Research In Human Lumbar Disc Degeneration In The Role And Mechanism Of Leptin

Background and purposeLow back pain (Low back pain, LBP) is one of the most common disorders of the musculoskeletal system, with an average annual incidence rate of about30%and a lifetime prevalence of about70%. Intervertebral disc degeneration (IDD) is considered to be a major cause of LBP and acute discogenic pain. With accelerating aging process of the social population, the number of patients with IDD is increasing. Nevertheless, the exact etiology and pathogenesis of the disc degeneration remain unknown. The possible etiological factors included the body weight, injury, infection, smoking, and genetic predisposition. Research showed that obesity was an established risk factor for Low back pain. The incidence of IDD in person with body mass index, BMI≥25kg/m2was4.3times higher, than person with BMI≤25kg/m2. Obesity is one of the independent causes of IDD. Leptin (16kDa) is a small molecule protein secreted mainly by adipose tissues. It can cause weight loss by adjusting appetite and promoting fat metabolism. Study showed that the serum leptin levels were positively associated with body weight, namely the expression of leptin in obese person increased. Several recent studies indicated that leptin could not only involve in the metabolism of fat but also in other physiological and pathological processes, such as immune regulation, thermogenesis, ovarian function, tumorigenesis, metastasis, proliferation and inflammatory cell reaction. Study showed that the expression of leptin was detected in synovial fluid, osteophyte and articular cartilage in patients with osteoarthritis. And studies have confirmed that the cultured human intervertebral disc cells can naturally secreted detectable levels of Leptin. Leptin and its specific receptor were detected in NP cells of prolapsed lumbar intervertebral disc. Study confirmed that leptin could stimulate proliferation of disc cells in vitro. These researches provide the basis for further study to investigate the mechanism of leptin in the pathogenesis of IDD. However, the role of leptin in the pathogenesis of disc degeneration is not yet clear. There were no published papers before. The purpose of this study was to investigate the role of leptin in IDD and its mechanism.MethodsThe human NP cells were dissected from patient disc specimens and then cultured in vitro. The levels of leptin were measured by real-time PCR and immunofluorescence. The proliferation of NP cells was detected by CCK8method after being plated and treated in different concentrations of leptin. We also observed the proliferation ability of NP cells with changes in time when treated with10ng/ml of Leptin. The expression of p-AKT, p-ERK1/2, p-STAT3in NP cells were detected by Western blot. The expression of cyclin D1, PCNA and Ki-67were detected by real-time RT-PCR, Western blot and immunofluorescence staining. We also observed whether the proliferation ability of NP cells, as well as the expression of cyclin D1, PCNA and Ki-67was inhibited after being traeted by AG490, Wortmannin and U0126, which were the signal pathway inhibitors of JAK, PI3K and MEK pathway. RNA and protein were extracted and separated from NP cells, in which the expression level of OBRa and OBRb were detected by Real-time RT-PCR and Western blot. We also conduct the correlation analysis with clinical data. Immunofluorescence and real-time PCR and Western blot were performed to investigate the effect of leptin on cytoskeleton protein, such as F-actin, β-actin, β-tubulin and vimentin. We transfected pRaichu-1237x plasmid, which encodes YFP-RhoA-PKN-CFP fusion protein into human nucleus pulposus cells and observe the changes of FRET signal of RhoA protein in10ng/ml Leptin. Expression of p-LIMK1and p-Cofilin was detected by Western blot method, and the change of F-actin by leptin stimulation in NP cells was detected immunofluorescence staining. Using RhoA einhibitor C3exoenzyme or ROCK inhibitor Y-27632, we observed the changes in p-LIMK1、p-Cofilin�'�F-actin. After stimulated by leptin, the expression of extracellular matrix of Collagen I, Collagen II, MMP-2and MMP-14were detected by real-time RT-PCR and Western blot method and the expression of p-JAK, p-p38, p-MEK was detected by Western blot method.ResultsWe successfully isolated and cultured human NP cells in vitro. The expression of CA21, cytokeratin19was significantly higher and the expression of IBSP, FBN1was significantly lower in NP cells. Leptin can promote the proliferation of NP cells and induce the expression of cyclin and D1, PCNA and Ki-67through the activation of p-AKT, p-ERK1/2, p-STAT3signaling pathway. These effects can be inhibited by AG490Wortmannin and U0126JAK, which were the JAK, PI3K and MEK signal pathway inhibitor respectively. Both the mRNA and protein of OBRa and OBRb were expressed in NP tissues, and the expression was individually different. OBRb expression was correlated with patients’body weight, but no correlation was found between the expression of OBRb and OBRa with age, disease types, duaration of low back pain and sex. After leptin stimulation, the cytosolic F-actin microfilaments were thicker and longer and the expression of F-actin and fluorescence intensity increased significantly. Moreover, the expression of β-actin also increased. The expression of Vimentin increased significantly without obvious change in the structure. The expression of β-tubulin had no obvious change. In10ng/ml of leptin, NP cells gradually retracted their pseudopodia and the FRET signal also increased gradually. The peak was reached at5min after the stimulation. Leptin can stimulate the phosphorylation of p-LIMkl and p-confilin-2and remodeling of the skeleton protein F-actin in NP cells. These effects can be inhibited by C3exoenzyme and Y-27632, which were the RhoA and ROCK signal pathway inhibitor. Leptin can induce the expression of p-JAK, p-p38, p-MEK and up-regulate the expression of Collagenl, MMP-2and MMP-14, while inhibiting the expression of Collagen2.ConclusionsThis study investigated the effects of leptin on human ICD and its possible molecular mechanism. Our study shows that both OBRa and OBRb were expressed in NP tissues, and the expression was individually different. OBRb expression was correlated with BMI. Leptin can promote the proliferation of NP cells through activation of JAK, PI3K and MEK signal pathway. Leptin can change the expression of skeleton protein F-actin, β-actin,β-tubulin and vimentin. Leptin remodel the skeleton protein F-actin through the activation of RhoA/ROCK and LIMK1/Cofilin signal pathway. Leptin can induce the expression of Collagenl, MMP-2MMP-14, while inhibiting the expression of Collagen2.