| 1.BackgroundOsteoarthritis(O A)is a type of joint disease that results from breakdown of joint cartilage and underlying bone,and commonly happened among old people.The most commonly involved joints are those near the ends of the fingers,at the base of the thumb,neck,knee and hips.Osteoarthritis can be classified into either primary or secondary depending on whether or not there is an identifiable underlying cause.The secondary is usually caused by factors like congenital disorders of joints,inflammatory diseases,injury to joints or ligaments and joint infection.There is no significant factor for primary.And studies have shown that there is a greater prevalence of the disease among people over 50 years old.Transforming growth factor beta 1(TGF-?1)is an important kind of cytokines.After maturation,it will be secreted into the extracellular environment and work with the specific receptor on the cell membrane,resulting in the reaction of downstream signals.TGF-?1 is able to regulate a cohort of cell activities,including proliferation,migration,and apoptosis.Evidences have shown that TGF-?1/Smad pathway plays an signficant role in the development of OA.Poly(ADP-ribose)polymerase-1(PARP-1)belongs to the PARP proteins family.It mainly works in the process of DNA damage repair.Studies have demonstrated that when the level of PARP-1 was decreased,the repair of single-strand DNA breaks would be inhibited.In the absence of PARP-1,when these breaks were encountered during DNA replication,the replication fork stalled and the double-strand DNA breaks accumulated.PARP-1 has also been shown to be over expressed in kinds of cancers.Consequently,inhibition of PARP-1 has been proved to be one of the effective way to cure caner.Researches have shown that PARP-1 was significantly related to the TGF-?1/Smad pathway,but their regulation was complicated.In this study,we identified that with the development of OA,the level of PARP-1 would increase.The mechanism was due the PARP-1's function on inhibition of Smad2/3 activity and improvement on Smad 1/5/8 activity.In the animal model,we also demonstrated that the OA on mice knee could be recovered after treatment of PARP-1 inhibitor.2.Purposes2.1 Aging causes chondrocyte dysfunction,therefore leads to the development of molecular mechanism of primary OA.2.2 To study the effects of imbalanced pathways TGF-pl/ALK5/Smad2/3 and TGF-?1/ALK1/Smadl/5/8 in the downstream of chondrocytes TGF-?1 on the occurrence and development of OA.2.3 To investigate the effects and molecular mechanism of the interaction of signaling pathways PARP-1 and TGF-?1/Smad in chondrocytes on the transcription of TGF-?1/Smad Signaling Pathway Gene and Chondrocytes.3.Materials and methods3.1 The construction of the carrier3.1.1 PCR reaction The specific primer sequence was designed according to the sequence of the target fragment,and the target fragment was obtained by PCR amplification with cDNA as template.Following the normal PCR reaction conditions,the annealing temperature and the extension time were adjusted according to the length of the primer and the size of the target fragment.The amplification rate of normal Taq PCR enzyme was 1 kb/min.3.1.2 Agarose gel electrophoresis Gel concentration,gel size depends on the length of the DNA sample sequence and the number of samples,the length of the electrophoresis can be adjusted on the length of the sample sequence and gel concentration.3.1.3 Digestion and ligation The expression plasmids used in the experiments were pGL3 and pcDNA3.1-eGFP.After the expected sequence was amplified by PCR,the former was digested with BamHI and Kpnl,and the latter was digested with Xbal and EcoRI.And then it was cloned by T4 DNA ligase into the same treated plasmid.3.1.4 Plasmid transformation and minipreparation The plasmids were transformed into the plasmids according to the procedure,plasmid minipreparation was done with plasmid minipreparation kits.3.2 Extraction and culture of primary rat knee cartilage cells The articular chondrocytes were extracted,passaged,frozen,and transfected strictly according to procedures.3.3 RNA extraction Total RNA samples were extracted by using the Trizol method.3.4 Reverse Transcription RNA was reverse transcribed into cDNA,as a template for PCR.3.5 Real-time quantitative PCR RT-PCR was performed on a Real-time PCR instrument using the SYBR Green qPCR Supermix Real-time PCR kit.3.6 Immunoblot assay All operation was performed according to the procedures of cell lysis,SDS-PAGE gel electrophoresis,transfer film,imaging and others.3.7 Immunoprecipitation experiments After the immunoprecipitation reaction,centrifuge at 3,000 rpm for 3 min at 4?and centrifuge the agarose beads to the bottom of the tube.Carefully aspirate the supernatant.The agarose beads were washed 3-4 times with 1 ml of lysis buffer.Finally 15?L of 2 × SDS loading buffer was added,boiled for 5 minutes;then Western blotting analysis was performed.3.8 Establishment of mouse knee osteoarthritis modelThe knee osteoarthritis model used in this study is the model of medial meniscus instability in mice.3.9 Sampling from mouse kneeThe complete knee specimens were taken,and desilicated after being fixed 24 hours.After the specimen was washed,the samples were treated by dehydration and transparent processing,and immersed with wax and embedded,eventually sectioned.3.10 Safranin fast green staining of paraffin sections of mouse knee jointsSafranin fast green staining was performed after Dewaxing and rehydration of paraffin sections.3.11 Immunohistochemistry of paraffin sections of mouse knee joints After dewaxing and rehydration of paraffin sections,immunohistochemistry was performed.3.12 Immunohistochemical stainingHistological assessment of articular cartilage injury in mice was conducted with the recommended method of OARSI(Osteoarthritis Research Society International)[1].After the mice were killed,the mouse knee was removed,fixed and decalcified and then embedded in paraffin per the method described above.Sections were sagitated from the lateral side of the joint,and the slices of each specimen were continuous and across the entire joint.The thickness of each slice was 5?m.After 10 continuous slicing,three sections were selected out of the 10 and set on one slide.About ten slides were prepared for each and then safranin fast green staining was performed for cartilage Injury score.The middle section could be set on the slide for immunohistochemistry.4.Results4.1 PARP-1 level increased in primary knee osteoarthritisThe degree of primary knee osteoarthritis in mice increased with age.PARP-1 is positively correlated with the severity of primary knee osteoarthritis.The expression of PAPR-1 in traumatic knee osteoarthritis didn't change.4.2 Expression of TGF-?1/Smad signaling pathway in primary knee osteoarthritisThe activity of Smad2/3 in primary knee osteoarthritis decreased and the activity of Smadl/5/8 in primary knee osteoarthritis increased.4.3 Effects of PARP-1 on TGF-?1/Smad signaling pathway and its molecular mechanism4.3.1 SiRNA inhibited the expression of PARP-1 in chondrocytesTargeted siRNA significantly inhibited the transcription and translation of PARP-1 and reduced its level in chondrocytes.4.3.2 The decrease of PARP-1 leaded to changes in TGF-?1/Smad signaling pathwaysCompared to the control group,the expression level of p-Smad2/3 significantly increased when the chondrocytes were simultaneously transfected with PARP-1 siRNA.The expression of Smad2/3 on the downstream target gene increased by detecting the expression of Smad2/3 pGL3-(CAGA)12-Luc plasmid when PARP-1 expression was down-regulated,in contrast,the expression level of p-Smadl/5/8 decreased with the decrease of PARP-1.4.3.3 The decrease in PARP-1 resulted in the expression of Collagen ? and PAI-1When the expression of PARP-1 was inhibited,the mRNA and protein expression of Collagen ? and PAI-1 in chondrocytes both significantly increased.4.3.4 The decrease in PARP-1 resulted in the inhibition of Smad2/3 complex multimerizationWhen the expression of PARP-1 decreased,the content of Smad2/3 protein precipitated by PAC antibody decreased,indicating that the degree of adenosine diphosphate oligomerization of Smad2 and Smad3 decreased,which was consistent with previously reported results.4.4 The effects of PARP-1 on primary knee osteoarthritis in mice4.4.1 Expression of PARP-1 and MMP-13 after treatment of PARP-1 inhibitorAfter treatment of PARP-1 inhibitor,there was not significant difference in the expression of PARP-1,compared with NS group.However,expression of MMP-13 was significant lower than that in NS group.Immunohistochemical staining of paraffin sections also showed the same results.4.4.2 PARP-1 inhibitor leaded to changes in TGF-?1/Smad signaling pathwaysAfter injection of the inhibitor,the expression level of p-Smad2/3 significantly increased.However,the expression level of p-Smadl/5/8 decreased.4.4.3 The decrease in PARP-1 resulted in the expression of Collagen II and PAI-1The mRNA and protein expression of Collagen ? and PAI-1 in chondrocytes both significantly increased after injection of PARP-1 inhibitor.4.4.4 PARP-1 inhibitor reduced the degree of arthritisIn the knee joint treated with PARP-1 inhibitor,the degree of arthritis significantly reduced,indicating that the treatment of knee osteoarthritis has a significant effect.5.DiscussionStudies have shown that PARP-1 plays an important role in the pathogenesis and progression of primary osteoarthritis.The content of PARP-1 was positively correlated with the degree of primary osteoarthritis.PARP-1 can regulate the expression of downstream genes by interacting with TGF-?1/Smad signaling pathway,and PARP-1 can also act directly on Smad2/3 complex to affect its regulation of target gene expression.At the cellular level,inhibition of PARP-1 expression resulted in higher expression of p-Smad2/3,lower expressiong of Smadl/5/8,and higher expression of Collagen ? and PAI-1.In the mouse experiment,the amount of PARP-1 in the knee joint had no significant changes by injection of the inhibitor;however,the expression of MMP-13 was significant higher compared with NS group,which was confirmed in the subsequent pathological examination.At the same time,the use of OARSI arthritis diagnostic criteria,also confirmed that the reduction in PARP-1 content can effectively alleviate the development of primary knee osteoarthritis. |
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