| ObjectiveThe pathogenesis for osteoarthritis (OA) is complicated, and now a lot of data shows that complement system plays key roles in OA pathogenesis. The alternative pathway which is the main pathway in complement system plays a key role in OA. Chondroitin sulfate (CS) is composed alternatively of D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) residues, and it has the capacity of attenuating OA expecially the low molecular weight chondroitin sulfae (LMWCS). However, there is no relative report about the roles of LMWCSs in treating OA via regulating complements till now. Meanwhile, different sources, structures and preparation methods may make CS has a variety of activities in attenuating OA. In order to investigate the relationship between LMWCSs and anti-complement activities and the roles of LMWCS in attenuating OA by regulating complement system, LMWCSs were prepared by depolymerizing shark, porcine and bovine cartilage CSs using four different methods. Hemolytic assay was employed to evaluate their anti-complement activities in vitro. The structure of LMWCSs and activity relationship was studied further and a LMWCS with better anti-complement activity was selected. Then the LMWCS with better anti-complement activity was evaluated both in vitro and in vivo to reveal the relationship between anti-complement activity and the attenuating OA effect of LMWCS. Finally, the near infrared spectroscopy (NIRS) was introduced to establish a rapid and high thoughput method for the screening of anti-comlement of CS samples.Methods1 Preparation and characterization of LMWCSsLMWCSs were prepared by depolymerizing shark, porcine and bovine cartilage CSs using oxidative degradation, enzymatic degradation, HC1 degradation and microwave-assisted alkali degradation methods. Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible (UV-Vis) spectroscopy, high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) methods were used to characterize the LMWCSs2 Anti-complement activity and the structure and activity relationship analysis of different LMWCSsHemolytic assay was employed to evaluate anti-complement activities of different LMWCSs. The structure and activity relationship was studied based on the the characterization results and the IC50 values which were caculated through hemolytic assay. The LMWCS with better anti-complement activity was selected for further investigation both in vivo and in vitro.3 Study on the effects of LMWCS on the chondrocytes in vitroAn ELISA method was introduced to investigate the anti-C3 depositon ability of LMWCS. The protective ability of LMWCS to chondrocytes was evaluated by normal human serum (NHS) mediated chondrocytes death method. The level of C3 mRNA of chondrocytes induced by LPS was investigated using RT-PCR method.4 Study on the effects of LMWCS on osteoarthritis in vivoAn OA mice model was established by destabilization of the medial meniscus (DMM) method. The results of attenuating OA by regulating the complement system through oral administration of LMWCS were investigated. The funtional wind-up was used to evaluate the pain caused by OA. The degree of attenuating OA was evaluated by Safranin-O method. ELISA and immunohistochemistry methods were used to investigate the level of C5b-9.5 The establishment of a fast screening method for the anti-complement activity of CS based on NIRSCS samples from different sources and molecular weights were collected. Reflective spectra were collected by near infrared spectrometer. Hemolytic assay was employed to evaluate the IC50 of all CS samples. A factorial experiment design was used to select the best pre-processing methods. Then UVE (uninformative variable elimination), CARS (competitive adaptive reweighted sampling) and rondom frog methods were used to select the useful variables to establish PLS (partial least square) regression model. And the PLS model was evaluated RMSEP (root-mean-squares error of prediction), RPD (residual predictive deviation) and Rp.Results1 Preparation and characterization of LMWCSsTwelve LMWCSs were successfully prepared by depolymerization of CSs from shark, porcine and bovine sources by four different methods. The FTIR spectra confirmed that LMWCSs had the similar backbones with CS. HPLC result illustrated the disaccharide compositions of LMWCSs were the same as the CS samples. The composition of LMWCSs indicated that HCl and microwave-assisted alkali method had great effect on the sulfate contents. UV-Vis results indicated the formation of A4,5-hexuronic acid caused by enzymatic method. NMR results showed that A4,5-hexuronic acid was formed in the non-reducing end of LMWCSs degraded by enzymatic methods. The structures of the non-reducing end of LMWCSs from others methods were the same to intact CSs. The Mw of LMWCSs was ranging from 1217 Da to 8085 Da.2 Anti-complement activity and the structure and activity relationship analysis of different LMWCSsThe biological activities among different preparation methods were compared. LMWCSs prepared by the oxidative degradation method had better activity than that by other three degradation methods. HCl and microwave-assisted alkali degradation led to loss of sulfate contents, which was important for the anti-complement activity of CS. In the enzymatic method, A4,5-unsaturated disaccharides was formed, which may decrease the anti-complement activity. The anti-complement activity of LMWCSs was better than that of non-degraded CS generally. Hence, the structures of ADi-2,6diS, ?Di-4,6diS and ?Di-6S were good for enhancing the anti-complement activity, while ADi-6S and ?Di-OS had adverse effect on anti-complement activity. LMWCS-S-O could have better effect in attenuating OA. Therefore, LMWCS-S-O and CS from porcine cartilage (CS-P) were selected for further investigation both in vivo and in vitro.3 The effects of LMWCS on the chondrocytes in vitroThe results of the ELISA method which was used to investigate the anti-C3 deposition capacity showed that LMWCS-S-O could inhibit the formation of C3b significantly at 400?g/mL in the alternative pathway. While CS-P showed significant inhibitory effect at 1.2 mg/mL. The morphology and Alcian blue methods were used to identify the chondrocytes. Cytotoxic effect of LMWCS-S-O and CS-P on the articular chondrocytes was determined using MTT assay. The results showed that both LMWCS-S-O and CS-P did not show cytotoxic effect on chondrocytes at 500 ?g/mL after 24 h. The results of NHS mediated chondrocyte death indicated that LMWCS-S-O could protect chondrocytes from death significantly at 400 ?g/mL. The RT-PCR results showed that LMWCS-S-O could inhibit the level of C3 mRNA in chondrocytes induced by LPS.4 The effects of LMWCS on osteoarthritis in vivoThe OA model was sucessfully established by DMM method. The functional wind-up results showed that LMWCS-S-O could attenuate the pain caused by OA. The histological results showed that LMWCS-S-O could inhibit the loss of chondrocytes. Both ELISA and immunohistochemistry results showed that LMWCS-S-O could low down the level of C5b-9 and attenuate OA by regulating complement system. CS-P also showed attenuating effect, but the result was not significant compared to model group.5 The establishment of a fast screening method for anti-complement of CS based on NIRSSixty CS spectra from different sources and relative molecular weights were collected. The reference values (IC50) were determined by hemolytic assay. The best pre-processing method, fisrt derivative (FD)-SNV-SG smoothing-mean center (MC), was selected through a factorial experiment design.47 useful variables were selected in 3112 variables and a PLS model was constructed. The Rc=0.983,Rp=0.969, RMSEP=0.376 mg and RPD=5.46, which indicated that this PLS model could be used for CS anti-complement activity screen in practice.Conclusions and significanceA series of LMWCSs from different sources were prepared by different methods and the stucture and activity relationship between LMWCS and anti-complement capacity was interpreted. The structures of ?Di-2,6diS, ?Di-4,6diS and ?Di-6S were good for enhancing the anti-complement activity, while ?Di-6S and ?Di-OS had adverse effect on anti-complement activity. LMWCS with high content of ?Di-6S, ?Di-2,6diS, ?Di-4,6diS and low Mw might have good capacity in attenuating OA. The attenuating OA effect of LMWCS through regulating complement system was studied both in vitro and in vivo. The results showed that LMWCS from shark had great potential in attenuating OA by regulating complement system. Finally, a rapid CS anti-complement activity screening method based on NIRS was constructed, and it is beneficial to realizing the fast screen for CS. |
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