Mechanism of ShuiJingDan in Treating Acute Gouty Arthritis Flares Based on Network Pharmacology and Molecular Docking.

Purpose: This study examined the underlying mechanisms of SJD's anti-inflammatory and analgesic effects on acute GA flares. Methods: This study used pharmacology network and molecular docking methods. The active ingredients of ShuiJingDan (SJD) were obtained from the Traditional Chinese Medicine Systems Pharmacology Analysis Platform (TCMSP), and the relevant targets of GA were obtained from the Online Mendelian Inheritance in Man (OMIM) database and Therapeutic Target Database (TTD). The core drug group-target-disease Venn diagram was formed by crossing the active ingredients of SJD and the relevant targets. Gene Ontology (GO) analysis was conducted for functional annotation, DAVID was used for Kyoto Encyclopedia of Genes, and Genomes pathway enrichment analysis, and R was used to find the core targets. The accuracy of SJD network pharmacology analysis in GA treatment was verified by molecular docking simulations. Finally, a rat GA model was used to further verify the anti-inflammatory mechanism of SJD in the treatment of GA. Results: SJD mainly acted on target genes including IL1B, PTGS2, CXCL8, EGF, and JUN, as well as signal pathways including NF-κB, Toll-like receptor (TLR), IL-17, and MAPK. The rat experiments showed that SJD could significantly relieve ankle swelling, reduce the local skin temperature, and increased the paw withdrawal threshold. SJD could also reduce synovial inflammation, reduced the concentrations of interleukin-1ß (IL-1ß), IL-8, and COX-2 in the synovial fluid, and suppressed the expression of IL1B, CXCL8, and PTGS2 mRNA in the synovial tissue. Conclusion: SJD has a good anti-inflammatory effect to treat GA attacks, by acting on target genes such as IL-1ß, PTGS2, and CXCL8.

Clock genes regulate skeletal muscle energy metabolism through NAMPT/NAD+/SIRT1 following heavy-load exercise.

The biological clock is an invisible "clock" in the organism, which can regulate behavior, physiology, and biochemical reactions. However, the relationship between clock genes and energy metabolism in postexercise skeletal muscle is not well known. The purpose of this study was to determine the mechanisms through which peripheral clock genes regulate energy metabolism in skeletal muscle. We analyzed the rhythm of mRNA expression of the clock genes Bmal1 and Clock in skeletal muscle following heavy-load exercise and measured related indicators of mitochondrial structure and function. We obtained the following experimental results. First, heavy-load exercise induced loss of circadian rhythm of Bmal1 between ZT0 and ZT24, and the circadian rhythm of Clock was not restored between ZT0 and ZT72. Second, analysis of mitochondrial morphology in group E showed abnormal swelling and ridge structure damage at ZT0, which recovered somewhat at ZT24 and ZT48, and the damage had essentially disappeared by ZT72. Third, the expression of NAMPT/NAD+/SIRT1 signaling axis proteins in group E was abnormal at ZT0, the content of NAMPT and the activity of SIRT1 significantly increased, and the content of NAD+ significantly decreased. Fourth, the expression of BMAL1 and PGC-1α in group E significantly increased, whereas the ATP and ADP content, as well as the activities of COXII and COXIV, were significantly changed. Finally, the colocalization of BMAL1 and SIRT1 in group E was significantly upregulated at ZT0. These results suggest that the skeletal muscle clock gene Bmal1 may regulate the energy metabolism level of skeletal muscle after exercise through the NAMPT/NAD+/SIRT1 signaling pathway.

Exercise improved bone health in aging mice: a role of SIRT1 in regulating autophagy and osteogenic differentiation of BMSCs.

Introduction: This study was designed to investigate the effect of running exercise on improving bone health in aging mice and explore the role of the SIRT1 in regulating autophagy and osteogenic differentiation of Bone marrow Mesenchymal Stem Cells (BMSCs). Methods: Twelve-month-old male C57BL/6J mice were used in this study as the aging model and were assigned to treadmill running exercise for eight weeks. Non-exercise male C57BL/6J mice of the same old were used as aging control and five-month-old mice were used as young controls. BMSCs were isolated from mice and subjected to mechanical stretching stimulation in vitro. Results: The results showed that aging mice had lower bone mass, bone mineral density (BMD), and autophagy than young mice, while running exercise improved BMD and bone mass as well as upregulated autophagy in bone cells. Mechanical loading increased osteogenic differentiation and autophagy in BMSCs, and knockdown of SIRT1 in BMSCs demonstrated that SIRT1-regulated autophagy involved the mechanical loading activation of osteogenic differentiation. Conclusion: Taken together, this study revealed that exercise improved bone health during aging by activating bone formation, which can be attributed to osteogenic differentiation of BMSCs through the activation of SIRT1-mediated autophagy. The mechanisms underlying this effect may involve mechanical loading.

Massage protects skeletal muscle from injury during long-term heavy-duty exercise via integrin ß1 and laminin 2 channels of basement membrane.

BACKGROUND: Massage is widely used in exercise-induced skeletal muscle damage (EIMD). It has been proven that massage can improve the morphology and function of damaged skeletal muscle in multiple ways. However, whether massage can protect skeletal muscles from injury during long-term heavy-duty exercise has not yet been determined. METHODS: In this study, a rat model of overuse injury was established by eccentric running for 4 weeks, and pressing at constant pressure and frequency and massage were used as intervention methods to explore whether massage could protect skeletal muscle from injury through upregulating integrin and the basement membrane laminin. RESULTS: The results showed that compared with the model group, the ultrastructure of skeletal muscle in the massage group was relatively complete and clear, and the maximum isotonic and tetanic contraction forces were significantly increased (P < 0.01). In addition, in the massage group, ß1 integrin expression was significantly increased, p-FAK protein expression was decreased, and the co-localization of ß1 integrin and the basement membrane laminin 2 was significantly increased (P < 0.01). CONCLUSION: Our study shows that during long-term heavy-duty exercise, massage can enhance the cell adhesion function mediated by integrin ß1 and laminin 2 to protect skeletal muscle from injury and prevent the occurrence of overuse injury.

Neuroprotection of exercise: P2X4R and P2X7R regulate BDNF actions.

The neurotrophin brain-derived neurotrophic factor (BDNF), which acts as a transducer, is responsible for improving cerebral stroke, neuropathic pain, and depression. Exercise can alter extracellular nucleotide levels and purinergic receptors in central nervous system (CNS) structures. This inevitably activates or inhibits the expression of BDNF via purinergic receptors, particularly the P2X receptor (P2XR), to alleviate pathological progression. In addition, the significant involvement of sensitive P2X4R in mediating increased BDNF and p38-MAPK for intracerebral hemorrhage and pain hypersensitivity has been reported. Moreover, archetypal P2X7R blockade induces mouse antidepressant-like behavior and analgesia by BDNF release. This review summarizes BDNF-mediated neural effects via purinergic receptors, speculates that P2X4R and P2X7R could be priming molecules in exercise-mediated changes in BDNF, and provides strategies for the protective mechanism of exercise in neurogenic disease.

[The role of clock gene BMAL1 in exercise-induced skeletal muscle injury recovery].

Objective: To investigate the role of clock gene BMAL1 in exercise-induced skeletal muscle injury recovery. Methods: Two hundred and eight 8-week-old SD rats were randomly divided into the control group (Group C, n=104) and the exercise group (Group E, n=104). Group E performed a 90-minute downhill run on the treadmill. After exercise, the gastrocnemius muscle of 8 rats in Group C and Group E were collected at 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h, 54 h, 60 h, 66 h and 72 h. The expression of skeletal muscle core clock gene, BMAL1 was measured by real-time fluorescence quantitative PCR. The parameters of fitting cosine curve were obtained by cosine analysis software circacompare (R package), and the change trend of rhythmic oscillation was analyzed. The ultrastructure of skeletal muscle fibers was observed by transmission electron microscope. The expressions of skeletal muscle BMAL1 and DESMIN were detected by Western blot; Immunofluorescence was used to observe the localization and contents of BMAL1 and DESMIN. Results: In Group C, three complete circadian rhythm cycles of mRNA BMAL1 were observed within 72 hours; in Group E, the circadian rhythm of BMAL1 mRNA disappeared at 0 h～24 h. Compared with Group C, the expression level of BMAL1 mRNA was significantly increased at 0 h, 6 h, 12 h, and 18 h after exercise in Group E (P＜0.05), and the expression of BMAL1 protein was significantly increased at 0 h and 12 h after exercise(P＜0.05), and recovered to the level of that in Group C from 24 h to 72 h(P＞0.05). The expression of DESMIN protein was decreased at 0 h and 12 h after exercise(P＜0.05), gradually increased at 24 h, increased significantly at 48 h(P＜0.01), and recovered to the control level at 72 h (P＞0.05). In Group E, BMAL1 and DESMIN were co-localized at 0 h, 12 h, and 24 h after exercise; the colocalization at 0 h～24 h showed a trend of first decreasing and then increasing, and the fluorescence intensity at 24 h reached the highest value. Conclusion: The post-exercise clock gene BMAL1 may be involved in the enhanced synergy of regulating the cytoskeletal protein DESMIN, it is thus related to the promotion of muscle fiber structure recovery.

FAM134B-mediated ER-phagy alleviates endoplasmic reticulum stress of rat soleus muscle in response to acute exercise.

ER-phagy is a selective endoplasmic reticulum (ER) autophagy mediated by ER-localized receptors, which ensures proper cellular homeostasis under stress. However, it remains unclear whether ER-phagy is involved in skeletal muscle response to exercise stress. Male 8-week-old Sprague-Dawley rats were subjected to an exercise protocol comprising a 90-min downhill run with a slope of -16° and a speed of 16 m/min. The soleus of the rats was sampled at 0, 12, 24, 48, and 72 h after exercise. After exercise, the sarcoplasmic/ER calcium ATPase (SERCA) content decreased, the protein disulphide isomerase (PDI) content increased, and ER stress (GRP78 and CRT) and autophagy (FAM134B and LC3)-related protein expression increased in the soleus muscle of rats, and gradually recovered with time. We also used pharmacological methods to simulate the effects of exercise stress on skeletal muscle cells to further explore the mechanism of ER-phagy in skeletal muscle cells. Thapsigargin was used to inhibit the SERCA pump of L6 myoblasts and successfully induce ER stress and activate ER-phagy. During this process, the ER-phagy receptor FAM134B anchors and fragments ER, and then binds with LC3 to form autophagosomes. These results suggest that ER-phagy is involved in the skeletal muscle cell response to exercise stress, which helps to maintain cellular ER homeostasis during exercise.