Urolithin A Alleviate Intervertebral Disc Degeneration In Vitro And In Vivo

Objective 1.To investigate the effect of Urolithin A on TNF-? induced extracellular matrix synthesis and catabolism,and its molecular mechanism.2.To establish the puncture-induced intervertebral disc degeneration model and study the effect of Urolithin A on intervertebral disc degeneration in vivo.Methods 1.In vitro,NP cells were isolated and cultured from 12 weeks male Sprague Dawley rats.Immunofluorescence staining of type II collagen,Alcian blue staining and Toluidine blue staining were used to identify the NP cells.We detected the effect of urolithin A with different concentrations on the proliferation of NP cells by using CCK-8 method.We tested whether urolithin A could protect NP cells from hydrogen peroxide(H2O2)-induced senescence using SA-?-galactosidase staining.The percentage of SA-?-gal positive cells were calculated.The expression levels of type II collagen,Aggrecan,MMP3,MMP13 were detected by qPCR and western blot.The effect of urolithin A on NF-?B,MAPKs and PI3K/Akt signaling pathway was detected by western blot.2.In vivo,we established puncture-induced intervertebral disc degeneration model by using 12 weeks male SD rats.Co7-8 and Co8-9 discs were punctured by 21 G needle.The depth was 5mm from skin,followed by rotation at 360° and holding for 30 s.Thirty rats were divided randomly into three equal groups(n=10 per group): sham-operated mice(control group),punctured and DMSO-treated mice(IDD group),and punctured and urolithin A-treated mice(UA group).The second day the rats were given food containing UA or DMSO for 4 weeks.UA was mixed with the rodent diet at a dose of 25mg/kg/day.X-rays and MRI were acquired before puncture and 4 weeks after puncture.We also used histological analysis of the HE staining,Alcian blue staining,safranin-O-Fast Green stain staining to evaluate the effect of UA on IDD.Results 1.UA had no significantly effect on the proliferation of NP cells and had no cytotoxic at the concentrations below 40?M.The proportion of SA-?-gal positive cells increased significantly after adding 50?M H2O2,whereas treated with 5?M UA and 10?M UA could reverse this change.UA could protect NP cells against H2O2-induced cellular senescence.UA treatment increased the mRNA and protein expression levels of TNF-?-induced inhibition of Collagen II and decreased the expression of MMP3 and MMP13,but the mRNA expression of aggrecan was not changed.In terms of molecular mechanism,UA inhibited TNF-?-mediated phosphorylation of ERK,JNK and Akt,while the effect on phosphorylation of p65 and p38 was not obvious.2.4 weeks after surgery,the X-rays showed a decrease in intervertebral disc height,the DHI decreased.MRI T2 image showed loss of water in the NP tissues and the Pfirrmann score was increased.The histological image of HE staining,Alcian blue staining,Safranin O-fast green staining showed that the content of NP tissues decreased,the fiber proliferated,the structure of AF was disordered and the extracellular matrix was significantly reduced.However,oral administration of UA could significantly ameliorated the above changes.ConclusionUA could effectively prevent IDD.In vitro UA could protect NP cells from senescence,inhibit the synthesis of ECM-degrading enzymes and promote the synthesis of collagen II by affecting MAPK and PI3K/Akt signaling pathway.In vivo UA could improve the DHI and decrease the Pfirrmann grade and histologic score.