Related Research Of Proximal Fibula Osteotomy For The Treatment Of Osteoarthritis

Knee osteoarthritis(KOA)is one of the important causes of disability and dyskinesia in the elderly.Its clinical manifestations are pain,effusion,stiffness and deformity.The pathogenesis of knee osteoarthritis is unclear,and it is urgent to find a treatment that can effectively reduce the load on the medial knee joint,reduce pain,and even reverse osteoarthritis.Our previous study showed that after proximal fibula osteotomy(PFO)treatment,the imaging performance of knee osteoarthritis patients has improved significantly,and knee function has also significantly recovered,confirming that PFO is a simple,cost-effective and effective surgical method for osteoarthritis.There are few related researches on the biological mechanism of PFO,and the research is mainly based on cadaver specimens and imaging data.So it is difficult to reveal the pathological mechanism and molecular biological mechanism behind it.Therefore,it is of great significance to establish a PFO mouse model to observe whether PFO can reduce osteoarthritis from the perspective of imaging and pathology.As we all know,the main cause of the occurrence and development of osteoarthritis is the progressive destruction of cartilage.Apoptotic necrosis of chondrocytes,acceleration of chondrocyte hypertrophy,and increased focal calcification of articular cartilage can ultimately lead to cartilage destruction.These biological processes are closely related to the pathogenesis of osteoarthritis.More and more studies have found that Lnc RNAs are differentially expressed in osteoarthritis tissues and play an important role in the occurrence and development of osteoarthritis.They can be used in epigenetic regulation,transcriptional regulation and post-transcriptional regulation.Lnc RNAs could regulate gene expression at multiple levels and participates in a variety of biological processes,including: cell proliferation,apoptosis,development,and immune response.It is of great significance to screen osteoarthritis-specific Lnc RNAs biomarkers and study the regulatory role of Lnc RNAs in chondrocyte inflammatory response.In this study,we established a proximal fibula osteotomy model in mice to observe the effect of proximal fibula osteotomy on delaying joint degeneration and inhibiting knee osteoarthritis.In order to further understand the molecular mechanism of osteoarthritis,we performed high-throughput sequencing on IL-1?-induced inflammatory chondrocytes,screened differentially expressed Lnc RNAs,and selected UHCI as the research object to explore its role of the inflammatory response mechanism in chondrocyte.Part?Proximal fibular osteotomy alleviates medial compartment knee osteoarthritis in a mouse modelObjective: The purpose of this study was to establish a mouse model of proximal fibular osteotomy(PFO),and to determine if PFO could delay degeneration of medial compartment of the knee joint in mouse model.Methods:1.An animal model of destabilization of the medial meniscus(DMM)was used to induce post-traumatic osteoarthritis(OA).2.PFO was performed to examine the effectiveness of PFO on protection against medial compartment knee osteoarthritis(KOA).Micro-CT was used to observe osteosclerosis development in the subchondral bone.3.Safranin O-fast green staining was used to evaluate the progression of articular cartilage destruction.4.The condylar–plateau angle(CPA)and anatomical femorotibial angle(a FTA)were measured to determine whether knee alignment was changed after PFO.Results:1.PFO treatment decreased osteophyte formation and osteosclerosis development in the subchondral boneWe found that PFO treatment decreased the osteophyte formation in the medial tibial plateau of DMM mice.Moreover,we observed that mice undergoing DMM developed serious osteosclerosis,which was remarkably decreased after PFO treatment,as compared with DMM group.To quantify the extent of osteosclerosis among the groups,we calculated the value of BV/TV(trabecular bone volume per total volume).The value [(48.57±1.13)%] of DMM mice was significantly higher than that [(59.10±1.80)%] of Sham mice at 2 weeks(P <0.01),while that [(52.60±1.11)%] of DMM+PFO mice was lower,as compared with DMM mice(P <0.05).Similarly,the value of Sham mice and DMM mice at 4 weeks was(47.34±1.32)% and(61.19±1.67)% respectively(P <0.001).Nevertheless,DMM+PFO mice displayed the value of(53.40±1.21)%,which was less than DMM mice(P <0.01).2.PFO treatment inhibited progression of articular cartilage destructionDMM mice developed serious arthritis characterized by loss of articular cartilage and osteosclerosis of subchondral bone.In contrast,DMM+PFO group had less progression of cartilage destruction.The OARSI scoring system was used to quantify the severity of the joint damage.And 2-week postoperative Sham mice and DMM mice displayed maximal arthritic scores of 0.40±0.19 and 2.20±0.37 respectively(P <0.01).However,the maximal scores of 2-week postoperative DMM+PFO mice were significantly lower at 1.00±0.27 as compared to DMM mice(P <0.05).Similarly,the maximal scores of 4-week postoperative mice were 0.60±0.19,3.00±0.32,and 1.80±0.37,respectively,and statistical significance was detected between Sham mice and DMM mice(P <0.001),and between DMM mice and DMM+PFO mice(P <0.05).Furthermore,the summed scores of mice undergoing DMM+PFO were also significantly lower than those of mice undergoing DMM at 2 or 4 weeks,and showed a similar trend with the maximal scores(data not shown).3.PFO treatment might alleviate osteoarthritis by reducing the change of knee alignmentThe anteroposterior films of mouse knees were selected from the image data of micro-CT.We found that the CPA of DMM mice was significantly increased compared to Sham mice(Sham:-2.32°±0.37°;DMM:-0.78°±0.47°,P <0.05).However,DMM+PFO mice displayed smaller angle of-2.14°±0.34°(P <0.05).Furthermore,DMM mice had the a FTA of 8.38°±0.45°,which was higher than that 6.85°±0.46° of Sham mice(P <0.05).The a FTA of DMM+PFO mice displayed decreased trend 7.54°±0.44° relative to DMM mice,however,there was no significant difference between them(P =0.22).Conclusion:1.PFO suppressed the tibial subchondral bone remodeling.2.PFO could inhibit the progression of articular cartilage destruction.3.PFO treatment might alleviate osteoarthritis by reducing the change of knee alignment.Part? High-throughput sequencing and verification of differentially expressed Lnc RNAs in IL-1? induced chondrocyte injury modelObjective: To find differentially expressed Lnc RNAs in the IL-1? induced chondrocyte injury model by high-throughput sequencing.Methods:1.IL-1? was used to stimulate human chondrocytes CHON-001 to make a model of human chondrocyte injury.The control group was given the same amount of PBS solution,cells were collected,and total RNA was extracted.2.Use the extracted Total RNA for quality control and processing to obtain the library for high-throughput sequencing.3.Lnc RNAs screening after quality control.4.Differential expression analysis and verification of selected Lnc RNAs.Results:1.Quality control showed Total RNA was compliance with sequencing requirements of Lnc RNAs.Six samples from the control group(Con group)and experimental group(IL group)were administered with IL-1?(10ng / m L)and the same amount of PBS solution for 12 hours,and then the cells were collected.After the total RNA was extracted by Trizol,it was detected by agarose gel eletrophoresis,Nano Drop ND-2000 spectrophotometer,and Agilent 2100,the results showed good RNA purity without significant degradation,and its purity and integrity met Lnc RNAs sequencing requirements.2.Screening of candidate Lnc RNAsAfter sequencing was completed and transcripts were obtained,a total of 231,844 transcripts were screened for Lnc RNAs.A total of 2649 known Lnc RNAs,4280 newly discovered Lnc RNAs,and 4158 TUCPs were found.Among the newly discovered Lnc RNAs,linc RNA accounted for 12.3%,anti-sense Lnc RNA accounted for 2.5%,and intronic Lnc RNA accounted for 85.1%.3.Differential expression analysis and verificationAccording to Q value <0.05,a total of 6 candidate transcripts were selected from the two groups,and a total of 21 differential Lnc RNAs were screened.Among them,12 Lnc RNAs were up-regulated in IL-1?-induced inflammatory chondrocytes and 9 Lnc RNAs were down-regulated.Eight differentially expressed Lnc RNAs were selected and their transcript IDs were: LNC?000346,LNC?000888,LNC?001916,LNC?003694,LNC?003814,ENST00000434309.1,ENST00000412788.5,ENST00000417089.6,qRT-PCR was performed on the expression levels of the eight selected Lnc RNAs.It was found that the expression trend of the above 8 Lnc RNAs in IL-1?-induced inflammatory chondrocytes was consistent with the sequencing results,which proved that the sequencing results were reliable.Conclusion:1.Total RNA was extracted from 6 samples in 2 groups,the quality control showed compliance with Lnc RNAs sequencing requirements;2.High-throughput sequencing and analysis were used to screen 21 differentially expressed Lnc RNAs induced by IL-1? in chondrocytes,of which 12 were up-regulated and 9 were down-regulated;3.The qRT-PCR method proved that the screening results were reliable and could be used for subsequent research.Part ? Regulatory function of Lnc RNA UHCI / Birc2 / NF?B signal axis in the human chondrocyte CHON-001 inflammatory responseObjective:To study the role of UHCI in regulating the inflammatory response of CHON-001 cells,and to preliminary explore its mechanism in regulating the regulation of inflammatory signaling pathways.Methods:1.UHCI was transfected with overexpression plasmid and Si RNA,respectively.Overexpression and knockdown efficiency were observed by qRT-PCR,and protein expressions of inflammatory factors IL-1? and IL-6 were observed by Western Blot.2.Observe the subcellular localization of UHCI in CHON-001 Cells by fluorescence in situ hybridization and immunofluorescence double staining.3.GO analysis of differentially expressed m RNAs shows m RNA enrichment in different biological processes,molecular functions and cellular components.4.KEGG pathway analysis of differentially expressed m RNAs shows the enrichment of m RNAs in different signaling pathways.5.Observe the changes of Birc2 protein expression after overexpression and knockdown of UHCI.6.Observation of NF?B activation after overexpression and knockdown of UHCI.7.Observe whether UHCI can regulate the changes of indicators of inflammation of Birc2 / NF?B signaling pathway in CHON-001 chondrocyte inflammation model.Results:1.UHCI could regulate the expression of IL-1? and IL-6 in chondrocytes.After over-expressing UHCI,Western Blot showed that the expression of IL-1? and IL-6 protein increased significantly compared with the control group;and after knocking down UHCI,the expression of IL-1? and IL-6 protein decreased significantly.2.Observe the UHCI cell localization by double staining of fluorescence in situ hybridization and collagen II immunofluorescence.Collagen II is mainly expressed in the cytoplasm,while UHCI is more abundant in the nucleus and is also distributed in the cytoplasm,but the abundance of cytoplasm is lower.It is suggested that UHCI may play a regulatory role in the nucleus and cytoplasm,and the mainly in the nucleus.3.Enrichment analysis of differentially expressed m RNA colocalizated with Lnc RNAs.GO term analysis showed that the modules with higher enrichment were:(1)biological processes: regulation of ribonuclease activity,cytokine-mediated signaling pathways,cell necrosis,etc.,(2)molecular functions: 2'-5 '-Oligoadenylate synthase activity,adenosyltransferase activity,double-stranded RNA binding,etc.,(3)Cell components: organelle ribosomal subunit,mitochondrial ribosomal subunit,etc.KEGG pathway enrichment analysis showed that the TNF signaling pathway,NF?B signaling pathway,apoptotic signaling pathway,and Fox O signaling pathway were highly enriched.4.Birc2 may be a target gene of UHCI.After overexpression of UHCI,qRT-PCR showed that the expression of Birc2 RNA was increased,and Western Blot showed that the expression of protein also increased accordingly.However,the expression of Birc2 RNA and protein both decreased after silence of UHCI.5.UHCI could regulate the expression of p65 and p50 of NF?B signaling pathway.After overexpression of UHCI,the expression of p65 and p50 proteins in the NF?B signaling pathway was significantly up-regulated,and after silence of UHCI,the expression of p65 and p50 proteins was significantly down-regulated.6.UHCI can regulate chondrocyte inflammatory response by activating the Birc2 / NF?B signaling pathway.After treatment of with IL-1?,Western Blot results showed that the expression of Birc2 protein was up-regulated,the expression of NF?B p65 and NF?B p50 were up-regulated accordingly,and the expression of the inflammatory factor IL-1? was increased.After knocking down UHCI,the expression of these proteins were inhibited,overexpression of UHCI could lead to further increase in the expression of the above proteins.Conclusion: UHCI could regulate the expression of inflammatory factors IL-1? and IL-6.The localization of UHCI in chondrocye is nucleus,UHCI may play a regulatory role through transcription or pre-transcription levels.Birc2 may be a target gene of UHCI,and UHCI may promote the activation of NF?B signaling pathway by regulating Birc2.UHCI plays an important role in the inflammatory response of chondrocytes.Knockdown of UHCI can inhibit the inflammatory response of chondrocytes.