Experimental Study Of Niacinamide Treatment For Rrabbit Intervertebral Disc Degeneration

1. Niacinamide protects rabbit intervertebral matrix in vitro.Objective To investigate the protective effects of Niacinamide on aggrecan and typeⅡcollagen of rabbit interverbral disc invitro. Methods Chiba's 10ng/ml IL-1βinduced intervertebral disc (IVD) degeneration model was adopted in this study, and various concentration of Niacinamide was added to the medium for intervention: no Niacinamide as group 1, 0.5mg/ml Niacinamide without IL-1βas group 2, IL-1βwithout Niacinamide as group 3, IL-1βand 0.5mg/ml Niacinamide as group 4, IL-1βwith 0.25mg Niacinamide as group 5 and IL-1βwith 0.05mg/ml Niacinamide as group 6. At the 1st and 2nd week of culture, Safranin O- Fast Green Staining, glycosaminoglycan (GS) content measuring and aggrecan core protein RT-PCR was carried out to detect how aggrecan was regulated by niacinamide. At 2nd week of culture, RT-PCR and immunohistochemical staining was carried out for typeⅡcollagen. And after 1 week of culture, immunohistochemical staining for inducible nitric oxide synthase (iNOS) and transforming growth factor-β1 was carried out. Results①After 1 week's culture, GS content of nucleus pulposus (NP) in group 2 and group 4 was raised 44.8% (P<0.01) and 68.3% (P<0.01) by 0.5mg/ml Nia as compared with group 1 and group 3 respectively. GS content of group 4, with 2 weeks of 0.5mg/ml Nia treatment, is higher than either group 3 (P<0.01) or group 1(P<0.01).②Safranin O- Fast Green Staining demonstrated that, with increasing of Nia concentration, staining density and histological structure of IVDs was better reversed.③RT-PCR showed that Nia increased core protein gene expression in normal IVDs, and up-regulated the expression in degenerated IVDs by a dose related mode.④Type Ⅱcollagen staining demonstrated that lamellar structure and continuity of collagen of treated groups was better reversed than the group3.⑤RT-PCR showed that expression of typeⅡcollagen of group 4 was significantly stronger than that of group 3 (P<0.01).⑥The rate of iNOS positive-staining cells of each group was 17.6%,10.9%,73.9%,19.3%,43.6% and 64.7% respectively. The positive rate of group 3 is siginificantly higher than group 3 (χ2=148.374,P<0.001) and the rate of group 4 is lower than group 3, 4 and 6 (χ2=86.752, P<0.001;χ2=16.935,P<0.001;χ2=57.225,P<0.001).⑦The rate of TGF-β1 positive-staining cells of group 3 is reduced as compared with group 1 (χ2=148.345,P<0.001). And the rate of group 4 is higher than either of group 3, 5 and 6 (χ2=56.686, P<0.001;χ2=7.685,P=0.008;χ2=39.143,P<0.001). Conclusion Niacinamide can raise content and expression of both aggrecan and typeⅡcollagen. And it is capable of inhibiting IL-1βinduced down-regulationg of aggrecan and typeⅡcollagen. The inhibition is related with its inhibition on iNOS expression and prection on TGF-β1 expression. Thus, Niacinamide offers a potential choice for IVD degeneration therapy.2. Niacinamide regulates cell apoptosis and energy metabolism related genes in cultured rabbit intervertebral discsObjective To investigate the regulatory effect of Niacinamide on IL-1βinduced cell apoptosis and energy metabolism related gene expression in intervertebral disc in vitro. Methods Chiba's 10ng/ml IL-1βinduced intervertebral disc (IVD) degeneration model was adopted in this study, and various concentration of Niacinamide was added to the medium for intervention: no Niacinamide as group 1, 0.5mg/ml Niacinamide without IL-1βas group 2, IL-1βwithout Niacinamide as group 3, IL-1βand 0.5mg/ml Niacinamide as group 4. After 1 week of culture, TUNEL staining and immunohistochemical staining for FAS,Caspase-3,Bcl-2,HIF-1α,GLUT-1 and VEGF was used to detect alternated cell apoptosis and expression of energy metabolism related genes. Results①The rate of TUNEL positive-staining cells of each group was 15.7%,17.0%,39.5% and 31.3% respectively. The rate of group 4 is much lower than group 3 (χ2=1.555,P=0.212).②The rate of Fas positive-staining cells of each group was 9.3%,8.5%,24.3% and 22.8% respectively. The rate of group 4 is close to group 3 (χ2=0.102,P=0.749).③The rate of Bcl-2 positive-staining cells of each group was 64.1%,73.7.%,49.3% and 58.8% respectively. The rate of group 4 is higher than group 3, but not significantly (χ2=2.832,P=0.092).④The rate of Caspase-3 positive-staining cells of each group was 3.4%,4.2%,17.6% and 10.3% respectively. The rate of group 4 is significantly lower than group 3 (χ2=5.063,P=0.024).⑤The rate of HIF-1αpositive-staining cells of each group was 26.7%,4.2%,70.6% and 45.3% respectively. The rate of group 4 is significantly lower than group 3 (χ2=29.792,P<0.001).⑥The rate of GLUT-1 positive-staining cells of each group was 25.7%,16.3%,56.3% and 54.4% respectively. The rate of group 4 is significantly lower than group 3 (χ2=0.135 , P=0.713).⑦The rate of VEGF positive-staining cells of each group was 10.1%,3.6%,68.4% and 46.7% respectively. The rate of group 4 is significantly lower than group 3 (χ2=39.002,P<0.001). Conclusion Niacinamide can inhibite IL-1βinduced intervertebral disc cell apoptosis. Niacinamide improves energy metabolism of cultured IVD tissue and alleviates IL-1βinduced disturbance of energy metabolism.3. Niacinamide regulates rabbit nucleus pulposus cell apoptosis and proliferation in vitroObjective To investigate regulatory effects of Niacinamide on rabbit nucleus pulposus cell apoptosis and proliferation in vitro. Methods Cultured NP cells were divided 6 groups and various Niacinamide and IL-1βwas added to the medium: group 1 without any drugs as control, 0.5mg/ml Nia as group 2, 10ng/ml IL-1βand group 3, 10ng/ml IL-1βand non-specific Caspase inhibitor Z-VAD-FMK as group 4, 10ng/ml IL-1βand 0.05mg/ml Niacinamide as group 5, 10ng/ml IL-1βand 0.5mg/ml Niacinamide as group 6. After 3 days of co-culture with the drugs, the cells were examined with Annexin V-PI staining, Caspase-3,Caspase-8,Caspase-9 function staining and MTT assay. Results①The apoptotic rate (Mean±SEM) of the groups were 2.67±1.08%,2.71±0.53%,20.37±1.57%,11.34±0.67%,18.17±0.74% and 9.42±1.08%. The rate of group 2 did not change much than group 1 (P=0.950). The rate of group 4, 5 and 6 discended than group respectively (P=0.001,P=0.172,P=0.001).②The positive rate of Caspase-3 function staining of group 5 (12.35±0.64%) and 6 (12.35±0.64%) decreased as compared with group 3 (17.14±0.72%, P<0.001, P<0.001).③The positive rate of Caspase-9 function staining of group 5 (15.13±1.45%) and 6 (10.17±2.50%) decreased as compared with group 3 (19.4±0.98%, P=0.014, P=0.004).④The positive rate of Caspase-8 function staining did not change much with either Niacinamide or IL-1β.⑤The absorbance of the groups was 0.972±0.064,1.023±0.086,0.519±0.021,0.637±0.028,0.956±0.013 and 0.541±0.017 respectively. The A of group 6 is significantly higher than the A of group 4 (P<0.001), while the A of group is not significantly higher than group 3 (P=0.096). Conclusions Niacinamide is capable of impove cell proliferation and inhibite apoptosis of NP cells in vitro. The inhibition of apoptosis mainly acts via inhibition of mitochondrial path way.4. Estabilishment of Controlable Pressure Induced Rabbit Lumbar Intervertebral Disc Degeneration Model and Study of Alternated Expression of Energy Metabolism Genes of the ModelObjective To establish controlable pressure induced rabbit lumbar IVD degeneration model. And to investigate alternated expression of energy Metabolism genes of the model. Methods Twenty Japanese white rabbits were selected for animal model establishment. A"controllable pressure-induced rabbit IVD degeneration model"was adopted to impose various pressured on rabbit IVDs in vivo to obtain slightly degenerated rabbit IVD. Thompson's gross grading system was used to access the degeneration degree of the IVDs and HE staining was used to check the histological changes followed with pressure damage. The survived animals whose IVDs were compressed successfully were divided into 4 groups randomly. The IVDs were treated with no pressure as control (group 1), with 10 kg axial load for 24 hours (group 2), 72 hours (group 3), and 24 hours with a 48 hours free for self-reparation (group 4). RT-PCR was used to detect the expression of HIF-1αand GLUT-1. Western Blot and immunohistochemical test were carried out for the content and distribution of VEGF. Results①After 72h's over load, the IVDs of group 2 reached gradeⅠas assess ed by Thompson grading system. HE staining revealed that, the lamellar structure of AFs in group 2 was not as clear of that of normal control, and abnormal karyotype appeared more frequently.②HIF-1α: A very low expression was detected in normal AF, while the expression in group 2 was raised over 20 times as compared with normal (t=25.022, P<0.001). The expression in group 3 and group 4 decreased as compared with group 2.③GLUT-1 expressed weakly in normal AF. The expression in group 2 raised a lot as compared with control (t=18.314, P<0.001) and the expression in group 3 rose slightly than group 2 (t=2.819, P=0.023). The expression in group 4 is close to that in group 2.④Little VEGF content was detected in normal AF, while the content rose significantly in the other 3 groups. Immunohistochemical staining showed more VEGF positive stained cells in outer AF than in inner AF. Conclusion The"controlable pressure induced rabbit lumbar IVD degeneration model"is able to cause rabbit IVD degenerate effectively. Continuous pressure can strongly up-regulate expression of energy metabolism gene: HIF-1α, GLUT-1 and VEGF in vivo. These genes play important roles in AF adaptation and reparation in over load-caused damage. Thus, Niacinamide may be effective for IVD degeneration therapy.5. Niacinamide Treatment for Overload caused Rabbit Intervertebral Disc in vivoObjective To investigate therapeutic effects of Niacinamide for pressure caused rabbit intervertebral disc degeneration. Methods The"controllable pressure-induced rabbit IVD degeneration model"was adopted for estabilishing overload caused rabbit IVD degeneration model. Twenty four Japanese white rabbits were randomly divided into 6 groups with various pressure added to their lumbar IVDs, and with or without about 50mg/kg Niacinamide for oral administration per day: group 1, 2 rabbits, no pressure as fake operation control group; group 2, 2 rabbits, Niacinamide for 1 week without pressure; group 3, 5 rabbits, 10kg for 1 week without Niacinamide; group 4, 5 rabbits, 10kg for 1 week followed by 1 week's free recovery; group 5, 5 rabbits, 10kg for 1 week followed by 1 week's recovery with Niacinamide treatment and group 6, 5 rabbits, 10kg for 1 week with 1 week's recovery, during which Niacinamide was given continuously. Thompson grading system was used to assess IVD degeneration degree, and HE staining was used to check the hitlogical changes of the samples. Safranin O- Fast Green staining and immunohistochemical staining for typeⅡcollagen was used to detect the alternated content and distribution of aggrecan and typeⅡcollagen. Results①There was no abnormal phenomenon in IVD samples in group 2 as compared with group 1. The 5 rabbits all grededⅡin group 3. There 4 rabbits gradedⅡand 1 rabbit gradedⅢin group 4. There 2 rabbits gradedⅠand 3 rabbits gradedⅡin group 5. And There 3 rabbits gradedⅠand 2 rabbits gradedⅡin group 6.②HE staining revealed that IVD of group 4 suffered the most severe degeneration. However, the continuity and integrality of lamellar structure of AF, the shape of lacune structure of NP and the appearance of cells in group 4 recovered obviously as compared with group 4.③Safranin O- Fast Green staining tensity of group 2 raised as compared with group 1. The staining tensity of group 5 and 6 rose as compared with group 4, among which the increase of NP was the most obvious (29.9% and 32.1%;P<0.01,P<0.01), and there was a slightly increase when comparing staining intensity of group 6 with group 5.④TypeⅡcollagen staining revealed that continuity of collagen lines of group 6 was better reversed as compared with group 4. The staining intensity of AF of group 6 increased 53.2% as compared with group 4 (P<0.01). Ane there was a slightly increase when comparing staining intensity of group 6 with group 5. Conclusion Niacinamide can help to allevate overload caused damage to IVD. And Niacinamide can benefit the recovery of damaged IVDs.