Melittin acupoint injection in attenuating bone erosion in collagen-induced arthritis mice via inhibition of the RANKL/NF-κB signaling pathway.

Background: Rheumatoid arthritis (RA) is an autoimmune disease leading to chronic joint inflammation. Bone erosion is the most serious pathological condition of RA and the main cause of joint deformities and disability. Melittin acupoint injection (MAI) is an effective traditional Chinese medicine (TCM) method for RA treatment. This study aimed to investigate the effect of MAI on RA bone erosion and to elucidate the underlying mechanism. Methods: A collagen-induced arthritis (CIA) mouse model was established as the experimental subject. MAI was administrated once every other day for 28 days to mice with CIA. The effects of MAI on joint diseases were assessed by body weight, arthritis index (AI) score, swollen joint count (SJC) score, and hind paw thickness. Ankle radiological changes were captured by micro-computed tomography (micro-CT) and histological changes were observed by pathological staining. Organ histological changes, spleen index, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (Crea) levels of serum were tested to evaluate the toxicity of MAI. Cytokine expression levels were confirmed by enzyme-linked immunosorbent assay (ELISA) to evaluate the immunity of CIA mice. Results: MAI administration markedly improved the clinical signs of CIA in mice, including hind paw thickness, AI, and the number of swollen paw joints (most of them P<0.05 or even <0.01). According to histopathological analysis, MAI ameliorated inflammatory cell infiltration, synovial hyperplasia, pannus formation, and bone erosion (all P<0.01). Micro-CT and tartrate-resistant acid phosphatase (TRAP) staining (P<0.01) also revealed that MAI could relieve bone erosion via reducing the formation of osteoclasts. Not only could MAI relieve the immunological boost [P<0.05 for the high-dose MAI (HM) group], but also it had no liver or kidney side effects (P>0.05). In addition, it decreased the serum levels of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) and increased the serum levels of IL-4 and IL-10 (the majority of P<0.05 or even <0.01). Transcriptome sequencing results indicated that MAI affected the expression of osteoclast differentiation pathway genes, which was connected with the receptor activator of the nuclear factor κB ligand/nuclear factor kappa B (RANKL/NF-κB) pathway. Conclusions: Based on our findings, MAI could suppress joint inflammation and inhibit RANKL/NF-κB-mediated osteoclast differentiation to rescue bone erosion in CIA mice, suggesting that MAI can be a potentially therapeutic substance for RA.

GuiLu-ErXian Glue extract promotes mesenchymal stem cells (MSC)-Induced chondrogenesis via exosomes release and delays aging in the MSC senescence process.

ETHNOPHARMACOLOGICAL RELEVANCE: The treatment of osteoarthritis (OA) patients is a challenging problem. Mesenchymal stem cells (MSCs) are multipotent cells and play key roles in regenerative medicine for cartilage degeneration. GuiLu-ErXian Glue (GLEXG) is an herbal remedy widely used in traditional Chinese medicine to treat joint pain and disability in elderly OA patients. However, the mechanisms of how GLEXG affects MSCs-induced chondrogensis remains to be elucidated. AIM OF THE STUDY: The aim of this study was to investigate the effects of GLEXG on MSC-derived chondrogenesis, both in vitro and in vivo and its potential mechanisms. METHODS: Using human MSC (hMSCs) as in vitro model, the effects of HPLC-profiled GLEXG water extract on chondrogenic differentiation were investigated by 3D spheroid cultures under chondrogenesis-inducing medium (CIM) condition. The chondrogenesis process was evaluated by measuring the sphere sizes, chondrogenesis-related genes expression by reverse transcription real-time PCR that targeted type II/X collagens, SOX9, aggrecan, and protein expression by immunostaining. Anti-TGF-ß1 neutralization antibody was used for mechanistic study. Mono-iodoacetate (MIA) induced OA joint was used to evaluate the effects of GLEXG on in vivo model. MSCs-derived exosomes were purified for proteomics study and senescence process was evaluated by cumulative population doublings and senescence-associated ß-Galactosidase staining. RESULTS: The results showed that GLEXG enhanced hMSCs chondrogenesis and upregulated RNA expression of type II/X collagen, SOX9 and aggrecan at 0.1 µg/mL, 0.3 µg/mL in vitro. In vivo, GLEXG at the dose of 0.3 µg intraarticular (i.a.) injection rescued the MIA-induced cartilage defect. Proteomics and ingenuity pathway analysis obtained from MSCs-released exosomes suggested that senescence pathway was less activated in GLEXG group than in vehicle group. Besides, GLEXG was able to increase cumulative population doubling and delayed hMSCs senescence process after four passages in cultures. CONCLUSION: we conclude that GLEXG promotes in vitro MSC-induced chondrogenesis possibly via exosomes release and delays aging in the MSC senescence process and that treatment with GLEXG (0.3 µg, i.a.) rescued cartilage defects in rat OA knee model.

Deep Learning-Based Universal Expert-Level Recognizing Pathological Images of Hepatocellular Carcinoma and Beyond.

Background and Aims: We aim to develop a diagnostic tool for pathological-image classification using transfer learning that can be applied to diverse tumor types. Methods: Microscopic images of liver tissue with and without hepatocellular carcinoma (HCC) were used to train and validate the classification framework based on a convolutional neural network. To evaluate the universal classification performance of the artificial intelligence (AI) framework, histological images from colorectal tissue and the breast were collected. Images for the training and validation sets were obtained from the Xiamen Hospital of Traditional Chinese Medicine, and those for the test set were collected from Zhongshan Hospital Xiamen University. The accuracy, sensitivity, and specificity values for the proposed framework were reported and compared with those of human image interpretation. Results: In the human-machine comparisons, the sensitivity, and specificity for the AI algorithm were 98.0, and 99.0%, whereas for the human experts, the sensitivity ranged between 86.0 and 97.0%, while the specificity ranged between 91.0 and 100%. Based on transfer learning, the accuracies of the AI framework in classifying colorectal carcinoma and breast invasive ductal carcinoma were 96.8 and 96.0%, respectively. Conclusion: The performance of the proposed AI framework in classifying histological images with HCC was comparable to the classification performance achieved by human experts, indicating that extending the proposed AI's application to diagnoses and treatment recommendations is a promising area for future investigation.

Computational Analysis of Naturally Occurring Aristolochic Acid Analogues and Their Biological Sources.

Aristolochic acids are known for nephrotoxicity, and implicated in multiple cancer types such as hepatocellular carcinomas demonstrated by recent studies. Natural products that are analogues to aristolochic acids have been constantly isolated from organisms; a larger chemical space of these compounds and a wider coverage of biological sources should be determined in consideration of the potential hazard of aristolochic acid analogues and the wide distribution of their biological sources in the nature. Therefore, we carried out an in silico research of naturally occurring aristolochic acid analogues and their biological sources, as a supplement to existing studies. The result shows a chemical space of 238 naturally occurring aristolochic acid analogues that are present in 175 species of biological sources including 44 traditional medicines. With the computational estimation for toxicity and the implication in hazard assessment of a biological source with the presence of aristolochic acid analogues, we propose that additional awareness should be raised to the public for avoidance of toxic species, especially those that are used as herbal medicines and easily accessible.

Virtual Screening for Reactive Natural Products and Their Probable Artifacts of Solvolysis and Oxidation.

Chemically unstable natural products are prone to show their reactivity in the procedures of extraction, purification, or identification and turn into contaminants as so-called "artifacts". However, identification of artifacts requires considerable investments in technical equipment, time, and human resources. For revealing these reactive natural products and their artifacts by computational approaches, we set up a virtual screening system to seek cases in a biochemical database. The screening system is based on deep learning models of predicting the two main classifications of conversion reactions from natural products to artifacts, namely solvolysis and oxidation. A set of result data was reviewed for checking validity of the screening system, and we screened out a batch of reactive natural products and their probable artifacts. This work provides some insights into the formations of natural product artifacts, and the result data may act as warnings regarding the improper handling of biological matrixes in multicomponent extraction.

Upregulation of Yy1 Suppresses Dilated Cardiomyopathy caused by Ttn insufficiency.

Truncating variants in TTN (TTNtv), coding for the largest structural protein in the sarcomere, contribute to the largest portion of familial and ambulatory dilated cardiomyopathy (DCM). TTN haploinsufficiency caused by TTNtv is suggested as the disease mechanism. However, it is unclear whether TTN insufficiency causes DCM. Moreover, it is unknown whether modulation of downstream pathways serves as a therapeutic strategy for DCM caused by TTN insufficiency. Here, we show that reduction of cardiac Ttn expression by adeno-associated virus mediated shRNA (Ttn shRNA) generated DCM in mouse, demonstrating impaired cardiac performance, enlarged left ventricle (LV) and reduced LV wall thickness. A screen of 10 dysregulated and selected genes identified that Yin Yang 1 (Yy1) significantly suppressed DCM caused by Ttn shRNA. Gene profiling by RNAseq showed Yy1 modulated cell growth related genes. Ttn insufficiency activated cardiomyocyte cell cycle reentry by upregulating of Ccnd1 and Ccnd2. Cardiomyocytes activated by Ttn insufficiency did not advance to S phase by EdU incorporation assay. Yy1 promoted cardiomyocyte cell cycle by further enhancing Ccnd1 and Ccnd2 and increasing DNA replication without undergoing cell division. Importantly, upregulation of Ccnd1 and Ccnd2 suppressed DCM caused by Ttn insufficiency. Our findings demonstrate that DCM caused by Ttn insufficiency can be treated by therapeutically promoting cardiac cell cycle.

Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of Bmp7 and Ctgf.

RATIONALE: Pathogenic variations in the lamin gene (LMNA) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA-related DCM. OBJECTIVE: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 (Yy1) in suppressing Lmna DCM. METHODS AND RESULTS: We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf. Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-ß (transforming growth factor-ß)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFß/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. CONCLUSIONS: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.

Anti-inflammatory activities of Guang-Pheretima extract in lipopolysaccharide-stimulated RAW 264.7 murine macrophages.

BACKGROUND: Guang-Pheretima, which is originated from Pheretima aspergillum, has been documented in academic Chinese herbal studies for nearly 2000 years for its prominent treating effects of various inflammatory diseases such as asthma, cough and fever. However, the anti-inflammatory activity and mechanism of Guang-Pheretima has been rarely reported. Hence, we investigated the inhibitory effect and the underlying mechanism of Guang-Pheretima aqueous extracts on inflammatory response in RAW 264.7 cells. METHOD: RAW 264.7 macrophages were pretreated with various concentrations of Guang-Pheretima decoction (GPD) or protein-free Guang-Pheretima decoction (PF-GPD) and subsequently stimulated with lipopolysaccharide (LPS) to trigger the inflammatory response. Productions of nitric oxide (NO) were determined by Griess reaction, and prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 were measured by enzyme-linked immunosorbent assays (ELISA). The protein expressions and messenger ribonucleic acid (mRNA) amounts of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were analyzed by Western Blot and Real-Time polymerase chain reaction (PCR), respectively. Finally, the translocation of nuclear factor (NF)-κB was observed by Western Blot. RESULTS: GPD of the experimental concentrations showed no anti-inflammatory activity. In contrast, PF-GPD at concentrations of 40-320 µg/mL significantly inhibited NF-κB activation and reduced the production of inflammatory mediators, such as NO, PGE2, TNF-α, as well as the related key synthases including iNOS and COX-2. Moreover, PF-GPD markedly suppressed the release of inflammatory cytokines, such as IL-1ß and IL-6. CONCLUSION: These results demonstrate the excellent anti-inflammatory properties of PF-GPD, and suggest that Guang-Pheretima may be used to treat and prevent certain inflammatory diseases.

Emodin promotes the arrest of human lymphoma Raji cell proliferation through the UHRF1­DNMT3A­∆Np73 pathways.

Emodin is an active constituent found in the roots and rhizomes of numerous Chinese medicinal herbs. It exerts antitumor activity against Dalton's lymphoma in vivo, although the detailed mechanisms by which emodin induces apoptosis remains to be elucidated. The present study aimed to analyze the mechanisms underlying the response to emodin treatment. Using lymphoma Raji cells, an emodin­induced cell proliferating inhibition model was first established, then flow cytometry, western blotting, reverse transcription­quantitative polymerase chain reaction and luciferase reporter assay were performed. It was found that emodin decreased the percentage of Raji cell viability, induced apoptosis, and increased the activation of caspase 3, caspase 9 and poly (ADP­ribose) polymerase through the downregulation of ubiquitin­like protein containing PHD and RING domains 1 (UHRF1). The emodin­induced downregulation of UHRF1 led to an increase in the level of DNA methyltransferase 3A, which in turn inhibited the activity of p73 promoter 2 and decreased the levels of NH2­terminally truncated dominant­negative p73. The treatment of Raji cells with emodin combined with doxorubicin led increased cell death of Raji cells, indicating that emodin may sensitize Raji cells to doxorubicin­induced apoptosis.

Du-Huo-Ji-Sheng-Tang and its active component Ligusticum chuanxiong promote osteogenic differentiation and decrease the aging process of human mesenchymal stem cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Postmenopausal osteoporosis is the most common bone disease worldwide. Information concerning the effects of herbal medicines on mesenchymal cell osteogenesis and senescence remains lacking. AIM OF THIS STUDY: This study was designed to investigate the effects of Du-Huo-Ji-Sheng-Tang (DHJST), a Chinese herbal medicine and its active component Ligusticum chuanxiong on osteogenic differentiation and the aging process of human mesenchymal cells (hMSCs). MATERIALS & METHODS: hMSCs were used as in vitro model and osteogenesis was induced by administration of either osteogenesis inducing medium (OIM) or dexamethasone-depleted OIM (DDOIM) for 1-week or 2 weeks and the results were evaluated by measuring the formation of mineralization nodules. The effects of the compound recipe DHJST and its active component L. chuanxiong on hMSCs osteogenesis-related gene expression was determined by real-time PCR that targeted bone morphogenetic protein-2 (BMP2), RUNX2, ALP, COL-1, osteopontin (OPN), and osteocalcin (OCN). Antibodies against BMP-related signaling pathway proteins, such as BMP-2, ERK, SMAD 1/5/8, and RUNX2, were also detected at the protein level by Western blotting. Finally, the cumulative growth curve and senescence of the hMSCs were evaluated in order to assess the aging process. RESULTS: L. chuanxiong increased osteogenic activity in hMSCs and up-regulated BMP-2 and RUNX2 gene expression via the activation of SMAD 1/5/8 and ERK signaling. Furthermore DHJST also showed a trend towards promoting the same effects in the same system. In the absence of dexamethasone, DHJST did activate SMAD 1/5/8 and ERK signaling and hence increased RUNX2 protein expression in hMSCs. In addition, both DHJST and L. chuanxiong delayed the hMSCs aging process by decreasing cell senescence. CONCLUSIONS: We concluded that DHJST and its active component L. chuanxiong are able to promote osteogenic activity and decrease hMSCs senescence as cells age.

Pheretima aspergillum decoction suppresses inflammation and relieves asthma in a mouse model of bronchial asthma by NF-κB inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE AND AIM OF THE STUDY: Guang-Pheretima, the live form of the earthworm Pheretima aspergillum, is a traditional Chinese medicine commonly used for the treatment of asthma, cough, stroke, epilepsy and other diseases due to its anti-inflammatory, anti-asthmatic, anti-seizure, thrombolytic and diuretic properties. Although Guang-Pheretima is effective in the relief of asthma, its pharmacological activity and the underlying molecular mechanisms are not fully understood. Hence, we investigated the effects of a Pheretima aspergillum decoction (PAD) against inflammation in a model of ovalbumin (OVA)-induced asthma in BALB/c mice, as well as the nuclear factor-κB (NF-κB) pathway involved in this process. MATERIALS AND METHODS: OVA was used to sensitize and challenge the airway of the mice, and PAD was administrated by gavage. We measured airway hyperresponsiveness (AHR) in the mice 24h following a final methacholine challenge with whole-body plethysmography. The bronchoalveolar lavage fluid (BALF), serum and pulmonary tissues were collected 48h after the last challenge. The levels of inflammatory factors and the related mRNAs were determined by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR), respectively. The number of differential inflammatory cells in the BALF was counted. Serum total and OVA-specific IgE levels were measured with ELISA. The activation of NF-κB signaling in the lung was detected by western blotting. In addition, the lung tissues were stained with hematoxylin and eosin or periodic acid Schiff stain for histopathological examination. RESULTS: PAD treatment significantly alleviated AHR in the asthmatic mice, decreased the mRNA and protein levels of IL-4, IL-5 and IL-13 and downregulated IgE. In addition, PAD treatment attenuated mucus secretion and infiltration of inflammatory cells in the lung while inhibiting the activation of NF-κB signaling. CONCLUSIONS: PAD effectively inhibited the activation of NF-κB signaling in the lungs of mice with OVA-induced asthma, and mitigated AHR and Th2 type inflammatory reactions. Therefore, PAD may serve as a drug candidate for asthma treatment.

Effectiveness of Music Listening in Patients With Total Knee Replacement During CPM Rehabilitation.

AIM: This study investigated the effects of music listening on the anxiety, heart rate variability (HRV), and joint range of motion (ROM) of patients undergoing continuous passive motion (CPM) after total knee replacement surgery. METHOD: An experimental design was used. Participants in the experimental group (n = 49) listened to music from 10 min before receiving CPM until the end of the session (25 min in total) on the first and second day following surgery, whereas participants in the control group (n = 42) did not listen to music but rested quietly in bed starting 10 min before and throughout CPM. RESULTS: Compared with the control group, the experimental group exhibited significantly lower anxiety levels (p < .05) and increased CPM angles (p < .05) during treatment and increased active flexion ROM (p < .05) upon discharge. The low-frequency (LF)/high-frequency (HF) power ratio, normalized LF HRV, and normalized HF HRV of the two groups differed significantly, indicating that the patients in the experimental group had greater parasympathetic activity compared with those in the control group. CONCLUSION: Music listening can effectively reduce patient anxiety and enhance the ROM of their joints during postoperative rehabilitation. Health-care practitioners should consider including music listening as a routine practice for postoperative rehabilitation following orthopedic surgery.

Manipulation therapy prior to diagnosis induced primary osteosarcoma metastasis--from clinical to basic research.

Osteosarcoma (OS) patients who suffer manipulation therapy (MT) prior to diagnosis resulted in poor prognosis with increasing metastasis or recurrence rate. The aim of the study is to establish an in vivo model to identify the effects of MT on OS. The enrolled 235 OS patients were followed up in this study. In vivo nude mice model with tibia injection of GFP-labeled human OS cells were randomly allocated into MT(+) that with repeated massage on tumor site twice a week and no treatment as MT(-) group. The five-year survival, metastasis and recurrence rates were recorded in clinical subjects. X-ray plainfilm, micro-PET/CT scan, histopathology, serum metalloproteinase 2 (MMP2), metalloproteinase 9 (MMP9) level and human kinase domain insert receptor (KDR) pattern were assayed in mice model. The results showed that patient with MT decreased 5-year survival and higher recurrence or metastasis rate. Compatible with clinical findings, the decreased body weight (30.5 ± 0.65 g) and an increased tumor volume (8.3 ± 1.18 mm3) in MT(+) mice were observed. The increasing signal intensity over lymph node region of hind limb by micro-PET/CT and the tumor cells were detected in lung and bilateral lymph nodes only in MT(+) group. MMP2 (214 ± 9.8 ng/ml) and MMP9 (25.5 ± 1.81 ng/ml) were higher in MT(+) group than in MT(-) group (165 ± 7.8 ng/ml and 16.9 ± 1.40 ng/ml, individually) as well as KDR expression. Taking clinical observations and in vivo evidence together, MT treatment leads to poor prognosis of primary osteosarcoma; physicians should pay more attention on patients who seek MT before diagnosis.

Identification of multiple ingredients for a Traditional Chinese Medicine preparation (bu-yang-huan-wu-tang) by liquid chromatography coupled with tandem mass spectrometry.

Bu-yang-huan-wu-tang (BYHWT) is a popular Traditional Chinese Medicine formula consisting of seven herbal medicines (Astragalus membranaceus, Angelica sinensis, Paeonia lactiflora, Ligusticum chuanxiong, Carthamus tinctorius, Amygdalus persica and Pheretima aspergillum), that has been used in China for centuries to overcome stroke-induced disability. To ensure the consistency of quality, a reliable analytical method is required, therefore, we developed a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the major constituents in BYHWT. The herbal ingredients consisting of the cycloartane-type triterpene glycosides of astragaloside I, astragaloside II and astragaloside IV; isoflavones of formononetin, ononin calycosin, calycosin-7-O-ß-d-glucoside; ligustilide and paeoniflorin were separated on a C18 column with gradient elution of methanol/10 mM ammonium acetate buffer-formic acid (100:0.1, v/v). This study was performed by a mass spectrometer using electrospray ionization (ESI) with positive ionization ions monitored in the multiple reaction-monitoring (MRM) mode. The linearity, accuracy, precision, limit of detection (LOD) and lower limit of quantification (LLOQ) were validated for this quantification method, and the sensitivity, reliability and reproducibility were all confirmed. The experiments provided a good method for analyzing BYHWT extracts. This study also quantitated the active components in various brands of commercially available products. The results indicated that the pharmaceutical industrial products of BYHWT exhibited considerable variation in their contents of the herbal compounds.

Synthesis and SAR of piperazine amides as novel c-jun N-terminal kinase (JNK) inhibitors.

A novel series of c-jun N-terminal kinase (JNK) inhibitors were designed and developed from a high-throughput-screening hit. Through the optimization of the piperazine amide 1, several potent compounds were discovered. The X-ray crystal structure of 4g showed a unique binding mode different from other well known JNK3 inhibitors.

3,5-Disubstituted quinolines as novel c-Jun N-terminal kinase inhibitors.

The structure-based design and synthesis of a novel series of c-Jun N-terminal kinase (JNK) inhibitors with selectivity against p38 is reported. The unique structure of 3,5-disubstituted quinolines (2) was developed from the previously reported 4-(2,7-phenanthrolin-9-yl)phenol (1). The X-ray crystal structure of 16a in JNK3 reveals an unexpected binding mode for this new scaffold with protein.