3.1 KBD SD rat models were successfully established.
KBD SD rat models were established at first. After 4 weeks of low-selenium feeding, blood GPX activity was measured by kits. It tells that a low-selenium condition was induced (Fig. 2B)
Rats were administrated with T2-toxin gastrically for another 4 weeks. Then 3 rats in both normal and model groups were sacrificed for confirmation of KBD induction. the knee joints of those rats were collected for HE staining. As Fig. 3 shows, KBD model rats’ cartilage and subchondral bone were injured, and more inflammation cells infiltrated the synovium.
The body weight was also recorded, low selenium fed only lead to a subduing reduction of weight(Fig. 2A). But after 2 weeks of T2-toxin administration, the model rats showed significantly lower weight than normal rats, and the gap was even wider in the 8th week.
Serum inflammation cytokines were detected by ELISA. (Fig. 2C-D) In the beginning, TNF-α and IL-1β levels were consistent in both model and control rats. But after 4 weeks of low selenium feeding, the TNF-α and IL-1β level increased significantly, and the gap was enlarged by another 4 weeks of T2-toxin treatment.
Based on the blood level of selenium and inflammation cytokine, body weight, and histology staining, the KBD rat models were successfully induced.
3.2 KBD rats are more sensitive to pain
Behavioral tests of mechanical paw withdrawal threshold(PWT) and thermal paw withdrawal latency(PWL) were performed once a week to observe KBD rats’ pain state. As Fig. 4 shows, as the induction of the KBD model goes, rats were recorded with smaller PWT and shorter PWL. In detail, the gap of PWT and TWL between normal and KBD rats reached statistical significance in the 7th and 3rd week separately. It means KBD rats are more sensitive to pain.
3.3 NGF mcAb Tnz attenuates KBD rat pain and inhibits inflammation.
After the KBD model was successfully built, KBD rats were injected with PBS and Tnz (2.5 mg/kg and 5.0mg/kg) subcutaneously once a week for 4 weeks. PWT and PWL were tested at intervals of 3 days, while serum levels of IL-1β and Tnf-α were detected by ELISA every 2 weeks.
As treatments continues, the PWT and PWL of rats injected with both doses of Tnz rose and reached a plateau around the 20th day. But the statistical significance was observed on the 16th and 10th day respectively. It’s worth mentioning that, rats treated with 5.0mg/kg of Tnz were recorded with obviously higher PWL than the rats who received a lower dose from the 16th to 25th day. But from the perspective of PWT, the superiority of 5.0mg/kg Tnz is slight. (Fig. 5A-B)
At the end of the 4th week, Serum IL-1β and Tnf-α levels of the rats treated with TNZ were significantly lower than those with PBS. Besides, rats treated with a 5.0mg/kg dose of Tnz exhibits weaker inflammation cytokine level than 2.5mg/kg, but this phenomenon is not significant. (Fig. 5C-D)
In general, Tnz can relieve the pain of KBD rats and bring down serum inflammation factor levels of it.
3.4 Tnz acts on the NGF/Trka pathway to effect.
To test the NGF/Trka signal’s level in DRG, rats in groups were sacrificed to gather their DRG. Next, the level of key proteins in rats’ DRG was tested by western blotting and immunofluorescence experiment.
As Fig. 6 shows, western blotting experiments told us that the protein level of CGRP, BDNF, and phosphorylation level of Trka was upregulated significantly in the DRG of KBD rats, while a decrease in those treated with Tnz, and a higher dose of Tnz leads to more obvious reduction.
To get more concrete proof, in the DRG of normal, KBD rats and those received 5.0mg/kg Tnz, the proteins level of BDNF and CGRP were also detected by IF. BNDF and CGRP were upregulated in DRG of KBD rats more than normal rats, in the meanwhile, 5.0mg/kg Tnz treatment substantially rescued abnormally increased BNDF, and CGRP in DRG. (Fig. 7)