Study On The Therapeutic Effect And Pharmacological Mechanism Of Osteoarthritis Based On Mesoporous Silica

Osteoarthritis is a chronic bone and joint disease caused by degeneration of joint cartilage,which can lead to swelling,pain,stiffness,limitation of movement and limb incapacity of the affected joints.Now that China has gradually entered an aging society,osteoarthritis will become an increasingly serious problem,placing a huge burden on individual families and the social health care system.Finding a means to effectively treat osteoarthritis is one of the major problems that researchers need to solve.Currently,the three main treatments for osteoarthritis are non-pharmacological,pharmacological and surgical.For the initial stage of osteoarthritis,non-pharmacological treatment is generally used,such as symptomatic treatment with heat and physical therapy to increase blood circulation,but cartilage lesions still exist,so they cannot be cured.According to the 2010 edition of the guidelines for the diagnosis and treatment of osteoarthritis,topical non-steroidal anti-inflammatory drugs are preferred for drug treatment,or NSAIDs are given appropriately to relieve pain and delay disease progression.For patients whose conservative treatment is ineffective and whose quality of life is significantly reduced,surgical treatment can be chosen,such as arthroscopic examination and treatment to clear the synovial membrane and artificial joint replacement to completely remove the damaged cartilage.Currently,the most common treatment for osteoarthritis is drug therapy,such as diacerein and hyaluronic acid,but diacerein has adverse effects such as diarrhea and abdominal pain,and hyaluronic acid is only suitable for patients with early to moderate osteoarthritis,and is not effective for patients with severe joint stenosis and joint destruction.With the rapid development of material science in recent years,more and more materials have been applied in the treatment of various diseases,showing good therapeutic effects.Therefore,finding a suitable material for the treatment of osteoarthritis is expected to be the key to improve the effectiveness of the treatment of osteoarthritis.Studies have shown that osteoarthritis is caused by the mechanical injury resulting in the death of chondrocytes and the release of large amounts of free nucleic acids,which cause abnormal recognition of Toll-like receptors（TLRs）,resulting in an inflammatory response.It has been demonstrated that increased levels of free DNA in patients with sepsis and rheumatoid arthritis cause TLRs to activate the inflammatory response,and therefore free DNA is used as a biomarker for clearance through the binding of nucleic acid polymers to free DNA,thereby reducing the inflammatory response in vivo.The ability of positive polymers such as polyethyleneimine（PEI）to charge-bind to nucleic acids has been demonstrated,and such materials are expected to be effective therapeutic modalities for the removal of free DNA to suppress the inflammatory response.Nucleic acid-binding nanomaterials（NABNs）not only have the ability to scavenge nucleic acids,but also have lower toxic effects,making them safer and more efficient nucleic acid scavengers.Although NABNs have been studied in diseases such as sepsis,their therapeutic effects and molecular mechanisms in bone joints are not yet clear.Therefore,in this thesis,nucleic acid-binding nanomaterials will be constructed and validated in an osteoarthritis model,and their targeting,efficacy,and safety will be investigated as well as their potential related mechanisms.Objectives:To synthesize nucleic acid-binding nanomaterials with different charge densities and conduct in vivo experiments in animal models of osteoarthritis to elucidate their therapeutic effects on osteoarthritis and related mechanisms,and to reveal the targeting,safety,and efficacy of nucleic acid-binding nanomaterials in osteoarthritis,aiming to provide new therapeutic tools and theoretical basis for the treatment of osteoarthritis patients.Methods and results:1.To clarify the scavenging effect of polyethyleneimine-modified silica as nucleic acid-binding nanomaterials on free nucleic acids.In this thesis,polyethyleneimine-modified silica（MSN-PEI）was synthesized and characterized morphologically,and the degradation of MSN was detected by TEM.The binding rate of MSN-PEI to DNA was measured by Quant-i TTM Pico Green TM ds DNA Assay Kit.HUVEC cells were selected and the cytotoxicity of MSN-PEI was investigated by the MTT method.HEK-Blue TM TLR 9 reporter cells were used to observe the effect of MSN-PEI in suppressing the inflammatory response.The antiinflammatory effect of MSN-PEI in vitro was investigated using RAW264.7macrophages secreting inflammatory factors.The results showed that MSN-PEI has a good nucleic acid binding ability and can effectively inhibit the inflammatory response.2.MSN-PEI maintains the phenotypic stability of chondrocytes in the DMM mouse model.DMM surgery was performed on mice to establish an osteoarthritis model.The therapeutic effect of MSN-PEI was examined by measuring the body weight and joint swelling of mice.The results showed that the degree of joint swelling in mice was significantly reduced after treatment with MSN-PEI,and there was no significant change in body weight.The above results showed that MSN-PEI has good safety and therapeutic effects.The cartilage content in the knee joints of each experimental group was observed by using the staining technique of saffron O/fast green,and the m RNA expression levels of ADAMTS 5,Col2a1,MMP-13,TLR 9 and Col-X in the cartilage tissues of the knee joints of each experimental group were evaluated by the immunohistochemical staining technique,and the lesions of the cartilage tissues of the knee joints of each experimental group were stained by HE staining technique,and the Elisa kit was applied to The levels of cf DNA,IL-6,IL-1β,TNF-α in the serum and joint fluid of each experimental group were evaluated.The results showed that MSN-PEI significantly down-regulated the m RNA level of ADAMTS 5 and significantly increased the cartilage content compared with the model group;the levels of MMP-13 and Col-X were significantly down-regulated.The results of knee joints of DMM mice examined using micro CT showed that the formation of bone redundancy was reduced in DMM mice after MSN-PEI treatment.The above results showed that MSN-PEI has good bone as well as cartilage protective effects.3.Study of the anti-inflammatory effect of MSN-PEI in vivoUsing the DMM mouse model,the mouse knee joint was stained by immunohistochemical technique to detect the TLR 9 content.The macrophage markers F4/80 and i NOS were detected by immunofluorescence technique.The results showed that the contents of TLR 9,F4/80,and i NOS were significantly decreased after MSNPEI treatment.The above results showed that MSN-PEI inhibited the activation of macrophages by suppressing the activation of TLR 9.A CIOA rat model was established using collagenase injection,and the contents of cf DNA,IL-6,IL-1β and TNF-α in serum and joint fluid were detected by enzyme-linked immunoassay.The results showed that the contents of cf DNA,IL-6,IL-1β and TNF-α in serum and joint fluid were significantly reduced after MSN-PEI treatment.The above results showed that MSN-PEI has a good anti-inflammatory effect in animals in vivo.4.Safety evaluation of MSN-PEI in vitro and in vivoThe experiments showed that the semi-inhibitory concentration（IC50）of MSNPEI in HUVEC cells was better than in other groups.By HE staining different materials on the heart,liver,spleen,lung and kidney of mice,it was found that the morphological changes of MSN-PEI group were the smallest,and by weighing the heart,liver,spleen,lung,and kidney of rats and mice,it was found that the weight of each group did not change significantly.The above results showed that MSN-PEI has good biosafety in vivo.Conclusion:In this study,MSN-PEI nucleic acid-binding nanomaterials were synthesized and revealed through cellular level and animal level aspects that the materials have therapeutic implications in osteoarthritis by binding free DNA and inhibiting TLR 9activation,which in turn inhibits the activation of macrophages and attenuates the release of inflammatory factors.Compared with the polymer alone,MSN-PEI has higher targeting and safety,and can play a better therapeutic role,providing new targets and new strategies for the treatment of osteoarthritis.