Heat shock protein 60 mediates chronic morphine antinociceptive tolerance through TLR4-p38MAPK in rats

Objective　To investigate the function and potential mechanisms of heat shock protein 60 (HSP60) in chronic morphine antinociceptive tolerance. Methods　Fifty male rats were randomly divided into five groups (n=10): control group, morphine group, siRNA-negative control (siRNA-NC)+morphine group, siRNA-HSP60+morphine group, lipopolysaccharide (LPS)+siRNA-HSP60+morphine group. The control group and the morphine group were injected intrathecally with 10 μl of saline or morphine; the siRNA-NC+morphine group and the siRNA-HSP60+morphine group were given 10 μl of siRNA-NC and siRNA-HSP60 respectively, and then intrathecal morphine; LPS+siRNA-HSP60+morphine group was given 10 μl siRNA-HSP60 and LPS, and then intrathecal injection of morphine. All groups were treated continuously for 7 days. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to evaluate the expression of HSP60 in the spinal cord tissues of rats. The effect of siRNA-HSP60 on pain tolerance was explored by hot-water tail-flick test. Immunohistochemistry assay was applied to detect the expression of OX-42 in the spinal cord tissues of rats. RT-PCR was used to detect the mRNA levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the spinal cord tissues of rats. Western blotting was used to measure the protein levels of TLR4, p-p38MAPK in the spinal cord tissues of rats. Result　Intrathecal injection of siRNA-HSP60 significantly decreased morphineinduced HSP60 expression (P<0.05). Compared with the control group, morphine group rats were less tolerant to pain (P<0.05), and HSP60 deficiency enhanced analgesic effect (P<0.05). Compared with the siRNA-NC+morphine group, the expression levels of OX-42, IL-1β and TNF-α in siRNA-HSP60+morphine group decreased significantly (P<0.05). In addition, the protein levels of TLR4 and p-p38MAPK in siRNA-HSP60+morphine group were lower than those in the siRNA-NC+morphine group (P<0.05). LPS treatment increased the expression of TLR4 and p-p38MAPK (P<0.05); LPS reversed the effect of HSP60 deficiency on morphine tolerance (P<0.05). Conclusion　HSP60 deficiency showed an obvious attenuation on morphine antinociceptive tolerance through inhibiting the microglia activation and the inflammatory response, which may be related to the inhibition of TRL4-p38MAPK signaling pathway. DOI: 10.11855/j.issn.0577-7402.2020.12.05