Study On The Mechanism Of Pterostilbene In Suppressing Chondrocyte Senescence And OA By Inhibiting PI3K/AKT/NF-κB Signaling Pathway

Objective: Osteoarthritis(OA)is the most widespread bone and joint disease in the world,which not only has a great impact on the affected individuals and families,but also carries a heavy burden to the health care system and social economy.OA is becoming more prevalent as the global population ages,the number of obese people rises and the number of patients with joint injuries increases.It is estimated that about 250 million people worldwide are currently affected by OA.But with such a heavy burden,most osteoarthritis patients do not obtain proper treatment.Osteoarthritis is a complex chronic disease that involves a variety of tissues in the joints.At present,although there are many therapy methods for OA,most of them are palliative and symptomatic treatment.Among the many risk factors of OA,aging is one of the most critical factors.Targeting aging changes in intraocular tissue may be a promising treatment for OA.As human life expectancy rises,health services are faced with an increasing number of patients with age-related diseases.Such as neurodegenerative diseases such as Alzheimer’s disease,cardiac hypertrophy,osteoporosis and so on.Numerous studies have demonstrated that these diseases are closely associated with the accumulation of senescent cells over time in affected tissues.OA is also one of the main diseases related to aging,and is a complex joint disease accompanied by wear,inflammation and aging and other pathological processes.The accumulation of senescent cells in OA articular cartilage was first proposed by Price equals in 2002,and is currently a hot topic in OA research.It is largely accepted that cellular senescence is a major driver of senescence and aging-related diseases.The aging process of chondrocytes is similar to the pathological process of most cells aging.Cellular senescence can be provoked by stress factors,including cytotoxic drugs,nutrient deprivation,mitochondrial dysfunction,and oncogene activation.Senescent cells will undergo irreversible growth arrest,but they do not lose metabolic activity and can secrete a variety of bioactive molecules,which are called "senop-associated secretory phenotype(SASP)".SASP plays a beneficial role in wound healing and stimulating the growth of adjacent cells.Nevertheless,as the aging degree rises,accumulated senescent cells will continue to secrete SASP,leading to chronic inflammatory states(" inflammaging "),which promote many of the pathological changes that are the hallmark of aging.Inhibition of SASP is one of the important anti-aging treatment strategies,and NF-κB signaling pathway plays the most critical role in regulating SASP secretion.Therefore,regulating NF-κB signaling pathway to inhibit the aging promoting effect of SASP may be a reasonable treatment method.At present,there are many kinds of natural extracts administered in the field of OA therapy.Because natural extracts themselves have many advantages such as low drug toxicity and good tolerance,these narcotics display satisfactory therapeutic effects.Pterostilbene(PTE)is a trans-stilbene-like compound,named Pterostilbene because it was initially discovered in Pterostilbene.It was subsequently discovered in blueberries,dragon’s blood,alfalfa and grapes.Modern pharmacological studies have demonstrated that Astragalus membranaceus has good bioavailability and has a variety of pharmacological activities,such as anti-tumor,anti-inflammatory,antioxidant,anti-aging and cardiovascular protection,etc.It is one of the heated compounds in current research.In recent years,there have been few relevant studies in the field of OA treatment.The hypotheses of this research are as follows: 1.Will the OA model provoked by joint instability enhance the aging degree of the knee joint in rats? And further extend the progression of OA? 2.Since the NF-κB signaling pathway plays an important role in the pathological regulation of aging,can Pterocarpus members inhibit chondrocyte aging by regulating THE NF-κB signaling pathway and then regulating SASP,thus playing a therapeutic role in OA?The objectives of this research are as follows: 1.To analyze the therapeutic effects of PTE on chondrocyte senescence and OA through animal experiments and cell experiments.2.To explore the role of NF-κB signaling pathway and its upstream and downstream pathways in the above therapeutic effects of PTE.Material and methods: 1.At present,chondrocyte senescence is a research hotspot in OA,and inhibition of chondrocyte senescence is considered as a promising therapy method for OA,among which drug reduction of senescence related phenotype(SASP)expression is one of the two main treatment methods to inhibit aging.In this study,the most important SASP(IL-6 and MMP-13)in OA were selected as research indexes.According to literature review,NF-κB signaling pathway is the main regulatory pathway of cell senescence related phenotype(SASP).In this study,PTE was selected as the acting drug to investigate whether it could inhibit chondrocyte senescence and thus treat OA by inhibiting PI3K/AKT/NF-κB/SASP signaling pathway.2.To test the effect of PTE on OA model of rats with osteoarthritis.The knee OA model of rats was made by cutting anterior cruciate ligament and medial meniscus tibial ligament(ACLT+DMM),which were divided into CG(SHAM)group,ACL+ DMM OA group and OA+PTE group.One week after the model was built,pterostilbene(20mg/kg)was intraperitoneally injected into OA+PTE every 2 days.The rats were sacrificed 6 weeks later,and the general appearance of the knee joint was observed.Knee lavage fluid was collected to determine IL-6 level.Subsequently,the knee joint was fixed in 4%paraformaldehyde for 48 h,and EDTA was decalcified.After the decalcification,the knee joint was dehydrated,transparent,immersed in paraffin,embedded and made into 5μm paraffin section.Appropriate sections were selected for HE,toluidine blue and Muscovy red solid green staining.Mankin and OARSI scoring criteria were used to score the knee joint of rats.Meanwhile,the positive expression of type II collagen,MMP-13,IL-6,p16 and p21 were detected by immunohistochemistry.3.To investigate the effect of PTE on IL-1 β-induced chondrocyte senescence in rats.In cell experiments,primary chondrocytes from rats were firstly extracted and identified by immunofluorescence.Different concentrations of PTE were added and MTS method was used to detect the primary cytotoxicity and cell viability of rats,and the working concentration of Pterostilbene was determined.10ng/ m L IL-1β was added to simulate OA inflammation to build cell aging model.Chondrocytes were treated with different concentrations of PTE,and the chondrocyte cycle in each group was detected by flow cytometry.The proportion of senescent cells(the proportion of SA-β-Gal staining positive cells),cell cycle and ROS levels in each group were compared.The m RNA expressions of type II collagen,MMP-13,IL-6,p16 and p21 were detected by PCR.Western blot was used to detect the protein expression of type II collagen,ADAMTS-5,MMP-13,IL-6,p16 and p21.4.Bioinformatic analysis proved that the NF-κB signaling pathway plays an important role in the pathological process of OA and aging.At the same time,PI3K/AKT signaling pathway,an upstream pathway of NF-κB closely related to OA,was screened out.Verify whether PTE can play a role in inhibiting SASP secretion through PI3K/AKT/NF-κB signaling pathway,thus playing an anti-inflammatory and anti-chondrocyte senescence role.They were divided into blank control group,IL-1β group,IL-1β+PTE(10μmol/L)and IL-1β+PTE(20μmol/L)groups.Western blot analysis of PI3 K,p-PI3 K,AKT,p-Akt,p65,p-p65.The nuclear translocation of NF-κB p65 in chondrocytes was detected by immunofluorescence.Molecular docking was used to verify the interaction between PTE and PI3 K protein.Results: 1.The knee joints of rats in each group were examined by X-ray,and pathological changes of cartilage were observed by paraffin section HE staining,toluidine blue staining and Safranine red-fast green staining.OA pathological scores of articular cartilage was performed by Mankin score and OARSI score.By comparison between the groups,it was found that there was no significant difference in radiographic score of the knee joints of rats in each group,and pathological study showed that the knee joints of rats in ACLT+DMM OA model showed mid-early OA performance 6 weeks later.Significant differences in Mankin score and OARSI score between the OA+PTE treatment group and the OA group indicated that PTE had a therapeutic effect on OA.2.The concentration of IL-6 in knee lavage fluid of OA rats was detected by ELISA,and it was found that the level of IL-6 in knee joint lavage fluid of OA rats modulated by ACLT+DMM surgery was significantly higher than that of control rats.The level of IL-6in knee lavage fluid of OA rats treated with PTE was significantly lower than that of untreated OA rats,the difference being statistically significant.3.Immunohistochemical staining showed that after PTE treatment,compared with OA group,the expression of type II collagen was increased,and the expressions of MMP-13 and IL-6 in chondrocytes in the treatment group were decreased,with significant statistical differences.Meanwhile,immunohistochemical staining results of p16 and p21 in knee cartilage of rats in each group showed that the expression of aging markers p16 and p21 in OA group was significantly raised compared with that in the sham operation group,and the difference was statistically significant,while the expression of p16 and p21 in chondrocytes of PTE treated rats was significantly reduced compared with that in OA group.And it has statistical significance.Correlation analysis showed that the positive cell rates of p16 and p21 were correlated with Mankin score and OARSI score.4.The extracted rat primary chondrocytes were identified by immunofluorescence staining of type II collagen,and the appropriate concentrations of PTE(10μmol/L and20μmol/L)were determined by MTS.After SA-β-Gal staining of senescent cells,IL-1βsignificantly increased the number of SA-β-Gal positive chondrocytes in rats,proving that IL-1β can increase the senescence degree of chondrocytes.After PTE treatment,the ratio of SA-β-Gal positive chondrocytes in the treatment group decreased,which was statistically significant.These results indicated that PTE could inhibit IL-1β-induced chondrocyte senescence.The proportion of G0-G1 phase chondrocytes was significantly increased and cell cycle arrest occurred under the induction of IL-1β,while G0-G1 phase chondrocytes were significantly decreased in the PTE treatment group.IL-1β significantly increased ROS levels in chondrocytes,while PTE significantly decreased ROS levels in inflammatory rat chondrocytes.PCR and Western blot were used to detect the expression of OA in IL-1β-treated chondrocytes.IL-1 β significantly increased the expression of ADAMTS-5 and MMP-13,and significantly reduced the expression of type II collagen.Meanwhile,IL-1β significantly increased the expression of IL-6,p16 and p21.The effect of IL-1β was inhibited by PTE.20μmol/L PTE significantly inhibited the expression of MMP-13,IL-6,ADAMTS-5,p16 and p21,and significantly raised the expression of type II collagen.5.Western blot analysis showed that IL-1β significantly activated PI3K/AKT and NF-κB signaling pathway and the ratios of P-PI3 K /PI3 K,P-AKT/AKT,p-p65 / p65 and p-IκB α were significantly raised compared with the control group.Meanwhile,PTE at10μmol/L and 20μmol/L could significantly inhibit the activation of PI3K/AKT and NF-κB signaling pathway.At the same time,immunofluorescence staining of p65 showed that nuclear inward migration of p65 occurred under the inflammatory effect of IL-1β,while p65 migrated outward to cytoplasm in the PTE group,suggesting that PTE played an antiinflammatory role.6.Molecular docking experiment results showed that PTE could interact with the three sites of PI3 K glu-628,ASP-584 and ARG-389.PTE could interact with the three residues of PI3 K protein(Glu-628,ASP-584 and ARG-389).And stably attached to the PI3 K inhibitory binding pocket with high affinity.In other words,PTE occupies the binding site,thus playing a role in inhibiting the phosphorylation of PI3 K.Conclusion: 1.In vivo experiments proved that aging markers were associated with the severity of OA,and Pterostilbene(PTE)could reduce the expression levels of OA inflammation and aging markers,and reduce the severity of OA in knee joints of rats.2.In vitro PTE can also reduce the senescence degree of chondrocytes in the IL-β-induced inflammatory OA model.3.PTE plays a role in the treatment of OA by inhibiting PI3K/AKT/NF-κB signaling pathway and reducing the expression of SASP.