Shenhuang plaster ameliorates the Inflammation of postoperative ileus through inhibiting PI3K/Akt/NF-κB pathway.

BACKGROUND: Although Shenhuang plaster (SHP) from traditional Chinese medicine prescriptions, has the potential to promote the recovery progression of postoperative ileus (POI), the underlying mechanism remains elusive. Along these lines, in this work, both in vivo and in vitro studies were conducted to systematically explore the regulatory effect and mechanism of SHP on the inflammatory response of the intestinal basal layer in the POI model mice. METHODS: Intestinal manipulation in mice was utilized for the POI model. The impact of SHP in response to POI was evaluated by carrying fluorescein-labeled dextran, histomorphology, immunohistochemistry, in combination with flow cytometry analysis and transcriptome RNA sequencing in vivo. Besides, the cytotoxicity of the SHP treatment on RAW264.7 cells was detected by cell counting kit-8 (CCK-8), the biological effects were assessed by polymerase chain reaction (PCR) and the potential influences on the PI3K/Akt/NF-κB pathway were identified through detecting the expression levels of P85, AKT, IKK and P65 by western blot in vitro. RESULTS: The implementation of the SHP treatment could significantly reduce the expressions of interleukin (IL)- 1ß and tumor necrosis factor (TNF)-α in the intestine, whereas the recovery of gastrointestinal motility is promoted. In addition, SHP can regulate the polarization of macrophages, indicating that the proportion of the M2 type is increased after the application of the SHP treatment. In addition, SHP inhibited the activity of PI3K/AKT/NF-κB signaling pathway-related proteins. CONCLUSION: SHP can significantly ameliorate the inflammatory response of POI and at the same time promote the recovery of gastrointestinal motility. Its mechanism may be mediated by the polarization of macrophages through the PI3K/AKT/NF-κB signaling pathway.

Therapeutic or prophylactic anticoagulation in acute isolated distal deep vein thrombosis: protocol for a prospective, multicentre, single-blind, randomised controlled trial (TOP-IDDVT).

INTRODUCTION: The efficacy and safety of anticoagulant treatment is not established for patients with acute symptomatic isolated distal deep vein thrombosis (IDDVT). In real-world clinical practice, both therapeutic and prophylactic anticoagulation are used for acute IDDVT. However, therapeutic anticoagulation is associated with higher risk of bleeding than prophylactic anticoagulation. Thus, this study aims to assess the efficacy and safety in patients with first acute symptomatic IDDVT treated with therapeutic or prophylactic anticoagulation using rivaroxaban. METHODS AND ANALYSIS: This study is a prospective, multicentre, single-blind, randomised controlled trial. Outpatients with a first, acute, symptomatic, objectively confirmed IDDVT in four centres from 1 August 2021 are recruited. Eligible patients are randomised in a 1:1 ratio to receive prophylactic anticoagulation (rivaroxaban 10 mg once a day for 3 months) or therapeutic anticoagulation (rivaroxaban 20 mg once a day for 3 months). All patients are followed for 6 months. The primary efficacy outcome is radiographically confirmed recurrent venous thromboembolism. The primary safety outcome is the incidence of major or clinically relevant non-major bleeding events. ETHICS AND DISSEMINATION: This study has been approved by the Ethics Committee of Zhongshan Hospital Fudan University (B2021-175R). Study results will be disseminated through peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04967573.

Anti-hypertensive and endothelia protective effects of Fufang Qima capsule on primary hypertension via adiponectin/adenosine monophosphate activated protein kinase pathway.

OBJECTIVE: To investigate the potential mechanism of the vascular remodeling effect and provide additional information about anti-hypertension activity of Fufang Qima capsule. METHODS: Spontaneous hypertensive rats (SHRs) were used to study the underlying mechanism of the anti-hypertension activity of QM. In this study, SHRs were randomly divided into 5 groups: model group, Telmisartan group (7.2 mg/kg, p.o.), and three QM groups (0.9298, 1.8596, and 3.7192 g/kg, p.o.). Wistar Kyoto rats (WKY) were used as normal control group. Blood pressure (BP), aorta, perivascular adipose tissue (PVAT) histology were investigated to evaluate the effect of QM. Nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) phosphorylation were measured. Adiponectin (APN) secretion, as well as APN signal pathway proteins including APN, adiponectin receptors (R1 and R2) and adenosine 5'-monophosphate-activated protein kinase (AMPK) were all analyzed. RESULTS: QM significantly reduced BP and ameliorated the vascular pathological change, i.e. intima media thicken and collagen fiber hyperplasia. Meanwhile, QM increased concentration of NO and the phosphorylation of eNOS in the aorta. The anti-hypertensive and endothelia-protective effect of QM could be attributed to activating APN/ AMPK pathway by up-regulating the expression of APN in PVAT and APN Receptor 2, AMPKα and phosphorylated AMPKα in the aorta. CONCLUSION: The QM alleviation effect mechanism for primary hypertension was via modulating the APN/AMPK signal pathway.

Osteoking Decelerates Cartilage Degeneration in DMM-Induced Osteoarthritic Mice Model Through TGF-ß/smad-dependent Manner.

Osteoarthritis (OA) is a common disease characterized by cartilage degeneration. In recent years much attention has been paid to Traditional Chinese Medicine (TCM) since its treatments have shown efficacy for ameliorating cartilage degradation with mild side effects. Osteoking is a TCM prescription that has long been used in OA treatment. However, the exact mechanism of Osteoking are not fully elucidated. In the current study, destabilization of the medial meniscus (DMM)-induced OA mice was introduced as a wild type animal model. After 8 weeks of administration of Osteoking, histomorphometry, OARSI scoring, gait analysis, micro-CT, and immunohistochemical staining for Col2, MMP-13, TGFßRII and pSmad-2 were conducted to evaluate the chondroprotective effects of Osteoking in vivo. Further in vitro experiments were then performed to detect the effect of Osteoking on chondrocytes. TGFßRIICol2ER transgenic mice were constructed and introduced in the current study to validate whether Osteoking exerts its anti-OA effects via the TGF-ß signaling pathway. Results demonstrated that in wild type DMM mice, Osteoking ameliorated OA-phenotype including cartilage degradation, subchondral bone sclerosis, and gait abnormality. Col2, TGFßRII, and pSmad-2 expressions were also found to be up-regulated after Osteoking treatment, while MMP-13 was down-regulated. In vitro, the mRNA expression of MMP-13 and ADAMTS5 decreased and the mRNA expression of Aggrecan, COL2, and TGFßRII were up-regulated after the treatment of Osteoking in IL-1ß treated chondrocytes. The additional treatment of SB505124 counteracted the positive impact of Osteoking on primary chondrocytes. In TGFßRIICol2ER mice, spontaneous OA-liked phenotype was observed and treatment of Osteoking failed to reverse the OA spontaneous progression. In conclusion, Osteoking ameliorates OA progression by decelerating cartilage degradation and alleviating subchondral bone sclerosis partly via the TGF-ß signaling pathway.

Factor Xa inhibitor rivaroxaban suppresses experimental abdominal aortic aneurysm progression via attenuating aortic inflammation.

OBJECTIVE: Rivaroxaban is a specific factor Xa (FXa) inhibitor for venous thromboembolism treatment. Recently, increasing evidence have reported the beneficial effects of rivaroxaban on treating cardiovascular disorders such as coronary and peripheral artery disease. However, its potential influence on abdominal aortic aneurysm (AAA) remains unclear. This study aims to investigate whether rivaroxaban treatment could attenuate experimental AAA progression and its related mechanisms. APPROACHES AND RESULTS: In human aneurysmal aorta, FXa protein expression was significantly upregulated. Further investigations identified a positive correlation among plasma FXa level, AAA severity (the maximal aortic diameter), and intra-aneurysmal thrombus percentage. In Ang II (angiotensin II)-infused ApoE-/- mice, the administration of high dose rivaroxaban (15 mg/kg/d) for 14 days significantly reduced the maximal aortic diameter, while low dose rivaroxaban (5 mg/kg/d) did not display such a protective role. Although rivaroxaban treatments reduced the incidence of AAA and thrombus formation, these differences did not reach statistical significance. Immunohistochemistry revealed a pronounced aortic remodeling including increased collagen content and enhanced elastin degradation in Ang II-induced AAAs, which was inhibited by high dose rivaroxaban treatment. Further analysis demonstrated that rivaroxaban exerted its protective effects by decreasing leukocyte infiltration, inflammatory cytokines expression, and matrix metalloproteinases (MMPs) expression in the aortic wall. The inhibitory effect of rivaroxaban on aneurysm development was also observed in calcium chloride-induced AAA model. Mechanistically, in human aortic endothelial cells, FXa stimulation increased the expression of inflammatory cytokines (interleukin (IL)-1ß, IL-6, IL-8, monocyte chemoattractant protein-1) and adhesive molecules, which were all reversed by the cotreatment of rivaroxaban. Subsequent monocyte-endothelial cell interaction was enhanced after FXa stimulation and was alleviated by rivaroxaban cotreatment. In addition, FXa induced a significantly heightened expression of MMP2 in human aortic endothelial cells, which was ameliorated by rivaroxaban coadministration. CONCLUSIONS: Rivaroxaban attenuated both angiotensin II- and calcium chloride-induced abdominal aortic aneurysm (AAA) progressions, through inhibiting aortic remodeling and inflammation. Rivaroxaban could be a promising therapeutic agent in attenuating AAA development by counteracting FXa-induced aortic wall inflammation.

Bushenhuoxue formula accelerates fracture healing via upregulation of TGF-ß/Smad2 signaling in mesenchymal progenitor cells.

BACKGROUND: Although Bushenhuoxue formula (BSHXF) is successfully used as a non-traumatic therapy in treating bone fracture in China, the molecular mechanism underlying its effects remains poorly understood. PURPOSE: The present study aims to explore the therapeutic effects of BSHXF on fracture healing in mice and the underlying mechanism. METHODS: We performed unilateral open transverse tibial fracture procedure in C57BL/6 mice which were treated with or without BSHXF. Fracture callus tissues were collected and analyzed by X-ray, micro-CT, biomechanical testing, histopathology and quantitative gene expression analysis. Tibial fracture procedure was also performed in Cre-negative and Gli1-CreER; Tgfbr2flox/flox conditional knockout (KO) mice (Tgfbr2Gli1ER) to determine if BSHXF enhances fracture healing in a TGF-ß-dependent manner. In addition, scratch-wound assay and cell counting kit-8 (CCK-8) assay were used to evaluate the effect of BSHXF on cell migration and cell proliferation in C3H10T1/2 mesenchymal stem cells, respectively. RESULTS: BSHXF promoted endochondral ossification and enhanced bone strength in wild-type (WT) or Cre- control mice. In contrast, BSHXF failed to promote bone fracture healing in Tgfbr2Gli1ER conditional KO mice. In the mice receiving BSHXF treatment, TGF-ß/Smad2 signaling was significantly activated. Moreover, BSHXF enhanced cell migration and cell proliferation in C3H10T1/2 cells, which was strongly attenuated by the small molecule inhibitor SB525334 against TGF-ß type I receptor. CONCLUSION: These data demonstrated that BSHXF promotes fracture healing by activating TGF-ß/Smad2 signaling. BSHXF may be used as a type of alternative medicine for the treatment of bone fracture healing.

Bushenhuoxue formula promotes osteogenic differentiation of growth plate chondrocytes through ß-catenin-dependent manner during osteoporosis.

BACKGROUND: Bushenhuoxue formula (BSHXF) has shown excellent clinical effects on the treatment of osteoporosis in China. The aim of this study is to determine the anti-osteoporosis effects and precise molecular mechanisms of BSHXF on mouse models. METHODS: Ten-week-old female C57BL/6 J mice were subjected to ovariectomy and provided a daily treatment of BSHXF. At 8 weeks post-surgery, the femurs were harvested for tissue analyses including µCT, histology, qRT-PCR and immunohistochemical (IHC) staining of ß-catenin, ALP and FABP4. To investigate the role of ß-catenin in the anti-osteoporosis effects of BSHXF, relative experiments mentioned above were performed in ß-catenin conditional knockout mice. RESULTS: Ovariectomized (OVX) mice presented severe bone loss and excessive fat accumulation in the chondro-osseous junction underneath the growth plate, with decreased expression of ALP and increased expression of FABP4. BSHXF significantly recovered the OVX-induced abnormal osteogenesis and adipogenesis with the activation of ß-catenin in growth plate chondrocytes. Further, we generated growth plate chondrocyte-specific ß-catenin knockout (ß-cateninGli1ER) mice that exhibited bone loss and fat accumulation in the chondro-osseous junction, similar to the OVX mice. However, BSHXF failed to rescue the osteoporosis-like phenotype in ß-cateninGli1ER mice, indicating the anti-osteoporosis effects of BSHXF act mainly through ß-catenin signaling. No significant restoration of ALP and FABP4 was observed in ß-cateninGli1ER mice after the treatment of BSHXF. CONCLUSIONS: BSHXF attenuates osteoporosis by promoting osteogenic differentiation of growth plate chondrocytes mainly in ß-catenin-dependent manner. BSHXF is considered as a new candidate for the treatment of osteoporosis.

Yougui pills exert osteoprotective effects on rabbit steroid-related osteonecrosis of the femoral head by activating ß-catenin.

OBJECTIVE: To investigate the effect and underlying mechanism of Yougui pills (YGPs) on steroid-related osteonecrosis of the femoral head (SONFH). METHODS: Male New Zealand white rabbits were divided into three groups: control group, SONFH group and YGPs group. Rabbit SONFH was induced by methylprednisolone (MPS) combined with lipopolysaccharide (LPS). At 6 weeks post induction, the femoral heads were harvested for tissue analyses, including histopathology, mechanical test of femoral heads, micro-CT, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry for osteocalcin (OCN), vascular endothelial growth factor (VEGF) and ß-catenin. Protein levels of cathepsin K (CTSK), phospho-glycogen synthase kinase-3 beta (p-Ser9 GSK-3ß) and total glycogen synthase kinase-3 beta (GSK-3ß) in femoral heads were also detected. Additionally, the serum TRAP activity was measured using enzyme-linked immunosorbent assay (ELISA). Finally, the effects of YGPs treatment on osteoclast differentiation and osteoblast formation were evaluated in vitro. RESULTS: The ratio of empty lacuna was markedly lower in YGPs group than SONFH group. Micro-CT evaluation indicated that YGPs has a preventive effect on bone loss in rabbit SONFH. YGPs treatment could suppress bone resorption by reducing TRAP+ osteoclast and serum TRACP5b levels in necrotic femoral heads. Moreover, YGPs treatment could promote bone formation by up-regulating the expression of OCN, VEGF and ß-catenin, while increasing load-bearing capacity of femoral heads. Interestingly, p-Ser9 GSK-3ß downregulation, and CTSK upregulation in necrotic femoral head could be reversed by YGPs treatment, which also effectively inhibited RANKL-induced osteoclast differentiation and promoted osteoblast formation in vitro. CONCLUSION: YGPs could suppress osteoclastogenesis and promote bone formation during SONFH in rabbits by activating ß-catenin.

Effect of Traditional Chinese Medicine Product, QiangGuYin, on Bone Mineral Density and Bone Turnover in Chinese Postmenopausal Osteoporosis.

Introduction. The aim of this study was to investigate the efficacy of herbal formula QiangGuYin (QGY) in postmenopausal women. Materials and Methods. A total of 240 participants from six clinical centers were randomly to receive alendronate 70 mg/week, QGY granules 20 g/day, and placebo. Primary end points were BMD changes over 6 and 12 months; secondary end points were bone turnover markers changes at 3, 6, 9, and 12 months. Safety was monitored by clinical adverse events reported during the follow-up. Results. Of 240 women recruited, 218 completed the study. Significant BMD increases from baseline were observed over 6 and 12 months at each observed part both in QGY and alendronate compared with placebo (p < 0.01). Alendronate-treated subjects had significant decreases in ß-CTX compared to QGY-treated subjects at each time point assessed (p < 0.01). Reduction in t-P1NP was only observed in the QGY group at 3 and 6 months (-23.81% and -3.07%, resp.). No significant difference was observed in the overall incidence of clinical adverse events among the alendronate group and the QGY group (5.0% versus 7.5%, p = 0.513). Conclusion. 1-Year treatment with QGY demonstrated a safe statistical increase in BMD and new balance may be rebuilt after 9 months. This trail is registered with ChiCTR-POC-16008026.

Identification of human serum protein targets of Qianggu Decoction () in primary type I osteoporosis based on tandem mass tag labeling and liquid chromatography-tandem mass spectrometry technology.

OBJECTIVE: To investigate the serum protein targets of Qianggu Decoction (, QGD) on treating osteoporosis by the proteomics analysis using tandem mass tag (TMT) and liquid chromatographytandem mass spectrometry (LC-MS/MS). METHODS: Twenty serum protein samples were recruited (10 patients with primary type I osteoporosis before and after QGD treatment) and the high abundance ratios protein was removed, two serum samples were extracted and labeled with TMT reagent. Then, mass spectrometric detection, identification of differentially expressed proteins and bioinformatics analysis of differentially expressed proteins were carried out. RESULTS: A total of 60 proteins were identified, within a 99% confidence interval, to be differentially regulated of which, 34 proteins were up-regulated and 26 proteins were down-regulated. Differentially expressed proteins analyzed by Gene Ontology (GO) annotation mainly get involved in 12 different biological processes, 7 types of cellular components, and 6 kinds of molecular functions. Angiotensinogen (AGT), stromelysin-1 (MMP3), heparanase (HPSE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were screened as candidate protein targets of QGD treatment, which were related to metabolic mechanism of bone remodeling and/or bone collagen of osteoporosis. By the utilization of the protein-protein interaction network analysis tool named STRING10.0, it showed that AGT, MMP3, HPSE and GAPDH were located in the key node of the protein-protein interactions network. Furthermore, AGT, MMP3, HPSE and GAPDH were found to be directly related to BMP, MAPK, Wnt, SMAD and tumor necrosis factor ligand superfamily member 11 (TNFSF11) families. CONCLUSIONS: The proteomics analysis by using TMT combined with LC-MS/MS was a feasible method for screening the potential therapeutic targets associated with QGD treatment. It suggests that AGT, MMP3, HPSE and GAPDH may be candidate protein targets of QGD treatment which can be used as therapeutic effect monitor and early diagnosis of primary type I osteoporosis.