Characteristics of Pain Changes in Rats with Nerve Injury within 24 Hours after One-Time Tuina Intervention.

Tuina, as an external treatment method of traditional Chinese medicine, has been proven to have an analgesic effect on peripheral neuropathic pain (pNP) in clinical and basic research. However, the optimal time point for the analgesic effect of tuina may vary according to different injury sensations, affecting the exploration of the initiation mechanism of tuina analgesia. The research used minor chronic constriction injury (minor CCI) model rats to simulate pNP and used the intelligent tuina manipulation simulator to simulate the three methods (point-pressing, plucking, and kneading) and three acupoints (Yinmen BL37, Chengshan BL57, and Yanglingquan GB34) for performing tuina therapy. The study evaluated the changes in pain within 24 h and the optimal time point for the efficacy of tuina analgesia in rats with minor CCI models by testing cold sensitivity threshold (CST), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL). Furthermore, the study evaluated IL-10 and TNF-α expression changes through Elisa detection. The results show that tuina has both immediate and sustained analgesic effects. For the three different injury sensitivity thresholds of CST, MWT, TWL, and two cytokines of IL-10 and TNF-α, the analgesic efficacy of tuina within 24 h after intervention is significantly different at different time points.

Exploring the Mechanism of Immediate Analgesia Induced by Tuina Intervention on Minor Chronic Constriction Injury in Rats Using LC-MS.

Purpose: This study aimed to investigate changes in metabolomic expression in the spinal dorsal horn (SDH) and thalamus during a Tuina session, aiming to elucidate the mechanism of immediate analgesia. Methods: The rats were randomly divided into three groups: the Sham group, the Model group, and the Tuina group. A minor chronic constriction injury (minor CCI) model was established in both the Model group and the Tuina group. The therapeutic effect of Tuina was determined using the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests. Differential metabolites of the SDH and thalamus were detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatic analysis was performed using CV, PCA, Venn, and KEGG. Results: The therapeutic effect of MWT and TWL after instant Tuina intervention was significant. The therapeutic effect of Tuina instant was significantly better compared to the Model group. In the Veen analysis, it was found that Tuina instantly regulates 10 differential metabolites in the SDH and 5 differential metabolites in the thalamus. In the KEGG enrichment analysis, we found that differential metabolites were enriched in 43 pathways in the thalamus and 70 pathways in the SDH. Conclusion: Tuina therapy may have analgesic effects by metabolizing neurotransmitters such as 2-Picolinic Acid, 5-Hydroxy-Tryptophan Glutathione Betaine-aldehyde-chloride Leucine Lysine Methionine Sarcosine Succinic Acid Histidine Acetylcholine and 5-Hydroxyindoleacetic Acid through the cAMP pathway. It also affects pathways of neurodegeneration-multiple diseases, butanoate metabolism, tyrosine metabolism.

The effect of tuina on ulcerative colitis model mice analyzed by gut microbiota and proteomics.

Tuina can effectively alleviate ulcerative colitis-related symptoms, but the mechanism of action is unknown. The purpose of this research is to explore potential pathways for the treatment of tuina through gut microbiota and proteomics techniques. Thirty-two male BALB/c mice were divided into four groups, the control, model, mesalazine, and tuina groups. The ulcerative colitis model was established by freely drinking a 3% dextran sulphate sodium solution for 7 days. The mesalazine group and the tuina group, respectively, received 7 days of mesalazine and tuina treatment. Subsequently, their body weights, feces properties, colon length, histomorphological changes, gut microbiota, and colon proteomics were determined. Body weights, disease activity index score, colon histological scores, and microbiota diversity were restored in the tuina group. At the phylum level, Firmicutes was increased and Bacteroidota decreased. At the family level, Lachnospiraceae increased and Prevotellaceae decreased. At the genus level, the Lachnospiraceae_NK4A136_group was increased. Proteomics detected 370 differentially expressed proteins regulated by tuina, enriched to a total of 304 pathways, including biotin metabolism, Notch signaling pathway, linoleic acid metabolism, and autophagy. Tuina can effectively improve the symptoms of weight loss, fecal properties, and colon inflammation in ulcerative colitis mice and restore the gut microbiota diversity, adjusting the relative abundance of microbiota. The therapeutic effects of tuina may be achieved by modulating the signaling pathways of biotin metabolism, Notch signaling pathway, linoleic acid metabolism, and autophagy.

Exploring the mechanism of immediate analgesic effect of 1-time tuina intervention in minor chronic constriction injury rats using RNA-seq.

Previous studies have proved and investigated the mechanism of the analgesic effect of tuina treatment on neuropathic pain. The purpose of this study was to analyze changes in gene expression in the dorsal root ganglia (DRG) and spinal dorsal horn (SDH) after 1-time tuina intervention to investigate the immediate analgesic mechanism by tuina. An improvement in nociceptive behavior in minor chronic constriction injury (CCI) rats after 1-time tuina was observed. 1-time tuina was more effective in the amelioration of thermal hyperalgesia, but no changes were found in the ultrastructure of DRG and SDH. Sixty-five differentially expressed genes (DEGs) modulated by tuina were detected in the DRG and 123 DEGs were detected in the SDH. Potential immediate analgesic mechanisms of tuina were analyzed by the Kyoto Encyclopedia of Genes and Genomes. DEGs were enriched in 75 pathways in DRG, and 107 pathways in SDH. The immediate analgesic mechanism of tuina is related to the calcium signaling pathway, thermogenesis, and regulation of lipolysis in adipocytes.

[YU Tian-yuan's experience in the clinical application of Danzhong (CV 17) for mental illness].

To summarize YU Tian-yuan's experience of applying Danzhong (CV 17) for mental illness in acupuncture and tuina. YU Tian-yuan uses Danzhong (CV 17) alone or in combination with other acupoints to treat mental illnesses such as insomnia, palpitation and chest distress. Professor YU emphasizes 4 tips when treating diseases, nourishing the heart to tranquilize by light stimulation; regulating spirit by combined stimulation; leaving the acupoints and holding on the meridian for a wide range of stimulation; using rubbing and pushing manipulation in several directions for regulating qi to soothe the chest. And in clinical practice, formed a unique therapy to treat mental illness.

A Review on the Immunomodulatory Mechanism of Acupuncture in the Treatment of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease with a high prevalence and canceration rate. The immune disorder is one of the recognized mechanisms. Acupuncture is widely used to treat patients with IBD. In recent years, an increasing number of studies have proven the effectiveness of acupuncture in the treatment of IBD, and some progress has been made in the mechanism. In this paper, we reviewed the studies related to acupuncture for IBD and focused on the immunomodulatory mechanism. We found that acupuncture could regulate the innate and adaptive immunity of IBD patients in many ways. Acupuncture exerts innate immunomodulatory effects by regulating intestinal epithelial barrier, toll-like receptors, NLRP3 inflammasomes, oxidative stress, and endoplasmic reticulum stress and exerts adaptive immunomodulation by regulating the balance of Th17/Treg and Th1/Th2 cells. In addition, acupuncture can also regulate intestinal flora.

Tuina for peripherally-induced neuropathic pain: A review of analgesic mechanism.

Peripherally-induced neuropathic pain (pNP) is a kind of NP that is common, frequent, and difficult to treat. Tuina, also known as massage and manual therapy, has been used to treat pain in China for thousands of years. It has been clinically proven to be effective in the treatment of pNP caused by cervical spondylosis, lumbar disc herniation, etc. However, its analgesic mechanism is still not clear and has been the focus of research. In this review, we summarize the existing research progress, so as to provide guidance for clinical and basic studies. The analgesic mechanism of tuina is mainly manifested in suppressing peripheral inflammation by regulating the TLR4 pathway and miRNA, modulating ion channels (such as P2X3 and piezo), inhibiting the activation of glial cells, and adjusting the brain functional alterations. Overall, tuina has an analgesic effect by acting on different levels of targets, and it is an effective therapy for the treatment of pNP. It is necessary to continue to study the mechanism of tuina analgesia.

A Review on the Mechanism of Tuina Promoting the Recovery of Peripheral Nerve Injury.

Tuina, as one of the characteristic external therapies of Traditional Chinese Medicine (TCM), has been used to treat the disease caused by peripheral nerve injury (PNI) for thousands of years. An increasing number of clinical trials and animal experiments have demonstrated that tuina can improve the symptoms and promote the recovery of damaged nerves. This review focuses on the mechanistic studies of tuina in promoting the recovery of PNI, which might provide a neurobiological foundation for the effects of tuina. Although many mechanisms underlying the effects of tuina on nerve repair have been identified, there are still many unknown problems, such as the key substance or way for tuina to work, so further investigation is warranted.

Evaluation and Analysis of Water Quality of Marine Aquaculture Area.

In the rapid development of marine aquaculture, the water quality of aquatic environments is regarded as a main limiting factor. Therefore, it is necessary to assess the water quality and environmental conditions in marine aquaculture areas and find out the main influencing factors regarding damage to the water quality environment. In the present research, pond aquaculture and cage aquaculture areas were sampled in May, August and November in 2018. Nine water quality indicators were detected, including pH, temperature, salinity, dissolved oxygen, molybdate-reactive phosphorus, chemical oxygen demand, chlorophyll a, inorganic nitrogen and antibiotic resistance genes (ARGs). Principal component analysis (PCA) was used to analyze the water quality conditions, spatial-temporal changes, and the driving factors in pond and cage aquaculture areas. The results showed that three main components were extracted from the pond aquaculture area, which explained 66.82% of the results, the most relevant factors are salinity, dissolved oxygen and ARGs. For the cage aquaculture area, three main components were extracted which can account for 72.99% of the results, the most relevant factors are chlorophyll a, salinity and dissolved oxygen. The comprehensive scores of the principal components indicated that the heaviest polluted months in pond and aquaculture areas were August and November, respectively. The water quality of the pond aquaculture area is mainly limited by the volume of the pond, while aquaculture activities and seasonality are the main factors for cage aquaculture. ARGs in cage culture areas showed more variety and frequency compared with pond culture areas, which indicated that terrestrial input might be one of the sources for ARGs occurrence. The results would be helpful for the relevant authorities to select water quality monitoring parameters in marine aquaculture areas.

A novel antimicrobial peptide, Ranatuerin-2PLx, showing therapeutic potential in inhibiting proliferation of cancer cells.

Antimicrobial peptides are a promising resource for developing novel antibiotic and even anticancer drugs. Here, a 28-mer polypeptide, Ranatuerin-2PLx (R2PLx), was identified from lyophilised skin secretions. The chemically synthetic replicates exhibited moderate and broadspectrum antimicrobial effect against various microorganisms including methicillin-resistant Staphylococcus aureus (MRSA, minimal inhibitory concentration = 256 µM). In addition, R2PLx was found to inhibit the proliferation of several tumour cells, especially showing more potent effect on prostate cancer cell, PC-3. The early cell apoptosis was observed in 6 h by Annexin V-FITC/propidium iodide staining, as well as the activation of Caspase-3 at 5 µM peptide concentration. R2PLx may therefore be promising for developing new therapeutic approach for cancer treatment. Moreover, the artificial deficiency of conserved rana-box loop or net positive charge in C-terminal domain notably reduced the biological activities of the truncated and substituted isoforms, respectively, suggesting for maintaining their biological potency of ranatuerin family requires both cysteine-bridged segment and cationincity within the loop domain in C-terminus.

Adenine alleviates iron overload by cAMP/PKA mediated hepatic hepcidin in mice.

Hemochromatosis is prevalent and often associated with high rates of morbidity and mortality worldwide. The safe alternative iron-reducing approaches are urgently needed in order to better control iron overload. Our unbiased vitamin screen for modulators of hepcidin, a master iron regulatory hormone, identifies adenine (vitamin B4) as a potent hepcidin agonist. Adenine significantly induced hepcidin mRNA level and promoter activity activation in human cell lines, possibly through BMP/SMAD pathway. Further studies in mice validated the effect of adenine on hepcidin upregulation. Consistently, adenine dietary supplement in mice led to an increase of hepatic hepcidin expression compared with normal diet-fed mice via BMP/SMAD pathway. Notably, adenine-rich diet significantly ameliorated iron overload accompanied by the enhanced hepcidin expression in both high iron-fed mice and in Hfe-/- mice, a murine model of hereditary hemochromatosis. To further validate this finding, we selected pharmacological inhibitors against BMP (LDN193189). We found LDN193189 strongly blocked the hepcidin induction by adenine. Moreover, we uncovered an essential role of cAMP/PKA-dependent axis in triggering adenine-induced hepcidin expression in primary hepatocytes by using 8 br cAMP, a cAMP analog, and H89, a potent inhibitor for PKA signaling. These findings suggest a potential therapeutic role of adenine for hereditary hemochromatosis.

Online hyphenation of extraction, Sephadex LH-20 column chromatography, and high-speed countercurrent chromatography: A highly efficient strategy for the preparative separation of andrographolide from Andrographis paniculata in a single step.

A novel isolation strategy, online hyphenation of ultrasonic extraction, Sephadex LH-20 column chromatography combined with high-speed countercurrent chromatography, was developed for pure compounds extraction and purification. Andrographolide from Andrographis paniculata was achieved only in a single step purification protocol via the present strategy. The crude powder was ultrasonic extracted and extraction was pumped into Sephadex LH-20 column directly to cut the nontarget fractions followed by the second-dimensional high-speed countercurrent chromatography, hyphenated by a six-port valve equipped at the post-end of Sephadex LH-20 column, for the final purification. The results yielded andrographolide with the amount of 1.02 mg and a purity of 98.5% in a single step, indicating that the present method is effective to harvest target compound from medicinal plant.

Effects of Gnaphalium affine D. Don on hyperuricemia and acute gouty arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: The Gnaphalium affine D. Don is used in China as a folk medicine to treat gout, anti-inflammatory, antitussive and expectorant activities. The aim of this study was to evaluate the potential of the extract of G. affine to treat hyperuricemia and acute gouty arthritis in animal model. MATERIALS AND METHODS: G. affine extract was evaluated in an experimental model with potassium oxonate (PO) induced hyperuricemia in mice which was used to evaluate anti-hyperuricemia activity and xanthine oxidase (XO) inhibition. Therapies for acute gouty arthritis was also investigated on monosodium urate (MSU) crystal induced paw edema model. RESULTS: G. affine extract showed expressive results on active in reducing serum uric acid (Sur) through effect renal mGLUT9 and mURAT1 mainly and inhibit XO activity in vivo. The extract of G. affine also showed significant anti-inflammatory activity and reduced the paw swelling on MSU crystal-induced paw edema model. Meanwhile, eight major compounds were identified by HPLC-ESI-QTOF-MS/MS. CONCLUSIONS: The extract of G. affine showed significant effect on evaluated models and therefore may be active agents for the treatment of hyperuricemia and acute gouty arthritis.

DDR2-CYR61-MMP1 Signaling Pathway Promotes Bone Erosion in Rheumatoid Arthritis Through Regulating Migration and Invasion of Fibroblast-Like Synoviocytes.

Regulation of matrix metalloproteinases (MMPs) by collagen in the fibroblast-like synoviocytes (FLSs) plays a critical role in joint destruction in rheumatoid arthritis (RA). Our previous study indicated that discoidin receptor 2 (DDR2) mediated collagen upregulation of MMPs. However, the precise underlying mechanism remains unclear. We report here that CYR61, a secreted, extracellular matrix-associated signaling protein which is capable of regulating a broad range of cellular activities, including cell adhesion, migration, proliferation, and apoptosis, is significantly upregulated in collagen II-stimulated RA FLS. Further studies found that collagen II-activated phosphorylated-DDR2 induces CYR61 through activation of transcription factor activator protein 1 (AP-1). The elevated CYR61, in turn, accelerates MMP1 production via ETS1 (ETS proto-oncogene 1). In addition, CYR61 significantly promotes FLS invasion and migration. Blockade of CYR61 by an adenovirus expressing CYR61 shRNA (Ad-shCYR61) in vivo remarkably ameliorated the severity of arthritis, reduced inflammatory cytokine secretion, and attenuated bone erosion as detected by micro-computed tomography (µCT), in collagen-induced arthritis (CIA) rats. Taken together, we uncovered the Collagen II-DDR2-AP-1-CYR61-ETS1-MMP1 loop in RA FLS. In which, CYR61 acts as a hinge to promote cartilage damage through regulating FLS invasion, migration, and MMP1 production and the inflammatory cascade in RA. Thus, CYR61 may be a promising diagnostic and therapeutic target for RA treatment. © 2016 American Society for Bone and Mineral Research.

A novel NF-κB/YY1/microRNA-10a regulatory circuit in fibroblast-like synoviocytes regulates inflammation in rheumatoid arthritis.

The main etiopathogenesis of rheumatoid arthritis (RA) is overexpressed inflammatory cytokines and tissue injury mediated by persistent NF-κB activation. MicroRNAs widely participate in the regulation of target gene expression and play important roles in various diseases. Here, we explored the mechanisms of microRNAs in RA. We found that microRNA (miR)-10a was downregulated in the fibroblast-like synoviocytes (FLSs) of RA patients compared with osteoarthritis (OA) controls, and this downregulation could be triggered by TNF-α and IL-1ß in an NF-κB-dependent manner through promoting the expression of the YingYang 1 (YY1) transcription factor. Downregulated miR-10a could accelerate IκB degradation and NF-κB activation by targeting IRAK4, TAK1 and BTRC. This miR-10a-mediated NF-κB activation then significantly promoted the production of various inflammatory cytokines, including TNF-α, IL-1ß, IL-6, IL-8, and MCP-1, and matrix metalloproteinase (MMP)-1 and MMP-13. In addition, transfection of a miR-10a inhibitor accelerated the proliferation and migration of FLSs. Collectively, our data demonstrates the existence of a novel NF-κB/YY1/miR-10a/NF-κB regulatory circuit that promotes the excessive secretion of NF-κB-mediated inflammatory cytokines and the proliferation and migration of RA FLSs. Thus, miR-10a acts as a switch to control this regulatory circuit and may serve as a diagnostic and therapeutic target for RA treatment.

Corn oil as a vehicle in drug development exerts a dose-dependent effect on gene expression profiles in rat thymus.

The purpose of this study was to investigate the effects of corn oil (CO), which is widely used as a vehicle for water-insoluble agents in drug development, on the gene expression profiles in rat thymus with microarray technique. Female Wistar rats were administered daily with normal saline (NS) and CO 2, 5 and 10 ml kg⁻¹ per day for 14 days, respectively. Then, the thymus samples of rats were collected for microarray test and histopathology examination. CD4⁺ and CD8⁺ lymphocytes in peripheral blood were also numerated to assess the effects on lymphocyte subpopulations. The microarray data showed that the numbers of differentially expressed genes in the 2, 5 and 10 ml kg⁻¹ CO groups were 0, 40 and 458, respectively, compared with the NS control group. The altered genes were mainly associated with immune response, cellular response to organic cyclic substance and regulation of fatty acid ß-oxidation. However, no abnormal changes in thymus weight, CD4⁺ and CD8⁺ lymphocytes counts and histopathological examination were observed in the three CO groups. These data showed that 10 ml kg⁻¹ CO, the usually recommended dosing volume as a vehicle in drug safety assessment, caused obvious dysregulated genes in rat thymus. Our study suggests that the appropriate dosing volume of CO gavage as a vehicle for water-insoluble agents in drug development should be 2 ml kg⁻¹ per day, if agent effects on thymus will be assessed in gene levels.

Aluminum ammonium sulfate dodecahydrate purified from traditional Chinese medicinal herb Korean monkshood root is a potent matrix metalloproteinase inhibitor.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases and key regulators for many physiological and pathological functions. The MMP inhibitors have been shown to modulate diseases such as cancer, inflammation, and cardiovascular diseases. In this paper we tracked the MMP inhibitory activities of the traditional Chinese medicinal herb Korean Monkshood Root. The purified active ingredient was identified by the elemental analysis, infrared spectrum (IR) and X-ray diffraction as aluminum ammonium sulfate dodecahydrate. This inorganic compound showed inhibitory activities toward a number of MMP family members. In particular, it has a strong inhibitory effect toward MMP-2 and MMP-9, with IC50 values of 0.54 and 0.50 µM, respectively. Further analysis suggested that the MMP inhibitory activity is mainly due to Al(3+). Cell viability assays using human fibrosarcoma HT1080 cells showed aluminum ammonium sulfate had minimal cyto-toxicity with a concentration up to 500 µM. However, within 50 µM, it exhibited significant inhibition of cell invasion. To our knowledge, there has been no previous report of inorganic form of the MMP inhibitor with strong inhibitory activity. Our results for the first time showed that aluminum ammonium sulfate is an inorganic form of MMP inhibitor with high potency, and can be used to interfere with MMP related cellular processes.

Preclinical safety evaluation of IFNalpha2a-NGR.

IFNalpha2a-NGR is an antitumor agent of bacterial origin. The report presents the preclinical toxicity studies with IFNalpha2a-NGR in mice, rats and monkeys. The single-dose toxicity study showed no effect on general signs, body weight, food consumption, ophthalmology, hematology and clinical chemistry and necropsy analysis. In repeated-dose toxicity studies, increase in HB was noted both in rat and monkey, showed that IFNalpha2a-NGR may not cause the suppression of hematopoiesis. Decrease in AST, A/G, GLU, T-Bil in rat and AST, TP, GLO in monkey were noted, accompanied by increase in TP and GLB in rat and BUN in monkey. All the clinical chemistry changes were mild, reversible and considered to be incidental, since no related abnormal parameters or results were found. Increase in spleen and thymus organ-to-body weight ratios and decrease in menses were mild, reversible and likely related to pharmacology activity of IFNalpha2a. Ames, chromosomal aberration and bone marrow micronulecus test were conducted and the results were negative. The degree of irritation caused by various concentration of IFNalpha2a-NGR was determined to be the same as that induced by normal saline. In conclusion, preclinical safety studies that IFNalpha2a-NGR was well tolerated at pharmacologically active doses in mice, rats and monkeys.