p = 0.572) in PISF between peri­implant disease group and control group. Similarly, salivary MPO did not show significant differences (SMD = 1.62; 95 % CI = -1.01, 4.24; p = 0.227). CONCLUSIONS: Our results supported that the level of local OS biomarkers was closely related to peri­implant diseases. GSH-Px, total MPO and MDA may be PISF biomarkers with good capability to monitor the development of peri­implant disease. CLINICAL SIGNIFICANCE: This study found significant differences in the levels of local OS biomarkers (GSH-Px, total MPO, and MDA) between patients with peri­implant diseases and healthy subjects, which may be ideal candidate biomarkers for predicting and diagnosing peri­implant diseases.

Biodegradable Zn-5Dy Alloy with Enhanced Osteo/Angio-Genic Activity and Osteointegration Effect via Regulation of SIRT4-Dependent Mitochondrial Function.

Zinc (Zn)-dysprosium (Dy) binary alloys are promising biodegradable bone fracture fixation implants owing to their attractive biodegradability and mechanical properties. However, their clinical application is a challenge for bone fracture healing, due to the lack of Zn-Dy alloys with tailored proper bio-mechanical and osteointegration properties for bone regeneration. A Zn-5Dy alloy with high strength and ductility and a degradation rate aligned with the bone remodeling cycle is developed. Here, mechanical stability is further confirmed, proving that Zn-5Dy alloy can resist aging in the degradation process, thus meeting the mechanical requirements of fracture fixation. In vitro cellular experiments reveal that the Zn-5Dy alloy enhances osteogenesis and angiogenesis by elevating SIRT4-mediated mitochondrial function. In vivo Micro-CT, SEM-EDS, and immunohistochemistry analyses further indicate good biosafety, suitable biodegradation rate, and great osteointegration of Zn-5Dy alloy during bone healing, which also depends on the upregulation of SIRT4-mediated mitochondrial events. Overall, the study is the first to report a Zn-5Dy alloy that exerts remarkable osteointegration properties and has a strong potential to promote bone healing. Furthermore, the results highlight the importance of mitochondrial modulation and shall guide the future development of mitochondria-targeting materials in enhancing bone fracture healing.

Discovery, Structure Correction, and Biosynthesis of Actinopyrones, Cytotoxic Polyketides from the Deep-Sea Hydrothermal-Vent-Derived Streptomyces sp. SCSIO ZS0520.

Three new actinopyrone derivatives, actinopyrones E-G (1, 3, and 4), together with three known analogues, PM050463 (2), actinopyrone D (5), and PM050511 (6), were isolated from Streptomyces sp. SCSIO ZS0520 derived from a deep-sea hydrothermal vent. Their structures, complete with absolute configurations, were elucidated using extensive spectroscopic analyses combined with Mosher's method, ECD calculations, and bioinformatics analyses. These findings corrected the absolute configurations of previously reported actinopyrone analogues 2, 5, and 6 at C-3, C-9, and C-10. Notably, compound 6 displayed notable cytotoxicity against six human cell lines with IC50 values of 0.26-2.22 µM. A likely biosynthetic pathway and annotations of protein function are proposed on the basis of bioinformatics analyses. Genes coding for methyltransferase and glycosyltransferase tailoring chemistries needed to generate final structures were notably absent from the biosynthetic gene cluster. Taken together, these results enable further bioengineering of the actinopyrones and related congeners as potential antitumor agents.

Establishment of hairy root culture of Rubia yunnanensis Diels: Production of Rubiaceae-type cyclopeptides and quinones.

Rubia yunnanensis is an important medicinal plant with various bioactive secondary metabolites. In order to reduce the dependence on wild populations of the species, we aim to establish in vitro culture system that can produce Rubiaceae-type cyclopeptides (RAs) and quinones. Agrobacterium rhizogenes-mediated transformation of stem segments of in vitro grown R. yunnanensis plants using four A. rhizogenes strains was studied and transformation conditions were optimized. Hairy roots appeared with the highest frequency (68.89%) when stem segments (with leaves) without pre-culture were immersed in A. rhizogenes A4 strain bacterial suspension for 30 min, co-cultured on Murashige and Skoog (MS) solid medium in the dark for three days, and afterwards incubated in darkness. PCR analysis of rolB and rolC genes confirmed transformed nature of six hairy root clones. The hairy roots grew rapidly, especially showing the highest accumulation of biomass in MS liquid medium compared to in vitro grown plants and calli. Histological observation of hairy root revealed anatomical difference in vascular cylinder, where the cells exhibited high mitotic activity characterized by vigorous growth. The UPLC-MS/MS analysis revealed that the amount of RAs in the hairy roots grown in ½MS liquid medium (4.611 µg g-1 DW) was higher than that in in vitro grown plants (0.331 and 4.096 µg g-1 DW for shoots and roots respectively) and calli (1.082 µg g-1 DW), but still far lower than that in the roots of seed-borne plants (80.296 µg g-1 DW). However, the hairy roots accumulated high level of quinones (2320.923 and 5067.801 µg g-1 DW for MS and ½MS liquid media respectively), of the same order of magnitude as the roots of seed-borne plants (7409.973 µg g-1 DW). Hairy root culture of R. yunnanensis, with high accumulation of biomass and production of quinones, may offer an attractive perspective for the production of the RAs and quinones that could be further optimized for pharmaceutical use.

The Influence of Small Organic Molecules on Coagulation from the Perspective of Hydrolysis Competition and Crystallization.

Most coagulation studies focus on pollutant removal or floc separation efficiency. However, to understand the mechanism of coagulation, it is necessary to explore the behavior of coagulation in terms of the interactions among the functional groups on the surface of the metal hydrolysis precipitates during the hydrolysis process. In this study, for the first time, aluminum sulfate (alum) was used to investigate such interactions over the whole process sequence of hydrolysis, coagulation, and crystallization with, and without (as a control), the presence of specific low molecular weight (LMW) (molecular weight < 1000 Da) organic compounds with different chemical bonds. It was observed that primary nanoparticles (NPs) of around 10 nm size were produced during the hydrolysis of alum. The presence of organic compounds was found to influence the coagulation performance by affecting the metal hydrolysis and the properties of the nanoparticles. At pH 7, ethylenediaminetetraacetic acid disodium salt (EDTA) delayed the time when the particles start to aggregate but increased the maximum size of the flocs, while citric acid caused the crystallization of amorphous hydrates and inhibited the coagulation performance. In contrast, glucose, benzoic acid (BEN), and tris(hydroxymethyl)aminomethane (THMAM) had no significant effect on the coagulation performance. Therefore, LMW organics can bond to the hydrolysis products of metal ions through key functional groups, such as carboxyl groups, and then affect the coagulation process. The experimental results show that the presence of LMW organics can change the surface properties and degree of crystallization of the primary NPs, thereby affecting the performance of coagulation.

Qualitative and quantitative analyses of quinones in multi-origin Rubia species by ultra-performance liquid chromatography-tandem mass spectrometry combined with chemometrics.

Rubia species are one of the important multi-origin phytomedicines having both economical and medicinal values in many countries. Quinones are the predominant bioactive constituents of these species. Therefore, accurate analysis of these quinones is critical to quality control, clinical, and commercial applications of Rubia species. In this study, a sensitive and efficient ultra-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) method in positive and negative multiple reaction monitor (+/-MRM) modes was firstly developed for the characterization of 45 quinones and the quantification of 24 quinones from Rubia plants. The chromatographic separation was achieved on a Waters ACQUITY UPLC® BEH C18 column by using a gradient elution with a mobile phase consisting of 0.1 % formic acid in water and acetonitrile. The results indicated that quinones in multi-origin Rubia samples were different, but 10 quinones existed in all samples. R. cordifolia, which is a unique official medicinal material in Chinese Pharmacopeia, contained the most number of quinones among all the Rubia species. R. podantha and R. sylvatica had the next highest number of quinones, implying that these species could be used as alternatives for R. cordifolia. Chemometric approaches were applied to evaluate the chemical relationship between the Rubia samples based on the constituent quinones. In this study, a UPLC-QqQ-MS/MS method in the MRM mode has been developed for the analysis of Rubia species-derived quinones, which not only contributes to quality control and discrimination of Rubia species, but also suggests the potential of these species in clinical and commercial applications.

An integrated multiple reaction monitoring strategy based on predicted precursor ions and characteristic product ions for global profiling Rubiaceae-type cyclopeptides in three Rubiaspecies.

Complexity and diversity of natural compounds make it a challenge for globally profiling constituents in multiple species plants, especially for minor new compounds. Rubiaceae-type cyclopeptides (RAs) are one kind of active constituents and characteristic components in Rubia plants, particularly Rubia cordifolia (RC), which is one kind of traditional Chinese medicines. In this study, a new multiple reaction monitoring strategy (PPCP-MRM) based on predicted precursor ions and characteristic product ions was developed to globally profile RAs in RC and its two main adulterants, including Rubia alata (also named Jinjiancao in Chinese) (RAJ) and Rubia podantha (RP). Moreover, characteristic components of these species have been found by targeted relative quantitative analysis of RAs by LC-MS. In total, 39 RAs have been structurally annotated based on fragment ions in MS2 data, including 19 new compounds. In addition, 7 RAs as the chemical markers were found to distinguish these three Rubia species. The results indicated that this PPCP-MRM integrated strategy is a powerful tool for comprehensive analysis of RAs and screening chemical markers for Rubia species discrimination, which would be useful for distinguishing Rubia adulterants. Furthermore, this developed strategy could also be a useful tool for analysis of other cyclopeptides.

Selection and Validation of Appropriate Reference Genes for Quantitative RT-PCR Analysis in Rubia yunnanensis Diels Based on Transcriptome Data.

Real-time quantitative polymerase chain reaction (RT-qPCR) has been widely applied in gene expression and transcription abundance analysis because of its high sensitivity, good repeatability, and strong specificity. Selection of relatively stable reference genes is a precondition in order to obtain the reliable analysis results. However, little is known about evaluation of a set of reference genes through scientific experiments in Rubia plants. Here, 15 candidate reference genes were selected from R. yunnanensis transcriptome database and analyzed under abiotic stresses, hormone treatments, and different tissues. Among these 15 candidate reference genes, heterogeneous nuclear ribonucleoprotein (hnRNP), TATA binding protein (TBP), ribosomal protein L5 (RPL5), malate dehydrogenase (MDH), and elongation factor 1-alpha (EF-1α) were indicated as the five most stable reference genes by four statistical programs (geNorm, NormFinder, BestKeeper, and RefFinder). Ultimately, the validity of reference genes was confirmed by normalizing the expression of o-succinylbenzoate-CoA ligase (OSBL) and isochorismate synthase (ICS) involved in the anthraquinone biosynthesis pathway in different tissues and hormone treatments. Meanwhile, four other putative genes involved in the anthraquinone biosynthesis pathway were also normalized with the selected reference genes, which showed similar expression levels with those given by transcriptome data. This work is the first research that aims at a systematic validation on the stability of reference genes selected from R. yunnanensis transcriptome data and will be conducive to analyze gene expression in R. yunnanensis or other Rubia species.

Colletopeptides A-D, Anti-inflammatory Cyclic Tridepsipeptides from the Plant Endophytic Fungus Colletotrichum sp. S8.

Four new hybrid peptide-polyketide cyclic tridepsipeptides, colletopeptides A-D (1-4), were isolated and characterized from the endophytic fungus Colletotrichum sp. S8 derived from the stems of Rubia podantha with the guidance of LC-UV-MS detection. Their structures were elucidated by extensive spectroscopic analysis and X-ray crystallography. Compounds 1-4 are rare natural 12-membered cyclic tridepsipeptides containing a 3,5,11-trihydroxy-2-methyl dodecanoic acid unit in their structures. 1-4 inhibited lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages with the IC50 values of 8.3, 38.7, 13.5, and 22.2 µM, respectively. 1 also inhibited the production of inflammatory factors IL-6 and TNF-α, and decreased the phosphorylation of NF-κB-associated proteins IκBα and p65.

Rubipodanin B, a New Cytotoxic Cyclopeptide from Rubia podantha.

Using the TLC cyclopeptide protosite detection method, a new cyclohexapeptide named rubipodanin B (1), together with 11 known Rubiaceae-type cyclopeptides (RAs), RA-X-OMe (2), RA-IV (3), RA-XI (4), RA-XIII-OMe (5), rubiyunnanin C (6), RA-I (7), RA-III (8), RA-V (9), RA-VII (10), RA-XII (11) and rubipodanin A (12), were obtained from the roots and rhizomes of Rubia podantha Diels. The structures were determined using various spectroscopic methods. Among them, 2 was firstly identified as a natural product, and 3-6 were firstly isolated from this species. Cytotoxicity and NF-κB signaling pathway activity of 1, 2, 4, 6, 7 and 9 were evaluated. All these compounds showed cytotoxic activities against three human tumor cell lines, MDA-MB-231, SW620 and HepG2, with the IC50 values between 0.015 and 10.27 µm, and only 7 and 9 possessed NF-κB inhibitory activities with the IC50 values of 2.42 and 0.046 µm, respectively, which demonstrated that 2-alanine amino acid plays a key role to maintain the RAs bioactivity.

Systematic characterization and quantification of Rubiaceae-type cyclopeptides in 20 Rubia species by ultra performance liquid chromatography tandem mass spectrometry combined with chemometrics.

Rubiaceae-type cyclopeptides (RAs) are one of characteristic constituents isolated from Rubia species, which are candidates of innovative anti-tumor drugs due to their significant bioactivity. However, approaches on the systematic characterization and quantification of RAs are still not available because of low contents and complicated purification. In this study, an ultra performance liquid chromatography coupled triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) method was established and validated for qualitative and quantitative analysis of 14 RAs (1-14) in 20 Rubia plants from China. The separation was achieved on a Waters ACQUITY UPLC® BEH C18 column (50 × 2.1 mm, 1.7 µm) using gradient elution with a mobile phase consisting of 0.1% formic acid in acetonitrile and water. Multiple reaction monitoring (MRM) in positive mode was used to enable the selective detection of RAs from the Rubia root and rhizome extracts within 10 min. This method was proved to be specific, sensitive, precise, and accurate with the limits of detection and quantification at 0.6-11.4 ng/mL and 1.9-34.2 ng/mL, respectively, and the relative standard deviation (RSD) of the overall intra-day and inter-day precision less than 5.24%. Satisfactory recovery was obtained from 83.80% to 111.77%, with the RSD less than 5.32%. Totally, 67 samples were then qualitatively and quantitatively analyzed by this method and 51 of them were proved to contain RAs. Thereinto, R. podantha and R. yunnanensis from Yunnan were the two most abundant species. Additionally, RAs were detected in 8 Rubia species for the first time. Then chemometric approaches were revealed to explain the relationship between samples based on their contents of RAs. This study demonstrated that the method was not only useful for RAs source discovery and chemotaxonomy of Rubia species, but also could be extended to standardization of RAs medical materials and their new drug research and development.

Multi-elemental analysis of Ziziphora clinopodioidesfrom different regions, periods and parts using atomic absorption spectrometry and chemometric approaches

ABSTRACTIn this study, ten trace elements in Ziziphora clinopodioidesLam., Lamiaceae, from different regions, periods and parts in Xinjiang were determined by atomic absorption spectrometry following microwave-assisted acid digestion. The decreasing sequence of elements levels was K > Ca > Mg > Fe > Cu > Zn > Na > Mn > Cd > Pb. Chemometric approaches, such as correlation analysis, principal component analysis, and hierarchical cluster analysis were applied to classify Z. clinopodioides according to its elements contents. Principal component analysis revealed 83.51% of the variance with the first four principal component variables. Hierarchical cluster analysis indicated five groups from the eighteen regions, and the result of classification can correspond to the geographical distribution for the most regions. Variation in the elements exhibited a decreasing trend, but of different types in the studied periods. Elemental contents distributed in leaves were higher than those in flowers and stems. Therefore, chemometric approaches could be used to analyze data to accurately classify Z. clinopodioides according to origins. This study provided some elemental information on chemotaxonomy, diversity, changing pattern, distribution, and metabolism of Z. clinopodioides at spatial and temporal levels, and could be used as a reference of planting and quality standards.

Development and validation of an high-performance liquid chromatography-diode array detector method for the simultaneous determination of six phenolic compounds in abnormal savda munziq decoction.

AIMS: Given the high-effectiveness and low-toxicity of abnormal savda munziq (ASMQ), its herbal formulation has long been used in traditional Uyghur medicine to treat complex diseases, such as cancer, diabetes, and cardiovascular diseases. SETTINGS AND DESIGN: ASMQ decoction by reversed-phase high-performance liquid chromatography coupled with a diode array detector was successfully developed for the simultaneous quality assessment of gallic acid, protocatechuic acid, caffeic acid, rutin, rosmarinic acid, and luteolin. The six phenolic compounds were separated on an Agilent TC-C18 reversed-phase analytical column (4.6 × 250 mm, 5 µm) by gradient elution using 0.3% aqueous formic acid (v/v) and 0.3% methanol formic acid (v/v) at 1.0 mL/min. MATERIALS AND METHODS: The plant material was separately ground and mixed at the following ratios (10): Cordia dichotoma (10.6), Anchusa italic (10.6), Euphorbia humifusa (4.9), Adiantum capillus-veneris (4.9), Ziziphus jujube (4.9), Glycyrrhiza uralensis (7.1), Foeniculum vulgare (4.9), Lavandula angustifolia (4.9), Dracocephalum moldavica L. (4.9), and Alhagi pseudoalhagi (42.3). STATISTICAL ANALYSIS USED: The precisions of all six compounds were <0.60%, and the average recoveries ranged from 99.39% to 104.85%. Highly significant linear correlations were found between component concentrations and specific chromatographic peak areas (R (2) > 0.999). RESULTS: The proposed method was successfully applied to determine the levels of six active components in ASMQ. CONCLUSIONS: Given the simplicity, precision, specificity, and sensitivity of the method, it can be utilized as a quality control approach to simultaneously determining the six phenolic compounds in AMSQ.

Phytochemical composition and antioxidant capacity of Cordia dichotoma seeds.

This study aims to determine the phytochemical composition and antioxidant activity of air-dried Cordia dichotoma seeds. Total polyphenolic content was analyzed via the Folin-Ciocalteu method. Total triterpenoid content and amino acids was analyzed colorimetrically. The rosmarinic acid content was examined using high-performance liquid chromatography tandem mass spectrometry. The ethanolic extracts contained polyphenolic compounds (1.0%), triterpenoids (0.075%), amino acids (1.39%), and rosmarinic acid (0.0028%). The results from this study indicate that C. dichotoma seeds are a rich source of polyphenolic compounds and amino acids, which can be used for quality assessment. The ethanolic extract of C. dichotoma seeds has good antioxidant capacity.