Caspase 3 SiRNA Inhibit Apoptosis Of Chondrocytes And Relieve Osteoarthritis In Vivo

【Background】Osteoarthritis(OA), a common chronic joint disease, is characterized by cartilaginous degeneration and consequential cartilage hyperplasia. Clinical manifestations of OA may include joint pain, stiffness, locking of joints, and sometimes local inflammation. OA together with cancer and cardiovascular disease were assigned as the three major diseases that seriously affect human health by The World Health Organization (WHO). The etiology of OA is still not clear; a variety of potential forces-hereditary, developmental, metabolic, traumatic, and mechanical-may initiate processes leading to loss of cartilage. The incidence rate of OA is related to aging. Research shows that among people over the age of sixty,50% of them are with OA symptom; the incidence rate is up to 80% over the age of seventy-five. The rate of disability by OA is 53%. China is entering the ranks of countries of aging and thus OA gradually become more severe disease, affecting people's life quality.Treatment of OA consists of general treatment, medication and surgery. The general treatment includes patient education, medical exercise and physical therapy. The medication is commonly used to treat the pain from OA. The medicine consists of analgesic drugs, non-steroidal anti-inflammatory drugs, steroid hormones and cartilage protective drugs. Surgical treatment for patients in serious condition, includes osteophyte excision, arthrodesis, arthroplasty, etc. However, the above treatments can not prevent OA from the disease progresses, so people have been actively looking for more effective treatment and preventive methods. The chondrocyte apoptosis was found playing an important role in OA. A lot of studies focus on how to effectively inhibit chondrocyte apoptosis according to chondrocyte apoptosis pathway, in particular by caspase (cysteine protease)-dependent pathway. A number of peptide or non-peptide compounds were synthesized to inhibit the activity of caspase family, moreover inhibit chondrocyte apoptosis. However, these compounds can only inactive caspase protease by physical or chemical ways. Within a complex environment of the cells, the compounds easily lead to instability and affect the inhibitory result. Therefore, it is important to discover more effective inhibitory chondrocyte apoptosis technology for the treatment of osteoarthritis. Gene silencing is one of the very promising technology.【Objective】:To inhibit apoptosis of chondrocytes and relieve osteoarthritis by blocking the apoptotic cascade reaction, gene silencing of caspase 3, and transduction of caspase 3 siRNA into chondrocytes in vitro and in vivo with lentivirus.[Methods]:According to the gene library and principles of design siRNA, three rattus caspase 3 siRNA were designed and synthesized, cloned into pSIH1-H1-copGFP expression plasmid. The sequence was confirmed by gene sequencing. pSIH1-H1-copGFP-caspase 3 siRNA was transfected into rat cell line HSC-T6 by lipofectamine. The most efficient sequence of silence was screened out by caspase 3 mRNA RT-PCR detection and caspase 3 protein western blot detection in HSC-T6 cells. pSIH1-H1-copGFP-caspase 3 siRNA lentivirus was generated in 293TN cells by pPACKH1TM Lentivector Packaging Kit. The chondrocytes were isolated from rat joint. Caspase 3 siRNA was transducted into chondrocytes by lentivirus. After the chondrocytes was transducted, the caspase 3 mRNA was tested by RT-PCR and the caspase 3 protein was tested by Western blot. Apoptosis was induced in both the transducted cells and untransducted cells by TNF-a. Cell apoptosis was assessed by flow cytometry, Annevin V/PI staining. Afterward, the OA animal model was made by Hulth surgery in SD rat. The caspase 3 siRNA lentivirus was injected into the knee joints of SD rats after day 8 and day 15 under the Hulth osteoarthritis surgery. The animals were euthanizied at 2nd,4th,8th weeks after surgery. The joint's sections were H&E and Safranin O stained. All samples were used for Mankin score.[Results]:(1) Gene sequencing results show that the caspase 3 siRNA sequence in the constructed pSIHl-Hl-copGFP-caspase 3 siRNA expression vector was as the same as the designed sequence.(2) Within the three caspase 3 siRNA sequence, the most efficient sequence was screened out to be caspase 3 siRNA1 by caspase 3 mRNA RT-PCR detection and caspase 3 protein western blot detection in HSC-T6 cells. The silencing efficiency was about 87%.(3) The caspase 3 siRNA-lentivirus particles can be generated by pPACKH1 TM Lentivector Packaging Kit, the virus titer was as high as 1.08×108ifu/ml.(4) caspase 3 siRNA can be transducted into chondrocytes by lentivirus, the transduction rate was over 95%; The expression of caspase 3 mRNA and the caspase 3 protein in transducted chondrocytes was lower than that of the normal chondrocytes (P＜0.01). The silencing efficiency was about 85%. When apoptosis was induced in the cells by TNF-a, the apoptosis rate of the negative siRNA-chondrocytes was 7 times higher that than that of caspase 3 siRNA-chondrocytes.(5) The caspase 3 siRNA can be transduced into cartilage in vivo. The histological result showed the cartilage layer defect and severe fibrosis, cartilage cells disarrangement, loss most of cartilage matrix glycosaminglycan(GAG) staining after 8 weeks of Hulth surgery in the control group, the Mankin score of this group was 10.0, assigned as mediate to late stage OA symptoms. On the other hand, the caspase 3 siRNA treatment group only showed uneven cartilage surface and mild fibrosis, cartilage cells almost in arrangment, normal GAG syaining, the Mankin score was 4.25, assigned as the early OA symptoms at the same time. The Mankin score between two groups in 8th weeks had significance difference. [Conclusion]:(1) the designed and synthesized of the caspase 3 siRNA sequence can effectively inhibit caspase 3 gene expression, the gene silence rate was 87%(2) The designed caspase 3 siRNA can be highly transduced into chondrocytes by lentivirus and inhibit caspase 3 expression of chondrocytes.The transduction rate was over 95%.(3) Caspase 3 gene silencing in chondrocytes can effectively antagonize TNF-a induced apoptosis.(4) Caspase 3 siRNA can be transfected into chondrocytes by lentivirus vetor intra-articular injections in rats in situ.(5) Evaluation through the rat animal experiments showed that caspase 3 siRNA can relieve osteoarthritis in vivo.