Effect of manipulation on cartilage in rats with knee osteoarthritis based on the Rho-associated protein kinase/LIM kinase 1/Cofilin signaling pathways.

OBJECTIVE: To investigate the effect of manipulation treatment on knee osteoarthritis rats and the effect on Rho-associated protein kinase (ROCK)/LIM-kinase1 (LIMK1)/Cofilin signaling pathway. METHOD: Fifty Specific pathogen Free Sprague-Dawley rats were randomly divided into five groups ( = 8 each): blank group, model group, manipulation group, celecoxib group, and manipulation combined with celecoxib group (MC group). The osteoarthritis model was established by injecting 0.2 mL 4% papain into the articular disc of the rats. After successfully establishing the model, we treated the manipulation group with pushing manipulation using one-finger-meditation to the Neixiyan (EX-LE4), Waixiyan (EX-LE5), Xuehai (SP10), Liangqiu (ST34), and Zusanli (ST36) acupoints for 10 min each time. Also, the celecoxib group was gavaged with 24 mg•kg•d celecoxib, while the MC group was treated using both of these two methods. After four weeks, the cartilage of the right femur was removed for hematoxylin-eosin staining of the cartilage tissue. The expressions of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in serum were observed using the enzyme-linked immunosorbent assay. Besides, we detected the expressions of ROCK, LIMK1, Phospho-LIM-kinase1 (Phospho-LIMK1), Cofilin, and Phospho-Cofilin by Western blot. RESULTS: Compared to the model group, the manipulation group, celecoxib group, and MC group all exhibited superior results concerning pathological morphologic changes of cartilage, as observed by hematoxylin-eosin staining and calculated using the Mankin score. Besides, in contrast to the blank group, the model group exhibited elevated serum levels of IL-1ß and TNF-α ( 0.01), while the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage were all higher ( 0.01). Also, the serum levels of IL-1ß and TNF-α in each treatment group were lower (0.01) than in the model group. Moreover, there were lower expressions of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the manipulation group and the MC group (< 0.01). Compared with the model group, the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different ( > 0.05). CONCLUSION: In this study, we established that manipulation has a better curative effect than celecoxib. Manipulation inhibits the development of cytoskeleton damage in cartilage and slows articular degeneration by regulating the expression of related proteins in the cytoskeletal signaling pathway.

Shuxuetong injection and its peptides enhance angiogenesis after hindlimb ischemia by activating the MYPT1/LIMK1/Cofilin pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuxuetong (SXT) injection is formulated by leech and earthworm, has been widely used in the treatment of thrombotic cardiovascular and cerebrovascular diseases with remarkable clinical efficacy. AIM OF THE STUDY: The purpose of this study is to investigate the protective mechanism of SXT injection on the mice model of hindlimb ischemia, and to evaluate the angiogenic effects of SXT injection and its main active substances. MATERIALS AND METHODS: Hindlimb ischemia was induced by left femoral artery ligation. After operation, the mice were injected with saline, 10 mg/kg/d cilostazol, 37.5 mg/kg/d SXT injection, 75 mg/kg/d SXT injection and 150 mg/kg/d SXT injection via tail vein for 4 weeks. Ischemia severity was assessed using laser Doppler perfusion imaging system. Tissue recovery and capillary density were evaluated by histological and immunofluorescent staining. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF-BB) expression were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. Human umbilical vein endothelial cells (HUVECs) proliferation was measured using a BrdU kit and the viability of HUVECs was performed by MTT assay. Migration of HUVECs was performed by the wound healing method and a modified transwell assay. Capillary tube formation by HUVECs was examined by using Matrigel assay. Western blotting was used to detect the expressions of p-Cofilin, p-MYPT1, and p-LIMK1. RESULTS: SXT injection treatment significantly restored the blood flow and reduced tissue injury in mouse gastrocnemius muscle. SXT injection treatment increased capillary density and promoted angiogenesis in hindlimb ischemia. Moreover, SXT injection enhanced the expression of VEGF-A and PDGF-BB at both mRNA and protein levels in ischemic tissue of mice. SXT injection and its main active peptides dramatically increased the migration and capillary tube formation of HUVECs. SXT injection and its peptides enhanced protein expressions of the phosphorylation of MYPT1, Cofilin, and LIMK1. DSYVGDEAQSKR, YNELRVAPEEHP, and IQFLPEGSPVTM may act as the active components of SXT injection. CONCLUSION: SXT injection promoted angiogenesis and improved function recovery in hindlimb ischemia mice by regulation of VEGF-A/PDGF-BB. Moreover, SXT injection and its active peptides induced cell migration and tube formation in HUVECs through activating the MYPT1/LIMK1/Cofilin pathway. This study provided experimental basis for SXT injection in the treatment of ischemic diseases and revealed the effective substance of SXT injection in regulating angiogenesis, providing better evidence for the clinical application of SXT injection.