Melatonin Induces Autophagy In Amyotrophic Lateral Sclerosis Mice Via Upregulation Of SIRT1

Objective: Although there are many studies to explore the pathogenesis of amyotrophic lateral sclerosis(ALS),its diagnosis and treatment methods,so far,there is still a lack of effective strategies and specific therapies for its treatment.Studies have shown that taking high doses of melatonin can prolong survival in ALS patients.However,so far,the role of melatonin on ALS is still unclear,and related studies are very lacking.In this section,we proposed to construct an ALS mouse model to explore the effects of different doses of melatonin on the survival time and motor function of ALS mice.Methods: SOD1*G93A transgenic mice were used as ALS model animals and non-transgenic C57BL/6 mice(wild type,WT)were used as controls.Both ALS mice and WT mice received 10 mg/kg,50 mg/kg,and 100 mg/kg of melatonin by oral administration from 70 days after the animal’s birth until the mouse death or until 180 days.The basic condition and health status of each group of animals were observed after 70 days,and the weight of all animals was measured every week from 70 days.The time of morbidity and death in all ALS animals was recorded.All animals were tested for motor function every week from 70 days after birth until they died or up to180 days.Results: 1)All ALS mice died within 180 d.Within 180 days,the average survival time of ALS mice without melatonin treatment was 143.60±11.93 days,and after melatonin treatment at 10,50 and 100 mg/kg doses,the survival time of ALS mice was extended to 151.40±8.36 days,158.80±13.48 days and 159.30±10.02 days,respectively.Both 50 and 100 mg/kg of melatonin significantly extended the survival of ALS mice.The average onset time of ALS mice was 99.60±4.67 days,while after melatonin treatment at 10,50 and 100 mg/kg doses,the onset time of ALS mice was100.40±5.27,102.20±4.51 and 99.20±6.16 days,respectively,in which the difference was not statistically significant.2)All mice gradually increased in body weight from 14 w to 16 w,while the average body weight of ALS mice was significantly lower than that of WT mice.Starting at 17 w,the body weight of all ALS mice began to gradually decrease to a minimum of 20 w.Among them,the body weight of untreated ALS mice decreased from a peak of 19.17±1.25 g to 13.15±1.68 g at 20 w.In addition,melatonin treatment significantly inhibited the degree of weight loss in ALS mice starting at 17 w in a dose-dependent manner.In ALS mice treated with 10,50 mg/kg melatonin,their body weights at 20 w were 15.42±0.63 g,15.43±1.06 g,16.16±1.21 g,respectively,which were significantly higher than those of untreated ALS mice.3)In WT mice,all animals could reach a latency time of 180 s at all time points.In untreated ALS mice,the latency time of the animal’s rod experiment starting at 18 w gradually decreased with the recommended time of week.By 22 w,the latency time of the remaining animals in the ALS group had dropped to 12.74±4.07 s.After being treated with different doses of melatonin,the transfer rod experimental latency of all ALS mice was significantly increased at each time point compared with the ALS group.Conclusion:1.Melatonin can be dose-dependent to prolong the survival time of ALS mice,but does not affect the time of onset.2.Melatonin can improve weight loss in ALS mice on a dose-dependent basis.3.Melatonin can improve motor function in ALS mice on a dose-dependent basis.Objective: Autophagy plays an important role in a variety of diseases,including neurodegenerative diseases,and studies have shown that there is also a significant dysfunction of autophagy in the process of ALS disease progression.However,so far,there have been limited reports of autophagy in ALS.In this part of the study,we intended to explore the cell autophagy level in spinal cord tissues of WT and ALS mouse animals at different stages of ALS onset through ALS animal models.We also investigated the effect of different doses of melatonin on SIRT1-mediated autophagy in spinal cord histiocytes.Methods: In this study,human SOD1*G93A transgenic mice were used as ALS model animals,and non-transgenic C57BL/6 mice(wild type,WT)were used as controls.Control mice are injected with an equal volume of normal saline.In exploring the effect of melatonin on spinal tissue autophagy levels in ALS mice at different periods,the animals were grouped as follows: 1)90-day WT mice;2)90-day WT mice +100 mg/kg melatonin;3)90-day ALS mice;4)90-day ALS mice +100mg/kg melatonin;5)120-day WT mice;6)120-day WT mice +100 mg/kg melatonin;7)120-day ALS mice;8)120-day ALS mice +100 mg/kg kg melatonin.Among them,the melatonin treatment group was treated by dose gastric irrigation from 70 days to90 days or 120 days in each group of mice.When exploring the effects of different doses of melatonin on spinal tissue autophagy levels in ALS mice,the animals were grouped as follows: 1)120-day WT mice;2)120-day ALS mice;3)120-day ALS mice +10 mg/kg;4)120-day ALS mice±50 mg/kg;5)120-day ALS mice± 100 mg/kg melatonin.Among them,the melatonin treatment group was treated by each group of mice from 70 days to 120 days according to the dose.To explore the effect of SIRT1 on spinal tissue autophagy levels in ALS mice,the animals were grouped into: 1)120-day WT mice;2)120-day ALS mice;3)120-day ASL mice±100 mg/kg melatonin;4)120-day ASL mice±si-SIRT1;5)120-day ASL mice±si-NC;6)120-day ASL mice±100 mg/kg melatonin±si-SIRT1.Among them,the melatonin treatment group was treated by each group of mice from 70 days to 120 days according to the dose.Treatment of si-SIRT1 in ALS mice was from 90 days to 120 days after birth with 100 μL of the lenti-si-SIRT1.RT-PCR and Western blotting were used to detect the expression of autophagy-related genes and proteins beclin1,LC3 II/I,p62 and SIRT1 in mouse spinal cord tissue.Immunofluorescence staining was used to detect the expression of beclin1 and SIRT1 in animal spinal cord tissue.Results: 1)At 90 days,100 mg/kg of melatonin in mouse spinal cord tissues of both WT and ALS mice significantly improved the expression of the key autophagy proteins beclin1 and SIRT1 in mouse spinal cord tissues.Compared with WT mice,spinal cord tissue autophagy levels in ALS mice were significantly higher than in WT mice at 90 days,indicating that spinal tissue autophagy in ALS mice was significantly activated before the onset of illness,and melatonin could further activate its autophagy levels.However,at 120 days,the expression of the key autophagy proteins beclin1 and SIRT1 in the MICE of the ALS group was significantly reduced,and the melatonin of 100 mg/kg could significantly inhibit this decreasing trend.Immunofluorescence results of spinal cord tissue also showed that the key proteins of autophagy beclin1 and SIRT1 were elevated in both ALS and WT animal spinal cord tissues at 90 days,and melatonin further enhanced its expression.At 120 days,the expression of beclin1 and SIRT1 in the spinal cord tissue in the ALS group was extremely low,but melatonin increased its expression to some extent.2)At 120 days,the expression of autophagy-related proteins SIRT1,beclin1,and LC3 II in ALS mice was significantly reduced,while the expression of p62 was significantly increased.After treating with different doses of melatonin,the expression of SIRT1,beclin1,and LC3 II increased significantly with the increase of melatonin dose,while the expression of p62 decreased significantly with the increase of melatonin dose,and its effect was dose-dependent.The results of RT-PCR also showed that the m RNA expression of autophagy-related genes SIRT1,beclin1 and LC3 II in ALS animals at 120 days was significantly lower than that of WT animals,while the m RNA expression of p62 was significantly increased.However,when different doses of melatonin were treated,the m RNA expression of autophagy-related genes SIRT1,beclin1 and LC3 II was significantly increased,while the m RNA expression of p62 was significantly reduced,and the effect was dose-dependent.3)The expression of SIRT1,beclin1 and LC3 II in ALS mice was significantly reduced compared with WT mice,while p62 was significantly higher than that in WT mice,and the melatonin(100 mg/kg)treatment significantly reversed the above effects.On the basis of melatonin treatment,the simultaneous use of si-SIRT1 to inhibit the expression of SIRT1 significantly reversed the promoting effect of melatonin on the autophagic proteins SIRT1,beclin1,LC3 II and the inhibition of p62.In addition,when si-SIRT1 alone inhibited its expression without melatonin treatment,the autophagy level in ALS mice was further reduced and significantly lower than that of ALS mice treated with si-NC-negative controls.Melatonin(100 mg/kg)treatment significantly increased the m RNA expression of the autophagy genes SIRT1,beclin1,and LC3 II,and inhibited the m RNA expression of p62,while inhibition of SIRT1 reversed the above effects.Conclusion:1.Spinal cord tissue cell autophagy levels were elevated at 90 days in ALS mice,and significantly decreased at 120 days;2.Melatonin can be dose-dependent to increase the level of spinal cord histiocyte autophagy in ALS mice;3.Inhibition of SIRT1 reverses the activation of melatonin on autophagy levels of spinal cord tissue cells in ALS mice.Objective: The molecular biology mechanism affecting the occurrence and development of ALS is currently unclear.Among them,autophagy plays an important role in the ALS process.In the process of autophagy,SIRT1 is generally believed to play an autophagic activation role,and the activation of autophagy pathways will further affect the activation process such as apoptosis pathways and inflammatory pathways.However,no studies have found that SIRT1 plays a role in the ALS disease process.Methods: In this study,human SOD1*G93A transgenic mice were used as ALS model animals,and non-transgenic C57BL/6 mice(wild type,WT)were used as controls.The mouse grouping protocols are: 1)120-day WT mice;2)120-day ALS mice;3)120-day ASL mice + 100 mg/kg melatonin;4)120-day ASL mice + si-SIRT1;5)120-day ASL mice + si-NC;6)120-day ASL mice + 100 mg/kg melatonin +si-SIRT1.Among them,the melatonin treatment group was treated by each group of mice from 70 days to 120 days according to the dose.Si-SIRT1 is treated with a tail ivy of 100 μL from 90 to 120 days after birth in ALS mice lenti-si-SIRT1.Expression of apoptotic-related proteins pro-caspase-3,cleaved-capspase-3,bax,and bcl-2 was detected by Western blotting.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of IL-6,IL-1β,TNF-α of animal spinal cord tissues.Results: 1)In the spinal cord tissue of ALS mice at 120 days,the expression of pro-apoptotic proteins including activated cleaved-caspase-3 and bax was significantly increased,while the expression of anti-apoptotic protein bcl-2 was significantly reduced in the spinal cord tissue of ALS mice at 120 days.After the 100mg/kg melatonin treatment,the expression of cleaved-caspase-3 and bax in the spinal cord tissue of ALS mice was significantly reduced compared with untreated animals,while the expression of anti-apoptotic protein bcl-2 was significantly increased,and inhibition of SIRT1 significantly inhibited the inhibitory effect of melatonin on apoptotic protein.2)In the spinal cord tissue of ALS mice at 120 days,the expression of inflammatory factors IL-6,IL-1β,TNF-α was significantly increased relative to WT mice.After treatment with 100 mg/kg of melatonin,the expression of inflammatory factors IL-6,IL-1β,TNF-α in the spinal cord tissues of ALS mice was significantly reduced.However,after using si-SIRT1 to inhibit its expression,inhibition of SIRT1 significantly reversed the inhibitory effect of melatonin on inflammatory factor secretion.Conclusion:1.Apoptosis pathways in spinal cord tissue of ALS mice are activated and the level of inflammatory factor secretion is significantly increased;2.Melatonin can significantly inhibit the apoptosis pathway and inflammatory factor secretion in spinal cord tissue of ALS mice;3.Inhibition of sirt1 expression can reverse the inhibitory effect of melatonin on apoptosis pathways and inflammatory factor secretion in spinal cord tissue of ALS mice.