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(n=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 m L 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^(-1)·d^(-1 )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-1β(IL-1β)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-1βand TNF-α(P<0.01),while the expression of ROCK,LIMK1,Phospho-LIMK1,Cofilin,and Phospho-Cofilin in cartilage were all higher(P<0.01).Also,the serum levels of IL-1βand TNF-αin each treatment group were lower(P<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(P<0.01).Compared with the model group,the expression of ROCK,LIMK1,PhosphoLIMK1,Cofilin,and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different(P>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.

Role of inhibiting LIM-kinase2 in improving erectile function through suppression of corporal fibrosis in a rat model of cavernous nerve injury

We evaluated whether LIM-kinase 2 inhibitor （LIMK2i） could improve erectile function by suppressing corporal fibrosis through the normalization of the Rho-associated coiled-coil protein kinase 1 （ROCK1）/LIMK2/Cofilin pathway in a rat model of cavernous nerve crush injury （CNCl）. Sixty 11-week-old male Sprague-Dawley rats were divided equally into five groups： sham surgery （S）, CNCl （I）, and CNCl treated with low-dose （L）, medium-dose （M）, and high-dose （H） LIMK2i. The L, M, and H groups were treated with a daily intraperitoneal injection of LIMK2i （2.5, 5.0, and 10.0 mg kg-1 body weight, respectively） for 1 week after surgery. The erectile response was assessed using electrostimulation at 1 week, postoperatively. Penile tissues were processed for Masson＇s trichrome staining, double immunofluorescence, and Western blot assay. Erectile responses in the H group improved compared with the I group, while the M group showed only partial improvement. A significantly decreased smooth muscle/collagen ratio and an increased content of fibroblasts positive for phospho-/IMK2 were noted in the I group. The M and H groups revealed significant improvements in histological alterations and the dysregulated LIMK2/Cofilin pathway, except for LIMK2 phosphorylation in the M group. The inhibition of LIMK2 did not affect the ROCK1 protein expression. The content of fibroblasts positive for phospho-LIMK2 in the H group returned to the level found in the S group, whereas it did not in the M group. However, the L group did not exhibit such improvements. Our data suggest that the inhibition of LIMK2, particularly with administration of 10.0 mg kg^-1 body weight LIMK2i, can improve corporal fibrosis and erectile function by normalizing the LIMK2/Cofilin pathway.

MicroRNA-134在缺氧预处理新生大鼠缺氧缺血脑组织中的表达及意义

目的:观察micro RNA-134(miR-134)在缺氧预处理新生大鼠缺氧缺血后不同时期脑组织中的表达差异及其表达的特点及意义。方法:采用7日龄SD大鼠制备新生大鼠缺血缺氧性脑损伤动物模型。完全随机分为单纯缺血缺氧组、假手术对照组、缺氧预处理组(各18只)。3组又各分为处理后0h、1d、7d组(n=6)。每组6只用于荧光实时定量聚合酶链反应(qRT-PCR)观察miR-134转录量的差异。结果:miR-134在各组脑组织转录的表达量,单纯缺血缺氧组0 h(5.061±0.761)、1d(4.120±0.685)、7d(2.873±0.397);缺氧预处理组0 h(3.341±0.575)、1 d(2.769±0.351)、7d(1.658±0.290);假手术组0 h(6.617±1.988)、1d(5,798±1.116)、7d(5.984±1.879)。miR-134在单纯缺血缺氧组及缺氧预处理组脑组织的表达显著比假手术组减少(P<0.01),在同一时间段上,缺氧预处理组比单纯缺血缺氧组miR-134的表达明显减少(P<0.01)。同样在缺血缺氧后,无论是单纯缺血缺氧组还是缺氧处理组,miR-134的表达随着0h、1、7d的推移表达渐减少(P<0.05)。结论:miR-134的表达在调控神经系统发育中可能起着重要作用,缺氧预处理对新生大鼠缺血缺氧性脑损伤存在保护作用。

A conserved region in the 3' untranslated region of the human LIMK1 gene is critical for proper expression of LIMK1 at the post-transcriptional level

LIM kinase 1 （LIMK1）, a cytosolic serine/threonine kinase, regulates actin filament dynamics and reorganization and is involved in neuronal development and brain function. Abnormal expression of LIMK1 is associated with several neurological disorders. In this study, we performed a conservation analysis using Vector NTI （8.0） software. The dualluciferase reporter assay and real-time quantitative RT-PCR were used to assess the protein and mRNA levels of the reporter gene, respectively. We found that a region ranging from nt ＋884 to ＋966 in the human LIMK1 3＇ untranslated region （UTR） was highly conserved in the mouse Limkl 3＇ UTR and formed a structure containing several loops and stems. Luciferase assay showed that the relative luciferase activity of the mutated construct with the conserved region deleted, pGL4-hLIMK1-3U-M, in SH-SY5Y and HEK-293 cells was only -60% of that of the wild-type construct pGL4-hLIMK1-3U, indicating that the conserved region is critical for the reporter gene expression. Real-time quantitative RT-PCR analysis demonstrated that the relative Luc2 mRNA levels in SH-SY5Y and HEK293 cells transfected with pGL4-hLIMK1-3U-M decreased to50% of that in cells transfected with pGL4-hLIMK1- 3U, suggesting an important role of the conserved region in maintaining Luc2 mRNA stability. Our study suggests that the conserved region in the LIMK1 3＇ UTR is involved in regulating LIMK1 expression at the post-transcriptional level, which may help reveal the mechanism underlying the regulation of LIMK1 expression in the central nervous system and explore the relationship between the 3＇-UTR mutant and neuroloqical disorders.

LIMK2 is required for membrane cytoskeleton reorganization of contracting airway smooth muscle

Airway smooth muscle(ASM)has developed a mechanical adaption mechanism by which it transduces force and responds to environmental forces,which is essential for periodic breathing.Cytoskeletal reorganization has been implicated in this process,but the regulatory mechanism remains to be determined.We here observe that ASM abundantly expresses cytoskeleton regulators Limk1 and Limk2,and their expression levels are further upregulated in chronic obstructive pulmonary disease(COPD)animals.By establishing mouse lines with deletions of Limk1 or Limk2,we analyse the length-sensitive contraction,F/Gactin dynamics,and F-actin pool of mutant ASM cells.As LIMK1 phosphorylation does not respond to the contractile stimulation,LIMK1-deficient ASM develops normal maximal force,while LIMK2 or LIMK1/LIMK2 deficient ASMs show approximately 30%inhibition.LIMK2 deletion causes a significant decrease in cofilin phosphorylation along with a reduced F/G-actin ratio.As LIMK2 functions independently of cross-bridge movement,this observation indicates that LIMK2 is necessary for F-actin dynamics and hence force transduction.Moreover,LIMK2-deficient ASMs display abolishes stretching-induced suppression of 5-hydroxytryptamine(5-HT)but not acetylcholine-evoks force,which is due to the differential contraction mechanisms adopted by the agonists.We propose that LIMK2-mediated cofilin phosphorylation is required for membrane cytoskeleton reorganization that is necessary for ASM mechanical adaption including the 5-HT-evoked length-sensitive effect.