Uncovering the Key miRNAs and Targets of the Liuwei Dihuang Pill in Diabetic Nephropathy-Related Osteoporosis based on Weighted Gene Co-Expression Network and Network Pharmacology Analysis.

BACKGROUND: Diabetic nephropathy-related osteoporosis (DNOP) is the most common comorbid bone metabolic disorder associated with diabetes mellitus (DM). The Liuwei Dihuang Pill (LWD) is a traditional Chinese herbal medicine widely used to treat diabetic complications, including diabetic nephropathy (DN). This study aimed to identify the biomarkers of the mechanisms of DNOP in LWD with systems biology approaches. METHODS: Herein, we performed an integrated analysis of the GSE51674 and GSE63446 datasets from the GEO database via weighted gene co-expression network and network pharmacology (WGCNA) analysis. In addition, a network pharmacology approach, including bioactive compounds, was used with oral bioavailability (OB) and drug-likeness (DL) evaluation. Next, target prediction, functional enrichment analysis, network analysis, and virtual docking were used to investigate the mechanisms of LWD in DNOP. RESULTS: WGCNA successfully identified 63 DNOP-related miRNAs. Among them, miR-574 was significantly upregulated in DN and OP samples. A total of 117 targets of 22 components associated with LWD in DNOP were obtained. The cellular response to nitrogen compounds, the AGERAGE signaling pathway in diabetic complications, and the MAPK signaling pathway were related to the main targets. Network analysis showed that kaempferol and quercetin were the most significant components. MAPK1 was identified as a potential target of miR-574 and the hub genes in the protein-protein interaction (PPI) network. The docking models demonstrated that kaempferol and quercetin had a strong binding affinity for Asp 167 of MAPK1. CONCLUSION: This study demonstrated that miR-574 may play important roles in DNOP, and the therapeutic effects of kaempferol and quercetin on LWD in DNOP might be mediated by miR-574 by targeting MAPK1. Our results provide new perspectives for further studies on the anti-DNOP mechanism of LWD.

Quercetin suppresses inflammatory cytokine production in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a chronic, progressive and systemic autoimmune disease mainly characterized by symmetric multijoint synovitis. Quercetin has anti-inflammatory, anti-oxidation and immune regulation activities, and therefore shows high medicinal value. The present study aimed to observe the effect of quercetin on fibroblast-like synoviocytes (FLSs) in RA. Rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) were pretreated with 50 nmol/l quercetin for 2 h and were then stimulated using TNF-α for 24 h for subsequent experiments. RAFLSs were transfected with short interfering (si)-X-inactive specific transcript (XIST), microRNA (miR)-485 mimic, miR-485 inhibitor or si-PSMB8 or combination. ELISA, PCR and western blotting was used to evaluate the effect of quercetin on RAFLSs treated with TNF-α. It was revealed that quercetin inhibited the production of inflammatory cytokines and the expression of XIST in RAFLSs induced by TNF-α. Bioinformatics analysis indicated that XIST acted as a sponge for miR-485 and that proteasome subunit ß type-8 (PSMB8) was a direct target of miR-485. Moreover, PSMB8 functioned as a suppressor in inflammatory cytokine production of RAFLSs induced by TNF-α. Overall, quercetin was observed to inhibit the production of inflammatory cytokines and the expression of XIST in RAFLSs induced by TNF-α. Moreover, XIST-silencing could suppress the inflammatory reaction by sponging miR-485 in cells treated with TNF-α. Altogether, quercetin could suppress the development of RA in vitro.

Expression of VEGF-A Signaling Pathway in Cartilage of ACLT-induced Osteoarthritis Mouse Model.

BACKGROUND: Anterior cruciate ligament transection surgery (ACLT)-induced OA model was often used to investigate the molecular mechanism of knee osteoarthritis (KOA). Researches have shown that vascular endothelial growth factor (VEGF) played an important role in OA. The present study aimed to investigate the pathological changes after ACLT surgery and reveal the expression characteristics of the VEGF-A/VEGFR2 signaling pathway in this model. METHODS: Moderate KOA model was established by ACLT, and 1, 2, 4, 8, and 12 weeks after surgery, hematoxylin-eosin (HE) and Safranin-O(S-O) staining were used to detect the pathological changes in mouse knee cartilage, and the matrix biomarkers A Disintegrin and Metalloproteinase with Thrombospondin Motifs 5(ADAMTS5), Collagen II (COL-II) were detected using immunohistochemistry (IHC), CD31 was detected by immunofluorescence (IF) to show the vascular invasion in cartilage, and proteins expression of VEGF-A pathway were detected by Western blot (WB). Meanwhile, the inflammatory biomarkers cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in cartilage were detected by WB. RESULTS: ACLT surgery can lead to degeneration of cartilage in mice, and the characteristics of the lesion were time-dependent. The ADAMTS5-positive cells increased while COL-II decreased in OA cartilage with time, and new blood vessels labeled by CD31 can be seen from 1 week in OA cartilage, and increased in 8 and 12 weeks. The expression of VEGF-A, VEGFR2, COX-2, and iNOS were higher than control groups, which were basically consistent with the degree of osteoarthritis. CONCLUSIONS: The degenerative degree of articular cartilage was time-dependent; angiogenesis and inflammation were important pathological changes of cartilage in KOA. The expression of the VEGF-A/VEGFR2 signaling pathway was basically correlated with the degree of KOA.

Therapeutic potential of Liuwei Dihuang pill against KDM7A and Wnt/ß-catenin signaling pathway in diabetic nephropathy-related osteoporosis.

The effects of Liuwei Dihuang pill (LWDH) on diabetic nephropathy-related osteoporosis (DNOP) are unclear. The present study aimed to evaluate the effects of LWDH on KDM7A and Wnt/ß-catenin signaling pathway in DNOP rats and the high glucose-induced MC3T3-E1 cells. A DNOP model was prepared by streptozotocin in 9-week-old male Sprague-Dawley (SD) rats to evaluate the effects of LWDH. The cell viability and differentiation capacity of high glucose-induced MC3T3-E1 cells were determined by CCK-8 assay, Alizarin Red staining, and alkaline phosphatase (ALP) staining, respectively. Furthermore, the expressions of KDM7A and Wnt1/ß-catenin pathway-related proteins were determined by Western blot analysis. Treatment of DNOP rats with LWDH could significantly ameliorate the general state, degradation of renal function, and renal pathological changes. LWDH decreased the levels of TNF-α, IL-6, IL-8, IL-1ß, ALP, and TRAP, and increased the calcium, phosphorus in serum, as well as decreased the level of the calcium and phosphorus in the urine. Besides, LWDH significantly improved bone mineral density (BMD), bone volume (BV), and the bone microstructure of DNOP rats. Moreover, LWDH increased the levels of the elastic modulus, ultimate load, and bending strength in the femurs. In MC3T3-E1 cells, serum-containing LWDH significantly increases in cell viability and osteoblastic differentiation capability. The expression of α-SMA, vimentin, KDM7A, Wnt1 and ß-catenin were significantly down-regulated, and the E-cadherin, H3K9-Me2, H3K27-Me2, BMP-4, BMP-7, Runx2, osteocalcin, and Col1a1 were significantly up-regulated with LWDH treatment. The present study shows that LWDH has a therapeutic effect on DNOP, in part, through down-regulation of KDM7A and Wnt/ß-catenin pathway.

Aquaporin-4 expression dynamically varies after acute spinal cord injury-induced disruption of blood spinal cord barrier in rats.

The blood-spinal cord barrier (BSCB) changes badly after spinal cord injury (SCI), and it is an important pathophysiological basis of SCI secondary damage. Aquaporin-4 (AQP4), one of the transmembrane proteins in spinal cord, has been shown to be closely related to the development of the BSCB and edema. We established a SCI model in rats using a free-falling weight drop device to subsequently investigate AQP4 expression. AQP4 messenger RNA (mRNA) and protein expression and immunoreactivity were detected in spinal cord tissue using reverse transcription-real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry and Western blot analysis. We found the water content and edema of the spinal cord were significantly higher than the control group after SCI, which was related to the growth of BSCB permeability; both reached their peak on the third day after injury. One, 3, 5, 7 days after injury, the immune response and protein expression in the model group increased from 1 to 3 days, with a plateau period from 3 to 5 days and a decline from 5 to 7 days, showing a significant difference compared with the sham group at each time point (P < 0.05), while the RT-qPCR results showed a decline of mRNA just after 3 days. In conclusion, after SCI, the water content of the spinal cord and the BSCB permeability increases, together with the excessive expression of AQP4, which reached a peak on the third day. AQP4 expression is closely relative to the permeability of BSCB and the water content of the spinal cord.

Effects of Clematis chinensis Osbeck mediated by low-intensity pulsed ultrasound on transforming growth factor-ß/Smad signaling in rabbit articular chondrocytes.

PURPOSE: Clematis chinensis Osbeck (CCO) is an essential herb that has been shown to promote the biological functions of cartilage cells. In this study, we aimed to explore whether and how low-intensity pulsed ultrasound (LIPUS) enhanced CCO delivery into chondrocytes and stimulated biological activity in vitro. METHODS: Chondrocytes were isolated from knee articular cartilage of 2-week-old rabbits and treated with LIPUS plus CCO or recombinant transforming growth factor beta 1 (TGF-ß1; 0.5 ng/mL), with or without anti-TGF-ß1 antibodies (10 µg/mL), for 3 days. Cell proliferation was assessed by Cell-Counting Kit-8 assays. Immunocytochemistry, western blotting, and quantitative polymerase chain reaction were applied to detect the expression of type II collagen and some molecules in the TGF-ß1 signal pathway. RESULTS: LIPUS plus 0.1 mg/mL CCO solution promoted chondrocyte proliferation and type II collagen and TGF-ß1 expression synergistically in vitro (P < 0.05). In addition, treatment with anti-TGF-ß1 antibodies blocked this effect (P < 0.01), but not completely. CCO plus LIPUS also showed more enhanced effects on promoting TGF-ß receptor II and Smad2 signaling and reducing Smad7 signaling than either intervention separately (P < 0.05). CONCLUSIONS: CCO plus LIPUS promoted extracellular matrix deposition by accelerating the TGF-ß/Smad-signaling pathway in chondrocytes.

[Clinical observation of percutaneous coblation nucleoplasty for the treatment of cervical spondylotic radiculopathy].

OBJECTIVE: To evaluate the clinical effects of percutaneous coblation nucleoplasty in treating cervical spondylotic radiculopathy and investigate its mechanism of action. METHODS: Form January 2015 to January 2017, 21 patients with cervical spondylotic radiculopathy were treated by percutaneous coblation nucleoplasty, including 8 males and 13 females with an average age of 49.6 years old ranging from 43 to 61 years old. The course of disease was for 1 to 6 months with a median age of 4 months. Three cases were single segment, 9 cases were double segments, 7 cases were three-segment, 2 cases were four-segment. Intervertebral disc pressure, VAS were compared before and after operation. Angular displacement(AD) and horizontal displacement(HD) were measured by image data and in order to evaluate the cervical stability. Modified MacNab criteria was used to assess clinical effects. RESULTS: All the patients were followed up from 1 to 12 months with an average of 8.6 months. Preoperative intervertebral disc pressure was (32.0±5.26) cmH2O and immediately after operation was (21.0±7.18) cmH2O, there was statistical significance between before and after operation(P=0.003). Preoperative angular displacement and horizontal displacement was (3.85±1.26) ° and (1.23±0.58) mm, six months after operation was (4.18±1.31) ° and (1.69±0.46) mm, respectively. There was no statistical significance before and after operation(P>0.05). Preoperative VAS scores were 7.49±0.53, postoperative at 3 days, 3, 6 months were 3.51±0.49, 2.63±0.61, 2.56±0.71, respectively, and postoperative obtained obvious improvement(P<0.05). According to modified MacNab criteria, 6 cases obtained excellent results, 7 good, 4 fair 3 poor at 3 days;10 cases obtained excellent results, 5 good, 3 fair, 2 poor at 3 months; 12 cases obtained excellent results, 6 good, 1 fair, 1 poor at 6 months after operation. Postoperative clinical effect at 6 months was better than 3 d, and 3 months(P<0.05), and postoperative at 3 months was better than 3 d(P<0.05). CONCLUSIONS: Percutaneous coblation nucleoplasty in treating cervical spondylotic radiculopathy can effectively relieve the pain of neck, shoulder and upper limb and can also relieve some associated symptoms such as headache and dizziness.

Jisuikang, a Chinese herbal formula, increases neurotrophic factor expression and promotes the recovery of neurological function after spinal cord injury.

The Chinese medicine compound, Jisuikang, can promote recovery of neurological function by inhibiting lipid peroxidation, scavenging oxygen free radicals, and effectively improving the local microenvironment after spinal cord injury. However, the mechanism remains unclear. Thus, we established a rat model of acute spinal cord injury using a modified version of Allen's method. Jisuikang (50, 25, and 12.5 g/kg/d) and prednisolone were administered 30 minutes after anesthesia. Basso, Beattie, and Bresnahan locomotor scale scores and the oblique board test showed improved motor function recovery in the prednisone group and moderate-dose Jisuikang group compared with the other groups at 3-7 days post-injury. The rats in the moderate-dose Jisuikang group recovered best at 14 days post-injury. Hematoxylin-eosin staining and transmission electron microscopy showed that the survival rate of neurons in treatment groups increased after 3-7 days of administration. Further, the structure of neurons and glial cells was more distinct, especially in prednisolone and moderate-dose Jisuikang groups. Western blot assay and immunohistochemistry showed that expression of brain-derived neurotrophic factor (BDNF) in injured segments was maintained at a high level after 7-14 days of treatment. In contrast, expression of nerve growth factor (NGF) was down-regulated at 7 days after spinal cord injury. Real-time fluorescence quantitative polymerase chain reaction showed that expression of BDNF and NGF mRNA was induced in injured segments by prednisolone and Jisuikang. At 3-7 days after injury, the effect of prednisolone was greater, while 14 days after injury, the effect of moderate-dose Jisuikang was greater. These results confirm that Jisuikang can upregulate BDNF and NGF expression for a prolonged period after spinal cord injury and promote repair of acute spinal cord injury, with its effect being similar to prednisolone.

Efficacy of massage therapy on pain and dysfunction in patients with neck pain: a systematic review and meta-analysis.

Objective. To systematically evaluate the evidence of whether massage therapy (MT) is effective for neck pain. Methods. Randomized controlled trials (RCTs) were identified through searches of 5 English and Chinese databases (to December 2012). The search terms included neck pain, neck disorders, cervical vertebrae, massage, manual therapy, Tuina, and random. In addition, we performed hand searches at the library of Nanjing University of Traditional Chinese Medicine. Two reviewers independently abstracted data and assessed the methodological quality of RCTs by PEDro scale. And the meta-analyses of improvements on pain and neck-related function were conducted. Results. Fifteen RCTs met inclusion criteria. The meta-analysis showed that MT experienced better immediate effects on pain relief compared with inactive therapies (n = 153; standardised mean difference (SMD), 1.30; 95% confidence interval (CI), 0.09 to 2.50; P = 0.03) and traditional Chinese medicine (n = 125; SMD, 0.73; 95% CI 0.13 to 1.33; P = 0.02). There was no valid evidence of MT on improving dysfunction. With regard to follow-up effects, there was not enough evidence of MT for neck pain. Conclusions. This systematic review found moderate evidence of MT on improving pain in patients with neck pain compared with inactive therapies and limited evidence compared with traditional Chinese medicine. There were no valid lines of evidence of MT on improving dysfunction. High quality RCTs are urgently needed to confirm these results and continue to compare MT with other active therapies for neck pain.

Effect of Jisuikang () on kinetic dysfunction in patients after spinal injury.

OBJECTIVE: To explore the effect of Jisuikang (, JSK) on kinetic dysfunction in patients after spinal injury. METHODS: Eighty-four patients with spinal injury were assigned equally, according to a randomizing digital table to the treated group and the control group. Conventional treatment was given to both groups, and JSK was additionally given to the treated group. Changes of various kinetic function concerning parameters including kinetic score, grades of spinal injury, effectiveness of the treatment and available recovery rate in patients allocated in the treated group and the control group were observed and compared in the way issued by Association of Spinal Injury of America (ASIA). RESULTS: Better effects were shown in the treated group than those in the control group in improving kinetic score (92.00+/-9.95 scores vs 83.76+/-24.12 scores), ASIA overall improvement rate (69.05% vs 45.24%) and grades of effectiveness (P<0.05). However, the difference of available recovery rate between the two groups was insignificant (P>0.05). CONCLUSION: JSK could prevent secondary alteration of spinal injury, promote the recovery and regeneration of nerve tissues, but could not restore the function of a necrotic spine.