Integrin α6 Upregulation In Nucleus Pulposus Cell Under High Oxygen Tension Attenuates Intervertebral Disc Degeneration

BackgroundLow back pain（LBP）is a kind of lumbosacral discomfort with or without radiating pain in the lower limbs.In China,it is the most common disease seen in the spine clinics,and it is also a musculoskeletal disease with the highest incidence in the world.More than70%of people around the world have experienced low back pain one or more time in their whole lives.This disease is more common in the elderly population,it does not only affect the patients’life quality,but also imposes a heavy burden on families and society.It is reported that the medical cost of LBP in the US needs more than 50 billion dollars per year.Intervertebral disc degeneration（IVDD）is a major cause of LBP.The importance of IVDD in LBP has been confirmed,but the mechanism of IVDD has not been fully elucidated.Therapeutic options are mainly symptomatic treatment,which can not reverse the development trend of IVDD.Currently,there is a lack of effective treatment methods.Therefore,it is of great importance to further study the mechanism of IVDD to obtain new intervention targets and therapeutic drugs.Intervertebral disc（IVD）consists of three main parts:cartilage endplate（CE）,annulus fibrosus（AF）and nucleus pulposus（NP）.IVD plays an important role in distributing the load,sharing the pressure,and providing mobility and stability for the spine.The microenvironment of IVD tissue is significantly different from that of other tissues,which is characterized by low nutrient supply,low pH and low oxygen.The distance between NP tissue and its nearest blood vessels can be up to 8mm,leading to hypoxia environment.NP cells are more adapted to the environment without blood vessels.Hypoxia is the most important physical and chemical environment for healthy IVD tissue.With IVD aging and degenerating,changes of oxygen concentration would activate or inhibit multiple homeostasis genes to support its survival and function.Loss of hypoxia microenvironment plays an important role in the pathogenesis of IVDD.A recent study shows that the expression of anabolism-related genes decreas at 5%and 21%oxygen concentration,while increase at 1%oxygen concentration,suggesting that hypoxia is the most suitable microenvironment for NP cell to maintain its phenotype.In other words,conventional normoxia（21%O₂）is the pathological environment with high oxygen tension（HOT）for NP cells.Other studies have shown that increasing oxygen tension caused by abnormal neovascularization into IVDs is closely related to IVDD.Current studies mainly focuse on the protective role of hypoxia on NP cells.However,few studies try to investigate how NP cell adapts itself to HOT.Integrin（ITG）is a kind of heterodimer transmembrane glycoprotein,which consists of bothα（120-185k D）andβ（90-110k D）subunits.18αand 8βsubunits sonsistitute 24different heterodimers in total.At present,a large number of studies have confirmed that integrins play an important role in the pathogenesis of IVDD.Integrinα5β1 is a key receptor in NP cells.Upregulated integrinβ1 can inhibit apoptosis.Fibronectin in the extracellular matrix（ECM）help maintain NP cell’s function by the integrinβ1/FAK pathway.CCN2 has also been shown to inhibit interleukin-1β-induced catabolism in NP cells by integrinα5β1 andαVβ3.All in all,integrins play an important role IVDD.In our preliminary experiment,integrinα6（ITGα6）which is considerablely affected by HOT was selected.In this study,we intend to explore the mechanism of ITGα6expression upregulated by HOT,the ITGα6 expression of IVDD tissue in humans and rat models,the possible mechanism of ITGα6 in regulating metabolism and its role in vivo study.Objectives1.To demonstrate that HOT upregulates ITGα6 expression by activating PI3K/AKT signaling pathway;2.To investigate the expression of ITGα6 in IVDD tissue both in humans and rat IVDD models;3.To investigate the possible mechanism of ITGα6 in regulating the metabolism and its role in vivo study.Experimental proceduresPart 11.To obtain NP cells.NP cells from 3-month-old male Sprague-Dawley（SD）rats were isolated,extracted,cultured,the second or third generation were used for the following experiments.2.To identify the integrin subunit gene most significantly affected by HOT.Real time reverse transcription-polymerase chain reaction（RT-PCR）was used to detect 12 known integrin subunits after HOT exposure for 24h.The ITGα6 expression increased dramatically.3.To confirm that HOT upregulated the expression of ITGα6 at protein level.The ITGα6 expression was further detected by Western blot and immunofluorescence after the NP cells were exposed to HOT for different periods.4.To investigate the effect of ITGα6 on degeneration of NP cells.ITGα6 was knocked down by small interfering RNA（siRNA）,and the degeneration of NP cells was detected by Western blot.5.To investigate HOT upregulated ITGα6 expression through PI3K/AKT pathway.Western blot was used to detect the ITGα6 expression to determine whether HOT upregulated its expression through PI3K/AKT pathway by activating or inhibiting this pathway.Part 21.To determine ITGα6 expression levels in human NP tissues with different grades of degeneration.Clinical NP tissue samples with different grades of degeneration were collected according to Pfirrmann grading system,and degeneration degree was confirmed by histological examination.Then,Western blot and immunofluorescence were used to detect the integrinα6 expression in different degeneration degree.Grades.2.To determine ITGα6 and HIF-1αexpression levels and apotosis in animal NP tissues with different grades of degeneration.The annulus needle puncture model of IVDD was established in rats,histological examination was used to confirm the success of modeling,Western blot and immunofluorescence were used to detect the ITGα6 and hypoxia inducible factor 1α（HIF-1α）expression in different degeneration stages,and TUNEL staining was used to detect apoptosis.Part 31.To investigate the effect of HOT and ITGα6 on apoptosis of NP cells.NP cells were pretreated with siRNA to knockdown integrinα6,and then cultured in serum-free condition under hypoxia or HOT according to experiment groups.Apoptosis of NP cells was detected by flow cytometry with Annexin V-FITC/PI saining and Western blot.Then the changes of mitochondrial membrane potential and apoptosis inducing factor（AIF）were detected.2.To inestigate the effect of HOT and integrinα6 on ROS products.The NP cells were pretreated with siRNA to knockdown integrinα6,and then stimulated by HOT.The Reactive oxygen species（ROS）were detected by flow cytometry and fluorescence microscope.3.To investigate the relationship between HIF-1αand ITGα6.The ITGα6 expression was detected by knocking down or overexpressing HIF-1α,and the expression of HIF-1αafter silencing integrinα6 was detected to clarify their relationship.4.To investigate the effect of locally silencing ITGα6 in IVDD.Chitosan was used to wrap siRNA（ITGα6 or NC）to form nanoparticles solution（siRNA-ITGα6/NC-CH）to silence ITGα6.Then siRNA-ITGα6-CH solution was injected into discs in IVDD rat model.Western blot was used to detect the knockdown effect,and Magnetic Resonance Imaging（MRI）,histological examination and immunofluorescence were used to detect the degeneration degree.Results1.HOT upregulates the ITGα6 expression in NP cells by activating PI3K/AKT signaling pathway.2.ITGα6 expression was increased in mild IVDD both in human and rat NP tissues.3.Activating of PI3K/AKT/ITGα6 pathway protects NP cells to reduce extracellular matrix protein synthesis caused by HOT.4.ITGα6 protects NP cells from HOT-induced apoptosis.5.ITGα6 prevents ROS products induced by HOT in NP cells.6.There is interaction and feedback between HIF-1αand ITGα6 in NP cells.7.Local knockdown of ITGα6 in vivo can accelerate IVDD in rat model.Conclusion1.The expression of ITGα6 was increased in the early stage of disc degeneration;2.The loss of hypoxia environment in NP tissue is a major cause of increasing ITGα6expression;3.ITGα6 could protect NP cells against HOT-induced apoptosis and oxidative stress,and protect NP cells from HOT-inhibited ECM proteins synthesis;4.Silencing of ITGα6 in vivo obviously accelerated needle puncture-induced IVDD.