Clematichinenoside protects blood brain barrier against ischemic stroke superimposed on systemic inflammatory challenges through up-regulating A20.

Suppression of excessive inflammation can ameliorate blood brain barrier (BBB) injury, which shows therapeutic potential for clinical treatment of brain injury induced by stroke superimposed on systemic inflammatory diseases. In this study, we investigated whether and how clematichinenoside (AR), an anti-inflammatory triterpene saponin, protects brain injury from stroke superimposed on systemic inflammation. Lipopolysaccharide (LPS) was intraperitoneally injected immediately after middle cerebral artery occlusion (MCAO) in rats. Rat microvessel endothelial cells (rBMECs) were exposed to hypoxia/reoxygenation (H/R) coexisting with LPS. The results revealed that AR suppressed the excessive inflammation, restored BBB dysfunction, alleviated brain edema, decreased neutrophil infiltration, lessened neurological dysfunction, and decreased infarct rate. Further study demonstrated that the expression of nucleus nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α) and interlukin-1ß (IL-1ß) were suppressed by AR via zinc finger protein A20. Besides, AR increased in vitro BBB integrity through A20. In conclusion, AR alleviated cerebral inflammatory injury through A20-NF-κB signal pathway, offering an alternative medication for stroke associated with systemic inflammatory diseases.

Anti-arthritic effects of clematichinenoside (AR-6) on PI3K/Akt signaling pathway and TNF-α associated with collagen-induced arthritis.

CONTEXT: Clematichinenoside (AR-6) is a triterpene saponin from an anti-arthritic herbal formula Wei-Ling-Xian in Chinese, which is an herbal medicine derived from the dried root and rhizome of Clematis chinensis Osbeck, C. hexapetala Pall., or C. manshurica Rupr. (Ranunculaceae). OBJECTIVE: To investigate the modulating effect and explored the potential mechanism of AR-6 in rheumatoid arthritis (RA), using collagen-induced arthritis (CIA) in a rat model. MATERIALS AND METHODS: CIA was evaluated by measuring body weight, paw swelling and organ index. Expression of TNF-α, PI3K and p-Akt in synovium tissue was measured by immunohistochemistry. Furthermore, expression of TNF-α mRNA, PI3K mRNA and p-Akt mRNA was measured with RT-PCR. RESULTS: The intragastric administration of AR-6 (32, 16 and 8 mg/kg), especially the high dose level of 32 mg/kg, significantly suppressed the swelling of hind paws of CIA rats (p < 0.01) and inhibited their body weight loss (p < 0.01). Based on histopathological observation, all AR-6 groups showed great amelioration compared with model group. Moreover, AR-6 significantly reduced the production of TNF-α, PI3K and p-Akt expression by immunohistochemistry (p < 0.01), and decreased TNF-α mRNA, PI3K mRNA and p-Akt mRNA in CIA rat synovium (p < 0.01). DISCUSSION: Our study indicates the mechanism of AR-6 is associated with PI3K/Akt signaling pathway and TNF-α. CONCLUSIONS: Such characteristics relating to AR-6 curing chronic inflammation of CIA, may be effectively applied to the therapeutic potential in patients with inactive RA.

Anti-inflammatory effects of Clematis chinensis Osbeck extract(AR-6) may be associated with NF-κB, TNF-α, and COX-2 in collagen-induced arthritis in rat.

The root of Clematis chinensis Osbeck has been used widely in rheumatoid arthritis in Chinese traditional medicine, and AR-6 is a triterpene saponin isolated from it. In this present study, we investigated the in vivo effects of oral AR-6 in chronic rat with collagen-induced arthritis (CIA) and possible molecular mechanism. CIA was induced by immunizing 56 female Sprague-Dawley (SD) rats with chicken typeIIcollagen (CII). Following eighteen days, the immunization rats with CIA were treated with AR-6 (32, 16, 8 mg/kg), cyclophosphamide (7 mg/kg), and TGP (Total Glucosides of Paeonia) (180 mg/kg) for 7 days, and rats without CIA were given the same volume of purified water. TNF-α and IL-1ß levels in peripheral blood will be measured by ELISA, and Western blot analysis will be used to detect the expression of NF-κB p65 subunits, TNF-α and COX-2, in synovial membrane. We found that therapeutic treatment with AR-6 markedly improves the paw swelling and histopathological changes. Moreover, the serum levels of pro-inflammatory cytokines TNF-α and IL-1ß were markedly lowered, and the expression of NF-κB p65 subunits, TNF-α and COX-2, in the synovial membrane of CIA rats was significantly inhibited in the AR-6-treated groups. These results enable to prove that AR-6 has a potential anti-inflammatory effect in CIA rats, and its mechanism may relate to the inhibition of the expression of NF-κB p65 subunits, TNF-α and COX-2.

Association of glycemic variability assessed by continuous glucose monitoring with subclinical diabetic polyneuropathy in type 2 diabetes patients.

AIMS/INTRODUCTION: Diabetic peripheral neuropathy is a common diabetes-related microvascular complication. The relationship between peripheral nerve function and glucose variability is unclear. We investigated the association of glucose variability with subclinical diabetic polyneuropathy in a large-scale sample of patients with type 2 diabetes. MATERIALS AND METHODS: We enrolled 509 individuals with type 2 diabetes who were screened for diabetic peripheral neuropathy and monitored using a continuous glucose monitoring system. Multiple glycemic variability parameters, including the mean amplitude of glycemic excursions, glucose standard deviation (SDgluc ) and glucose coefficient of variation were calculated from 3-day glucose profiles obtained from continuous glucose monitoring. All participants underwent nerve conduction studies, and the composite Z-scores for nerve conduction parameters were calculated. RESULTS: Multivariate logistic regression analyses showed that SDgluc and the conventional risk factor hemoglobin A1c (HbA1c) were independently associated with abnormal nerve function, and the corresponding odds ratios (95% confidence interval) were 1.198 (1.027-1.397, SDgluc ) and 1.182 (1.061-1.316, HbA1c), respectively. The composite Z-score of nerve conduction velocity and response amplitude obviously decreased with greater SDgluc , and the composite Z-score of distal latency significantly increased with increasing tertiles of SDgluc (all P trend <0.05). After adjusting for age, sex, body mass index, diabetes duration and HbA1c, SDgluc was independently associated with nerve conduction velocity (ß = -0.124, P = 0.021). CONCLUSIONS: The SDgluc is a significant independent contributor to subclinical diabetic polyneuropathy, in addition to conventional risk factors including diabetes duration and HbA1c.

ATF5, a putative therapeutic target for the mitochondrial DNA 3243A > G mutation-related disease.

The mitochondrial DNA m.3243A > G mutation is well-known to cause a variety of clinical phenotypes, including diabetes, deafness, and osteoporosis. Here, we report isolation and expansion of urine-derived stem cells (USCs) from patients carrying the m.3243A > G mutation, which demonstrate bimodal heteroplasmy. USCs with high levels of m.3243A > G mutation displayed abnormal mitochondrial morphology and function, as well as elevated ATF5-dependent mitochondrial unfolded protein response (UPRmt), together with reduced Wnt/ß-catenin signaling and osteogenic potentials. Knockdown of ATF5 in mutant USCs suppressed UPRmt, improved mitochondrial function, restored expression of GSK3B and WNT7B, and rescued osteogenic potentials. These results suggest that ATF5-dependent UPRmt could be a core disease mechanism underlying mitochondrial dysfunction and osteoporosis related to the m.3243A > G mutation, and therefore could be a novel putative therapeutic target for this genetic disorder.

[Study on the dynamic variation of active components in Geranium carolinianum from different collection time].

OBJECTIVE: To determine the contents of the active components, gallic acid and ellagic acid in Geranium carolinianum from different collection time and to define the best collection time for this herb. METHODS: The contents of gallic acid and ellagic acid in each samples of Geranium carolinianum were determined by HPLC. The HPLC method was performed on a Diamonsil C, 8 column (150 mm x 4.6 mm, 5 microm) with acetonitrile-0.1% H3PO4 as mobile phase. The flow rate was 1.0 mL/min, the detection wavelength was 274 nm and the column temperature was 25 degrees C. RESULTS: The calibration curve of gallic acid and ellagic acid were linear in the range of 0.075-5.00 microg (r = 0.9995) and 0.05-2.00 microg (r = 0.9995), respectively. The average recovery of gallic acid and ellagic acid were 99.88% (RSD = 1.19%) and 99.08% (RSD = 2.81%), respectively. CONCLUSION: The content of gallic acid and ellagic acid in Geranium carolinianum both began to increase in flowering stage and increased to the maximum value in immature-fruit stage.

TIR generated by continuous glucose monitoring is associated with peripheral nerve function in type 2 diabetes.

AIMS: Continuous glucose monitoring (CGM)-derived time-in-range (TIR) of 3.9-10 mmol/L is associated with diabetic retinopathy in type 2 diabetes (T2DM), but its relationship to peripheral nerve function has not been previously investigated. To explore the association between the TIR and nerve conduction study parameters in patients with T2DM, we performed a cross-sectional analysis. METHODS: A total of 740 patients with T2DM were enrolled in this study. All of the participants were divided into tertiles according to the TIR (TIR low: ≤53%; TIR medium: 54-76%; TIR high: ≥77%). Composite Z-scores of nerve conduction velocity (CV), latency, and amplitude were calculated. The linear correlation between the TIR and composite nerve function Z-score was evaluated and risk assessment was analysed using binary logistic regression. RESULTS: The composite Z-score of the CV and amplitude increased with higher TIR and the composite Z-score of latency significantly decreased as the TIR tertiles increased (all P trend < 0.05). After adjusting for age, diabetes duration, height, weight and other confounding factors, higher TIR was associated with a higher composite Z-score of CV (ß = 0.230, P < 0.001), amplitude (ß = 0.099, P = 0.010), and lower composite Z-score of latency (ß = -0.172, P < 0.001). The risk of TIR tertiles and low composite Z-score of CV remained significant even after adjustment of HbA1c (TIR medium: OR = 0.48, P = 0.001; TIR high: OR = 0.41, P < 0.001). CONCLUSIONS: Higher TIR tertiles were independently associated with better peripheral nerve function. CGM-derived TIR may be a promising approach to screen patients for further assessment of possible diabetic peripheral neuropathy.

Illuminating NAD+ Metabolism in Live Cells and In Vivo Using a Genetically Encoded Fluorescent Sensor.

Understanding of NAD+ metabolism provides many critical insights into health and diseases, yet highly sensitive and specific detection of NAD+ metabolism in live cells and in vivo remains difficult. Here, we present ratiometric, highly responsive genetically encoded fluorescent indicators, FiNad, for monitoring NAD+ dynamics in living cells and animals. FiNad sensors cover physiologically relevant NAD+ concentrations and sensitively respond to increases and decreases in NAD+. Utilizing FiNad, we performed a head-to-head comparison study of common NAD+ precursors in various organisms and mapped their biochemical roles in enhancing NAD+ levels. Moreover, we showed that increased NAD+ synthesis controls morphofunctional changes of activated macrophages, and directly imaged NAD+ declines during aging in situ. The broad utility of the FiNad sensors will expand our mechanistic understanding of numerous NAD+-associated physiological and pathological processes and facilitate screening for drug or gene candidates that affect uptake, efflux, and metabolism of this important cofactor.

Triterpenoid saponins from the roots of Clematis chinensis Osbeck.

A new triterpenoid saponin named clematichinenoside AR(2), along with the six known compounds, was isolated and characterized from Clematis chinensis Osbeck (Ranunculaceae), a commonly used traditional Chinese medicine with anti-inflammatory and anti-rheumatoid activities. The structure of the new saponin was elucidated as 3-O-beta-[(O-alpha-L-rhamnopyranosyl-(1 --> 6)-O-beta-D-glucopyranosyl-(1 --> 4)-O-beta-D-glucopyranosyl-(1 --> 4)-O-beta-D-ribopyranosyl-(1 --> 3)-O-alpha-L-rhamnopyranosyl-(1 --> 2)-alpha-L-arabinopyranosyl)oxy]olean-12-en-21alpha-hydroxy-28-oic acid-O-alpha-L-rhamnopyranosyl-(1 --> 4)-O-beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranosyl ester (1) by spectral analysis and chemical methods. The effects of two major saponins (clematichinenosides AR and AR(2)) on the secretion of TNF-alpha in murine peritoneal macrophages induced by lipopolysaccharides were further investigated. The result indicated that a majority of triterpenoid saponins of this herb may be useful in the exploration of lead compounds for the treatment of some autoimmune diseases.

Conserved roles of glucose in suppressing reactive oxygen species-induced cell death and animal survival.

Carbohydrate overconsumption increases blood glucose levels, which contributes to the development of various diseases including obesity and diabetes. It is generally believed that high glucose metabolism increases cellular reactive oxygen species (ROS) levels, damages insulin-secreting cells and leads to age-associated diabetic phenotypes. Here we find that in contrast, high glucose suppresses ROS production induced by paraquat in both mammalian cells and the round worm C. elegans. The role of glucose in suppressing ROS is further supported by glucose's ability to alleviate paraquat's toxicity on C. elegans development. Consistently, we find that the ROS-regulated transcription factor SKN-1 is inactivated by glucose. As a result, the ROS/SKN-1-dependent lifespan extension observed in paraquat-treated animals, mitochondrial respiration mutant isp-1 and germline-less mutant glp-1 are all suppressed by glucose. Our study reveals an unprecedented interaction of glucose with ROS, which could have significant impact on our current understanding of glucose- and ROS-related diseases.

De Novo Mutation of m.3243A>G together with m.16093T>C Associated with Atypical Clinical Features in a Pedigree with MIDD Syndrome.

BACKGROUND: The syndrome of maternally inherited diabetes and deafness (MIDD) is typically caused by the m.3243A>G mutation and widely considered maternally inherited. In our study, we aimed to investigate the heredity way of the m.3243A>G among pedigrees with MIDD and discover novel mitochondrial DNA mutations related to atypical clinical phenotypes. METHODS: Heteroplasmy levels of the m.3243A>G mutation in peripheral blood, saliva, and urine sediment of 31 individuals from 10 unrelated pedigrees were measured by pyrosequencing. Clinical evaluations including endocrinological, audiological, and magnetic resonance imaging (MRI) examinations, mitochondrial function evaluation in peripheral blood mononuclear cells (PBMCs), and whole mitochondrial DNA (mtDNA) sequencing were performed among the spontaneous mutant pedigrees. RESULTS: Among the 10 unrelated MIDD pedigrees, we found that the de novo m.3243A>G mutation occurred in the family 1957 (F1957). The proband (F1957-II-1) and her son (F1957-III-1) both manifested diabetes with mild bilateral sensorineural hearing loss (SNHL) and abnormal brain MRI, and F1957-III-1 also complained of severe nausea and vomiting. Mitochondrial function evaluation in PBMCs revealed an increased level of ROS generation and decreased levels of ATP and mitochondrial membrane potential (ΔΨm) in the two m.3243A>G carriers. Whole mtDNA sequencing also revealed a de novo heteroplasmic substitution at m.16093T>C in both the proband and her son. CONCLUSIONS: Our study showed that de novo m.3243A>G mutation accompanied by other point mutations may occur in the very early embryonic or germ cell stage without maternal inheritance, bringing about both typical and atypical clinical features.

Protective effects of Clematichinenoside AR against inflammation and cytotoxicity induced by human tumor necrosis factor-α.

Clematichinenoside AR (AR), a major active ingredient extracted from traditional Chinese herb Clematis chinensis Osbeck, has been demonstrated to possess anti-inflammatory and immune-modulatory activities in the treatment of experimental rheumatoid arthritis (RA). The therapeutic potential of AR was supposed to be closely correlated to its ability against tumor necrosis factor-α (TNF-α). Therefore, we aimed to explore the protective effects of Clematichinenoside AR against inflammation and cytotoxicity induced by human TNF-α. AR treatment significantly decreased IL-6 and IL-8 secretion, and attenuated MMP-1 production in human RA-derived fibroblast-like synoviocyte MH7A cells stimulated by recombinant human TNF-α (rhTNF-α). AR might antagonize rhTNF-α-induced responses in MH7A cells through inhibiting p38 and ERK MAPKs signal activation. In TNF-α-sensitive murine fibroblast L929 cells, AR treatment attenuated the proliferation inhibition ratio induced by rhTNF-α/ActD and antagonized rhTNF-α-induced cytotoxicity. The cellular and nuclear morphological alterations in apoptotic characteristics induced by rhTNF-α/ActD in L929 cells were observed to be attenuated by the pretreatment with AR under a phase-contrast and fluorescence microscopy, respectively. The Annexin V-FITC/PI double-staining assay was performed to confirm that AR pretreatment obviously decreased the cell death. The antagonistic effects of AR against rhTNF-α-induced cytotoxicity might be potentially attributed to the degeneration of reactive oxygen species and the increasing of mitochondrial membrane potential, along with the suppression of durative phosphorylation of c-Jun N-terminal kinase (JNK). Collectively, our results indicated that AR antagonizes the inflammatory and cytotoxic activities induced by human TNF-α effectively in vitro, which provided further evidence for a novel mechanism underlying AR for treating RA correlating with excessive TNF-α production.

Psychological and behavioral characteristics of suicide attempts and non-suicidal self-injury in Chinese adolescents.

BACKGROUND: Suicide attempts (SA) and non-suicidal self-injury (NSSI) are prevalent in adolescents and important risk factors of suicide death. Both SA and NSSI are associated with multiple psychosocial, behavioral, biological and genetic factors. This study examined similarities and differences in psychological vulnerability and internalizing and externalizing problems between adolescents with SA and NSSI. METHODS: Participants consisted of 11,831 students and had a mean age of 14.97 (SD = 1.46) years. Students completed a structured questionnaire to report their demographic information, psychological characteristics, internalizing and externalizing problems, SA and NSSI. Based on the history of NSSI and SA in the last year, the sample was divided into four groups: non-self-harm (NSH), NSSI only, SA only, and NSSI+SA. Multivariate analyses of covariance and post-hoc pairwise comparisons were performed for multiple comparisons. RESULTS: Compared with NSH group, adolescents with either NSSI or SA scored significantly higher on trait anger, impulsiveness, hopelessness, internalizing and externalizing problems. NSSI+SA group and SA only group scored significantly higher than NSSI only group but both did not score significantly different on most psychological and behavioral variables. LIMITATIONS: Limitations include reliance on self-reported measures and cross-sectional survey. CONCLUSIONS: Psychological and behavioral profiles between adolescents with SA and NSSI are similar but are more severe in suicide attempters. The findings highlight the necessity of assessing psychological and behavioral problems for prevention and early intervention of adolescent self-harm.

Clematichinenoside AR induces immunosuppression involving Treg cells in Peyer׳s patches of rats with adjuvant induced arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Clematichinenoside AR (AR) has been defined as a major active ingredient of triterpenoid saponins extracted from Clematidis Radix et Rhizoma, which is a traditional Chinese herbal medicine that has long been used in the treatment of rheumatoid arthritis (RA). To further explore the mechanism of AR in the treatment of RA, we investigated whether its immunomodulatory effects are related to Treg-mediated suppression derived from Peyer׳s patches (PPs) in adjuvant induced arthritis (AIA) rat model. MATERIALS AND METHODS: AR (8, 16, 32 mg/kg) was orally administered daily from Day 18 to Day 31 after immunization. The effect of AR on AIA rats was evaluated by hind paw swelling and histopathological examination. Percentages of CD4(+)CD25(+)Foxp3(+) T regulatory cells were determined by flow cytometry. Levels of IL-10, TGF-ß1, IL-17A and TNF-α were measured by ELISA. Expressions of Foxp3 and RORγ in synovium were detected using immunohistochemical analysis. RESULTS: AR treatment significantly reduced paw swelling of AIA rats, and histopathological analysis confirmed it could suppress severity of established arthritis. AR treatment upregulated the percentages of CD4(+)CD25(+)Foxp3(+) Treg cells among CD4+ T cells in PPs lymphocytes, and increased the levels of IL-10 and TGF-ß1 secreted from ConA-activated PPs lymphocytes, whereas decreased the levels of IL-17 A and TNF-α. Similar tendency of circulating CD4(+)CD25(+)Foxp3(+) Treg cells percentages and serum cytokine levels were observed. Moreover, AR decreased the expression levels of Foxp3 and RORγ in joint synovial membrane. CONCLUSIONS: In conclusion, these results suggested AR has a potent protective effect on the progression of AIA, probably by augmenting CD4(+)CD25(+)Foxp3(+) Treg cells in PPs to induce immunosuppression, and modulating the balance between Treg cells and Th17 cells systemically. These findings may help to develop AR as a potent immunosuppressive agent for the treatment of RA.

Effect and mechanism of AR-6 in experimental rheumatoid arthritis.

The root of Clematis chinensis Osbeck has been used widely in rheumatoid arthritis in Chinese traditional medicine and AR-6 is a triterpene saponin isolated from it. In this present study, we investigated in vivo effects of oral AR-6 in chronic rat adjuvant-induced arthritis (AA) and in vitro effect in macrophage and synoviocytes cells. Arthritic scores and serum inflammatory mediators were evaluated 19 days after AA induction by endermic injection of Freund's complete adjuvant in Sprague-Dawley(S-D) rats. Oral administration of AR-6 to arthritic rats resulted in a clear decrease of clinical signs compared to untreated controls. The synoviocyte and macrophage response ex vivo were then analyzed. Anti-arthritic effects of AR-6 correlated with significant decrease of NO and TNF-alpha produced by peritoneal macrophages, ex vivo and in vitro. AR-6 also significant decreased the proliferation of synoviocyte. These data indicate that AR-6 is a potential anti-inflammatory therapeutic and preventive agent.