GSK-3ß inhibition alleviates arthritis pain via reducing spinal mitochondrial reactive oxygen species level and inflammation.

Pain is the main symptom of osteoarthritis, which severely reduces the patients' quality of life. Stimulated neuroinflammation and elevated mitochondrial oxidative stress are associated arthritis pain. In the present study, arthritis model was established by intra-articular injection of complete Freund's adjuvant (CFA) on mice. Knee swelling, pain hypersensitivity and motor disability were observed in CFA-induced mice. In spinal cord, neuroinflammation was triggered and presented as severe infiltration of inflammatory cells and up-regulated expressions of glial fibrillary acidic protein (GFAP), nuclear factor-kappaB (NF-κB), PYD domains-containing protein 3 (NLRP3), cysteinyl aspartate specific proteinase (caspase-1) and interleukin-1 beta (IL-1ß). Mitochondrial function was disrupted and characterized as elevated expressions of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), dihydroorotate dehydrogenase (DHODH) and cytochrome C (Cyto C), and reduced expressions of Bcl-2 and Mn-superoxide dismutase (Mn-SOD) activity. Meanwhile, as a potential target for pain management, glycogen synthase kinase-3 beta (GSK-3ß) activity was up-regulated in CFA induced mice. To explore potential therapeutic options for arthritis pain, GSK-3ß inhibitor TDZD-8 was intraperitoneally injected for three days on CFA mice. Animal behavioral tests found that TDZD-8 treatment elevated mechanical pain sensitivity, suppressed spontaneous pain and recovered motor coordination. Morphological and protein expression analysis indicated that TDZD-8 treatment decreased spinal inflammation score and inflammatory related protein levels, recovered mitochondrial related protein levels, and increased Mn-SOD activity. In summary, TDZD-8 treatment inhibits GSK-3ß activity, reduces mitochondrial mediated oxidative stress, suppresses spinal inflammasome response, and alleviates arthritis pain.

Glycogen synthase kinase-3ß inhibition decreases inflammation and relieves cancer induced bone pain via reducing Drp1-mediated mitochondrial damage.

Bone is the preferential site of metastasis for breast cancer. Invasion of cancer cells induces the destruction of bone tissue and damnification of peripheral nerves and consequently induced central sensitization which contributes to severe pain. Herein, cancer induced bone pain (CIBP) rats exhibited destruction of tibia, mechanical allodynia and spinal inflammation. Inflammatory response mainly mediated by astrocyte and microglia in central nervous system. Our immunofluorescence analysis revealed activation of spinal astrocytes and microglia in CIBP rats. Transmission electron microscopy (TEM) observations of mitochondrial outer membrane disruption and cristae damage in spinal mitochondria of CIBP rats. Proteomics analysis identified abnormal expression of proteins related to mitochondrial organization and function. Intrathecally, injection of GSK-3ß activity inhibitor TDZD-8 significantly attenuated Drp1-mediated mitochondrial fission and recovered mitochondrial function. Inhibition of GSK-3ß activity also suppressed NLRP3 inflammasome cascade and consequently decreased mechanical pain sensitivity of CIBP rats. For cell research, TDZD-8 treatment significantly reversed TNF-α induced mitochondrial membrane potential (MMP) deficiency and high mitochondrial reactive oxygen species level. Taken together, GSK-3ß inhibition by TDZD-8 decreases spinal inflammation and relieves cancer induced bone pain via reducing Drp1-mediated mitochondrial damage.

SIRT1 activation by SRT1720 attenuates bone cancer pain via preventing Drp1-mediated mitochondrial fission.

Bone cancer pain (BCP) is the pain induced by primary bone cancer or tumor metastasis. Increasing evidence and our previous studies have shown that mammalian silent information regulator 2 homolog (SIRT1) is involved in periphery sensitization and central sensitization of BCP, and the underlying mechanism of SIRT1 in bone cancer pain may provide clues for pain treatment. Dynamin-related protein 1 (Drp1) is an essential regulator for mitochondrial fission. In this research, BCP model rats were established by injecting MRMT-1 rat mammary gland carcinoma cells into the left tibia of female Sprague-Dawley rats and validated by tibia radiographs, histological examination and mechanical pain test. As a result BCP rats exhibited bone destruction and sensitivity mechanical pain. BCP increased inflammatory cells infiltration and apoptosis, reduced SIRT1 protein expression and phosphorylation, and elevated Drp1 expression in spinal cord. An agonist of SIRT1 named SRT1720 intrathecal treatment in BCP rats increased SIRT1 phosphorylation, reduced the up-regulated Drp1 expression, and reversed pain behavior. SRT1720 also regulated Bcl-2/BAX and cleaved caspase-3 expressions, and inhibited mitochondrial apoptosis in spinal cord of BCP rats. For in vitro research, SRT1720 treatment decreased Drp1 expression in a dose-dependent manner, blocked CCCP-induced mitochondrial membrane potential change, consequently reduced apoptosis and promoted proliferation. These data suggest that SIRT1 activation by SRT1720 attenuated bone cancer pain via preventing Drp1-mediated mitochondrial fission. Our results provide new targets for therapeutics of bone cancer pain.

[Effect of tanshinone II(A) on expression of different components in renin-angiotensin system of left ventricles of hypertensive rats].

OBJECTIVE: To investigate the effect of tanshinone II(A) on the expression of different components in the renin-angiotensin system of left ventricles of renal hypertensive rats. METHOD: The renal hypertension model was established in rats by the two-kidney-one-clip (2K1C) method. In the experiment, all of the rats were randomly divided into four groups (n = 15 per group) before the operation: the sham-operated (Sham) group, the hypertensive model (Model) group, the low-dose tanshinone II(A) group and the high-dose tanshinone II(A) group. At 5 week after the renal artery narrowing, the third and fourth groups were administered with 35 mg kg(-1) x d(-1) and 70 mg x kg(-1) x d(-1) of tanshinone II(A), respectively. The blood pressure in rats was determined by the standard tail-cuff method in each week after the operation. After the drug treatment for 8 weeks, all the rats were put to death, and their left ventricles were separated to determine the ratio of left ventricle weight to body weight (LVW/BW), the myocardial collagen content, and the expressions of different components in myocardial RAS, including angiotensin converting enzyme (ACE), angiotensin converting enzyme 2 (ACE2), angiotensin 1-type receptor (AT1R), Mas receptor mRNA expression and angiotensin II (Ang II) and angiotensin (1-7) [Ang (1-7)] content. RESULT: Compared with the sham group, the hypertensive model group exhibited a markable increase in the content of Ang II and Ang (1-7) and the mRNA expressions of ACE, ACE2, AT1R and Mas (P < 0.01). However, the treatment with tanshinone II(A) showed the does dependence, inhibited left ventricle hypertrophy, decreased myocardial Ang II content and the mRNA expression of ACE and AT, R in renal hypertensive rats (P < 0. 01) , further increased the myocardial Ang (1-7) content and the mRNA expression of ACE2 and Mas (P < 0.01) , but without any change in the blood pressure of hypertensive rats. CONCLUSION: The treatment with tanshinone II(A) could inhibit left ventricle hypertrophy of renal hypertensive rats. Its mechanism may be partially related to the expression of different components in the renin-angiotensin system for regulating myocardial tissues.

[The functional changes in L-type Ca2+ channel of hypertrophied cardiomyocytes in neonatal rats induced by angiotensin II].

OBJECTIVE: To investigate the molecular and functional changes in L-type Ca2+ channel of hypertrophied cardiomyocytes in neonatal rats induced by angiotensin II (Ang II). METHODS: The in vitro model of cardiomyocyte hypertrophy was established in cultured cardiomyocytes from neonatal rats. Whole cell patch clamp was used to measure the L-type Ca2+ currents. Semi-quantitative RT-PCR was used to determine the mRNA expression of L-type Ca2+ channel alpha1C subunits. RESULTS: In the hypertrophied cardiomyocytes induced by Ang II, I(Ca, L) densities were increased, whereas the features of I(Ca,L) activation, inactivation or recovery from inactivation were not affected. Meanwhile, Ang II increased the mRNA expression of L-type Ca2+ channel alpha1C subunits in cardiomyocytes. All these actions of Ang II could be blocked by the angiotensin II 1 type receptor blocker losartan. CONCLUSION: During cardiomyocyte hypertrophy induced by Ang II, there are significant changes in the molecule and function of L-type Ca2+ channels, which are mediated by the angiotensin II 1 type receptor.