Gross saponins of Tribulus terrestris attenuate rheumatoid arthritis by promoting apoptosis of fibroblast-like synoviocytes and reducing inflammation by inhibiting MAPK signalling pathway.

Among the numerous treatment options for rheumatoid arthritis (RA), the promotion of synoviocyte apoptosis and inhibition of inflammation are considered the most effective. However, the potential pro-apoptotic effects of gross saponins of Tribulus terrestris (GSTT), which are natural saponins derived from the herb Tribulus terrestris L., on rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) and their essential molecular mechanisms remain unclear. The aim of the present study was to investigate the influence of different concentrations of GSTT on RA-FLSs using various assays, including cell counting kit-8 (CCK-8), reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and western blot analysis. These assessments were conducted to evaluate the cell viability, changes in the levels of inflammatory cytokines, apoptosis rates and alterations in protein expression related to this process. In vivo, arthritis clinical score, haematoxylin and eosin (HE) staining and ELISA were used to assess paw inflammation, histopathology and serum inflammatory cytokine changes. Our findings demonstrated that GSTT substantially promotes the apoptosis of RA-FLSs and reduces pro-inflammatory cytokine levels. GSTT also reduced the Bcl-2/Bax ratio and inhibited JNK and p38 phosphorylation. Furthermore, GSTT exhibits positive effects on RA by improving clinical scores, reducing synovial inflammatory infiltration and lowering serum pro-inflammatory cytokine levels. Therefore, by promoting the apoptosis of RA-FLSs and suppressing inflammation through the inhibition of the MAPK signalling pathway, GSTT is a promising therapeutic intervention for RA.

Expression of SGTA correlates with neuronal apoptosis and reactive gliosis after spinal cord injury.

Small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA) is a novel TPR-containing protein involved in various biological processes. However, the expression and roles of SGTA in the central nervous system remain unknown. We have produced an acute spinal cord injury (SCI) model in adult rats and found that SGTA protein levels first significantly increase, reach a peak at day 3 and then gradually return to normal level at day 14 after SCI. These changes are striking in neurons, astrocytes and microglia. Additionally, colocalization of SGTA/active caspase-3 has been detected in neurons and colocalization of SGTA/proliferating cell nuclear antigen has been detected in astrocytes and microglial. In vitro, SGTA depletion by short interfering RNA inhibits astrocyte proliferation and decreases cyclinA and cyclinD1 protein levels. SGTA knockdown also reduces neuronal apoptosis. We speculate that SGTA is involved in biochemical and physiological responses after SCI.

Temporal-spatial expressions of Spy1 in rat sciatic nerve after crush.

As a novel cell cycle protein, Spy1 enhances cell proliferation, promotes the G1/S transition as well as inhibits apoptosis in response to UV irradiation. Spy1 levels are tightly regulated during mammary development, and overexpression of Spy1 accelerates tumorigenesis in vivo. But little is known about the role of Spy1 in the pathological process of damage and regeneration of the peripheral nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of Spy1. Spy1 expression was elevated gradually after sciatic nerve crush and peaked at day 3. The alteration was due to the increased expression of Spy1 in axons and Schwann cells after SNC. Spy1 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, Spy1 largely localized in axons in the crushed segment, but rarely co-localized with GAP43. These findings suggested that Spy1 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.

Changes in Ataxin-10 expression after sciatic nerve crush in adult rats.

Ataxin-10 is a cytoplasmic protein that belongs to the family of armadillo repeat proteins and the ataxin proteins are ubiquitously expressed in nervous tissue. A loss of Ataxin-10 in primary neuronal cells causes increased apoptosis of cerebellar neurons. Knockdown of ATXN10 with siRNA in HeLa cells results in cytokinesis defects-multinucleation. Because of the essential role of Ataxin-10 in nervous system and cellular cytokinesis, we investigated the spatiotemporal expression of Ataxin-10 in a rat sciatic nerve crush (SNC) model. After never injury, we observed that Ataxin-10 had a significant up-regulation from 3d, peaked at day 5 and then gradually decreased to the normal level at 4 weeks. At its peak expression, Ataxin-10 expressed mainly in Schwann cells and macrophages of the distal sciatic nerve segment from injury, but had few co-localizations in axons. Besides, the peak expression of Ataxin-10 was in parallel with proliferating cell nuclear antigen (PCNA), and Ataxin-10 co-labeled with PCNA. Thus, all of our findings suggested that Ataxin-10 may be involved in the pathophysiology of sciatic nerve after SNC.

Spatiotemporal expression of SKIP after rat sciatic nerve crush.

Ski-interacting protein (SKIP) is a highly conserved protein from yeast to Human. As an essential spliceosomal component and transcriptional co-regulator it plays an important role in preinitiation, splicing and polyadenylation. SKIP can also combine with Ski to overcome the G1 arrest and the growth-suppressive activities of pRb. Furthermore SKIP has the capacity to augment TGF-ß dependent transcription. While the distribution and function of SKIP in peripheral nervous system lesion and regeneration remain unclear. Here, we investigated the spatiotemporal expression of SKIP in an acute sciatic nerve crush model in adult rats. Western Blot analysis revealed that SKIP was expressed in normal sciatic nerves. It gradually increased, reached a peak at 1 week after crush, and then returned to the normal level at 4 weeks. Besides, we observed that up-regulation of SKIP was approximately in parallel with Proliferating cell nuclear antigen (PCNA), and numerous Schwann cells (SCs) expressing SKIP were PCNA and Ki-67 positive. Collectively, we hypothesized peripheral nerve crush induced up-regulation of SKIP in the sciatic nerve, which was associated with SCs proliferation.

Upregulated expression of ebp1 contributes to schwann cell differentiation and migration after sciatic nerve crush.

Ebp1, an ErbB3-binding protein, is the human homologue of the cell cycle-regulated mouse protein p38-2G4. Ebp1 was reported to inhibit the proliferation and induce the differentiation of human cancer cells. Its p48 isoform contributes to neuronal differentiation and growth factor specificity. However, the expression and role of Ebp1 in peripheral system lesions and repair are still unknown. Herein, we investigated the spatiotemporal pattern of Ebp1 expression following sciatic nerve crush. After crush, the level of Ebp1 protein was elevated gradually, peaked at day 5, and then declined to the normal at 4 weeks, which was similar to the expression of Oct-6. Furthermore, using double immunofluorescent staining, we found Ebp1 had a colocalization with S100 and Oct-6 in 5-day injured tissues. In vitro, we observed enhanced expression of Ebp1 during the process of cyclic adenosine monophosphate (cAMP)-induced Schwann cells differentiation. Interestingly, Ebp1-depleted SCs did not show significant morphologic change after the treatment of cAMP. Also, we observed a colocalization between Ebp1 and Cyclin D1 and that Ebp1-specific siRNA-transfected SCs had a decreased migration. Taken together, we speculated that Ebp1 was upregulated in the sciatic nerve after crush, which was involved in the differentiation and migration of Schwann cells.

Increased expression of Gem after rat sciatic nerve injury.

Gem belongs to the Rad/Gem/Kir subfamily of Ras-related GTPases, whose expression is induced in several cell types upon activation by extracellular stimuli. Two functions of Gem have been demonstrated, including regulation of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. Because of the essential relationship between actin reorganization and peripheral nerve regeneration, we investigated the spatiotemporal expression of Gem in a rat sciatic nerve crush (SNC) model. After never injury, we observed that Gem had a significant up-regulation from 1 day, peaked at day 5 and then gradually decreased to the normal level. At its peak expression, Gem expressed mainly in Schwann cells (SCs) and macrophages of the distal sciatic nerve segment, but had few colocalization in axons. In addition, the peak expression of Gem was in parallel with PCNA, and numerous SCs expressing Gem were PCNA positive. Thus, all of our findings suggested that Gem may be involved in the pathophysiology of sciatic nerve after SNC.

Changes in the Foxj1 expression of Schwann cells after sciatic nerve crush.

Foxj1 is a member of the Forkhead box family of transcription factors expressed in multiple tissues during development and a major regulator of cilia development. It was reported that Foxj1 has a significant up-regulation after traumatic brain injury and plays an important role in central nervous system injury and repair. However, its expression and function in the peripheral nervous system lesion are not well understood. In this study, we investigated the spatiotemporal expression of Foxj1 in a rat sciatic nerve crush model. After never injury, we observed that Foxj1 had a significant up-regulation from 1 day, peaked at day 3 and then gradually decreased to the normal level at 4 weeks. At its peak expression, Foxj1 expressed mainly in Schwann cells (SCs) of the distal sciatic nerve segment from injury, but had few co-localizations in axons. Besides, the peak expression of Foxj1 was in parallel with proliferating cell nuclear antigen (PCNA), and numerous SCs expressing Foxj1 were PCNA positive. Collectively, we hypothesized that peripheral nerve crush-induced up-regulation of Foxj1 in the sciatic nerve was associated with Schwann cells proliferation.

Temporal and spatial expression of KIF3B after acute spinal cord injury in adult rats.

The KIF3 subunit KIF3B was proved to be associated with mitosis. It has been known to be engaged in intracellular transport of neurons. To elucidate the certain expression and biological function in central nervous system, we performed an acute spinal cord contusion injury model in adult rats. Western blot analysis indicated a marked upregulation of KIF3B after spinal cord injury (SCI). Immunohistochemistry revealed wide distribution of KIF3B in spinal cord, including neurons and glial cells. Double immunofluorescent staining for proliferating cell nuclear antigen and phenotype-specific markers showed increases of KIF3B expression in proliferating microglia and astrocytes. Our data suggest that KIF3B may be implicated in the proliferation of microglia and astrocytes after SCI.

Dynamic changes of Jab1 and p27kip1 expression in injured rat sciatic nerve.

Jun activation domain-binding protein (Jab1) is a multifunctional protein that participates in affecting signaling pathway, controlling cell proliferation and apoptosis, and regulating genomic instability and DNA repair, and acts as a key subunit of COP9 signalosome. p27kip1, a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, was shown to inhibit the enzymatic activity of cyclin-CDK complexes, resulting in cell-cycle arrest at G1. Recent studies have shown that Jab1 directly binds to p27kip1 and induces nuclear export and subsequent degradation in a variety of human cancers, while the association and function of Jab1 and p27kip1 in nervous system lesion and regeneration remain unclear. Here, we performed a sciatic nerve injury model in adult rats and studied the dynamic changes of Jab1 and p27kip1 expression by Western blot. Sciatic nerve crush (SNC) resulted in a significant upregulation of Jab1 and a downregulation of p27kip1. Besides, we observed that Jab1 was expressed widely in Schwann cells (SCs) and had few co-localization in axons by double immunofluorescence staining. In addition, the peak expression of Jab1 was parallel with proliferating cell nuclear antigen (PCNA), and numerous SCs expressing Jab1 were PCNA-positive. Results obtained by co-immunoprecipitation and double labeling further showed their interaction in the sciatic nerve. Thus, these results suggested that Jab1 and p27kip1 may be involved in the pathophysiology of sciatic nerve after SNC.

Changes in the BAG1 expression of Schwann cells after sciatic nerve crush.

Bcl-2-associated athanogene-1 (BAG1), a co-chaperone for Hsp70/Hsc70, is a multifunctional protein, which has been shown to suppress apoptosis and enhance neuronal differentiation. However, the expression and roles of BAG1 in peripheral system lesions and repair are still unknown. In this study, we investigated the dynamic changes in BAG1 expression in an acute sciatic nerve crush model in adult rats. Western blot analysis revealed that BAG1 was expressed in normal sciatic nerves. BAG1 expression increased progressively after sciatic nerve crush, reached a peak 2 weeks post-injury, and then returned to the normal level 4 weeks post-injury. Spatially, we observed that BAG1 was mainly expressed in Schwann cells and that BAG1 expression increased in Schwann cells after injury. In vitro, we found that BAG1 expression increased during the cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation process. BAG1-specific siRNA inhibited cAMP-induced Schwann cell differentiation. In conclusion, we speculated that BAG1 was upregulated in the sciatic nerve after crush, which was associated with Schwann cell differentiation.