Notch Signaling Pathway Mediates Microglial Activation In The Mechanism Of Protective Effects Of Minocycline On Diabetic Neuropathic Pain

Diabetic neuropathic pain(DNP)is one of the most common complications of diabetes,mainly characterized by numbness,burning,stabbing pain,tenderness,spontaneous pain,hyperalgesia.Itinitiates from distal extremities symmetrically.According to statistics,there are approximatelyover 50% of diabetic patients eventually develop into DNP,accounting for 10% in all kinds of neuropathic pain.Because pain and allodynia often leads to discomfortable and anxiety,some patients with depression and suicidal tendency.Meanwhile,diabetic foot amputation has become the main cause of non-traumatic amputation,seriously affecting the quality of life ofpatients family with heavy social burden.Thus,DPN becomes the most pressing complication.The pathogenesis of DPN is not clear by far.Pain relief is usually used clinically butthe effect is limited and can't stop disease progressing.Recently,the study of DNP mainly related to hyperglycemia stress,metabolic disorders,oxidative stress,inflammation,but has yetmade the breakthrough.Our previous studies have found that microglia activation and apoptosis may be involved in the occurrence and development of DNP.At the same time,research on antibacterial drug minocycline discovered its additional function of nerve protective effect,making it an potential new drug against DNP.Part 1: To investigate the effect of minocycline on diabetic neuropathic pain ofrats.Methods: The animal model of diabetes was built by intraperitoneally injection of STZ 65 mg.Kg-1.After STZ induction,minocycline of 40 mg.Kg-1 and 80 mg.Kg-1 was administered.Blood glucose levels,nerve functional index(NCV)and behavioral indicators including tail flick latency(TFL),thermal withdrawal latency and mechanical withdrawal threshold(MWT)were observed after STZ induction(T1),2w after induction(T2),4w after induction(T3),8w after induction(T4).After 8 weeks of the execution,spinal cord tissue was collected for pathological slicingand immunofluorescence staining to observe the positive distribution of microglia markers Iba-1.Results: through a single dose of STZ intraperitoneally,diabetic animal model was established successfully.After2 w of the induction,TFL,TWL,MWT,NCV of diabetic group weresignificantly lower than control group(P<0.01),andhyperalgesia symptom continued to 8th w.Microglia markers Iba-1 expression were increased.After treatment with minocycline(Con + Mino40 and Con + Mino80),TFL,TWL,MWT,and NCV wereameliorated,as well as the Iba-1 expression level in dorsal horn(P<0.05).Conclusion: minocycline can relieve pain behavior,improve the nerve conduction velocity and inhibitmicroglia activation.and best minocycline dosage of40 mg kg-1 is selected.Part 2: minocycline relieves DNP through anti-inflammatory,antioxidant,and antiapoptotic mechanisms.Methods: Con group,DM group,Con+Mino,DM+Mino group rats were raised for 8 weeks before execution,lumbar enlargement of spinal cord was collectedfordetermine oxidative stress and lipid peroxidation parameter SOD by colorimetric assay of,MDA and nitrite concentration,inflammatory factors level of TNF alpha,IL-1 by ELISA method,and the levels of neuronal apoptosis in spinal cord dorsal horn through immunohistochemical method.Results: compared with normal control group,spinal cord SOD expression of diabetic rats were significantly reduced,and MDA,nitrite,TNF-a,and IL-1 were significantly higher,a large number of neuronal apoptosis positive cells were determine(P<0.01).The minocycline treatment group appeared higher SOD expression than DM group,while MDA,nitrite,TNF-a and IL-1,and neuronal apoptosis were decreased.Conclusion: oxidative stress,inflammation and apoptosis may participate in the DNP initiation and development,all of which can be restrained by minocycline with the basic effect of microglia inactivation,suggesting the nerve protective effect on DNP.Part 3: The effect of Notch signaling pathway in protective effects of minocycline on DNP.Methods: microglia inhibitor minocycline and(or)Notch signaling pathway inhibitor DAPT were administered separately on DNP rats.Behavioral changes(TFL,TWL,MWT)and expression of Notch-1,NICD,Hes-1 and Iba-1 were measuredby western blot.Results: TFL,TWL,MWT of DAPT group and Mino group were improved significantly than DM group(P<0.05),among which DAPT+Mino group were most significantly improved(P<0.01).Western blot results further confirmed that minocycline and DAPT administration significantly decreased expression of Notch-1,NICD,Hes-1 and Iba-1(P< 0.01).Conclusion: minocycline has therapeutic effect on diabetic neuropathic pain through inhibiting the activation of Notch signaling pathway.Consequently,minocycline plays a role on relieving diabetic neuropathic pain,including improving nerve pain threshold and conduction velocity,with the pathogenesis of overcoming oxidative stress,lipid peroxidation,inflammationand microglia activation.The underlying mechanism may be through the inhibition of microglia activation.Notch signaling pathway may mediate the activation of spinal microglia,thus it is possible that minocycline plays protective effcts on diabetic neuropathic pain by inhibiting Notch signal pathway ofspinal microglial cells.