| Cadmium is a common heavy metal contaminant with neurotoxic effects.Mitochondria are important target organelles of cadmium toxicity.It has been found that Sirtl/PGC-1αpathway is involved in the development of neurological diseases by affecting mitochondria.Our previous studies have shown that cadmium can damage neuronal cells by affecting the quality of mitochondria,but the role of Sirtl/PGC-1a pathway in cadmium-induced mitochondrial damage in neuronal cells remains unclear.Puerarin has neuroprotective effect and can maintain mitochondrial homeostasis.Our previous study has found that puerarin can attenuate cadmium-induced neuronal cells damage by stimulating cadmium excretion,inhibiting oxidative stress and apoptosis.However,whether puerarin can alleviate cadmiuminduced mitochondrial damage mediated by Sirtl/PGC-1α pathway in neuronal cells has not been studied.In this study,rat,rat pheochromocytoma cell（PC 12 cells）and primary rat cerebral cortical neurons were used to explore the protective effect of puerarin on Sirtl/PGC1α pathway-mediated mitochondrial damage induced by cadmium in neuronal cells of rats in vivo and in vitro.1.In vivo studies 24 six-week-old SD rats were prefed for 1 week,randomly grouped and treated in the following way,control group（Con）,puerarin group（Pur）,cadmium group（Cd）,cadmium and puerarin co-treatment group（Cd+Pur）.The rats in Pur group,Cd+Pur group underwent gavage of puerarin dissolved in sodium carboxymethyl cellulose at a dose of 200 mg/kg body weight daily,while the rats in the Con group and Cd group were given sodium carboxymethyl cellulose by gavage at the same dose and lasted for seven weeks.The rats in the Cd group,Cd+Pur group received drinking water containing 75 mg/L cadmium plus the gavage from the fourth week and lasted for four weeks.After the experiment,the cerebral cortices of rats were dissected and collected for subsequent experiments.The Cd concentrations were detected by flam atomic absorption spectrometry.Mitochondrial ultrastructure was observed by transmission electron microscopy.ATP level was detected by luminometer.The ratio of NAD+/NADH,the activity of MnSOD and the level of H2O2 were detected by colorimetry.The transcription levels of mitochondrial oxidative stress-related genes were detected by qRT-PCR.The expression levels of mitochondrial damage-related proteins were detected by Western blot.The results are as follows:①Compared with the Con group,the Cd concentrations of cerebral cortices in the Cd group increased significantly（P<0.05）.Compared with the Cd group,the Cd concentrations of cerebral cortices in the Cd+Pur group decreased significantly（P<0.05）.②Compared with the Con group,there were disruption and shadow of mitochondrial cristae and vacuoles in the mitochondrial matrix in the Cd group.Compared with the Cd group,the arrangement of the mitochondrial cristae was more regular,and the degree of vacuolization in the mitochondrial matrix was low in the Cd+Pur group.③Compared with the Con group,the level of ATP and the ratio of NAD+/NADH in the Cd group decreased significantly（P<0.05）.Compared with the Cd group,the ATP level and NAD+/NADH ratio in the Cd+Pur group increased but had no significant difference（P>0.05）.④Compared with the Con group,the MnSOD activity decreased significantly（P<0.05）,the H2O2 level,the transcription levels of UCP1,UCP2,UCP3,GST,CAT and Gpx mRNA in Cd group increased significantly（P<0.05）.Compared with the Cd group,the MnSOD activity increased significantly（P<0.05）,the H2O2 level,the transcription levels of GST,CAT and Gpx mRNA in the Cd+Pur group decreased significantly（P<0.05）.⑤Compared with the Con group,the protein expression levels of Sirtl and PGC-1α in the Cd group decreased significantly（P<0.05）.Compared with the Cd group,the protein expression levels of Sirtl and PGC-1α in the Cd+Pur group increased significantly（P<0.05）.⑥Compared with the Con group,the protein expression level of Drp1 increased significantly（P<0.05）,while the Mfn2 expression decreased significantly（P<0.05）in Cd group.Compared with the Cd group,the protein expression level of Drp1 decreased significantly（P<0.05）,while the Mfn2 expression increased significantly（P<0.05）in the Cd+Pur group.⑦Compared with the Con group,the protein expression levels of Pink1,Parkin and LC3II in mitochondria in the Cd group increased significantly（P<0.05）.Compared with the Cd group,the protein expression levels of Pink1,Parkin and LC3II in mitochondria in the Cd+Pur group decreased significantly（P<0.05）.The above results indicate that puerarin alleviates the mitochondrial damage induced by cadmium in the cerebral cortices of rats by alleviating mitochondrial structural damage,mitochondrial dysfunction,mitochondrial oxidative stress,the inhibition of Sirtl/PGC-1α pathway,abnormal expression of mitochondrial dynamic proteins and Pink 1/Parkin-mediated mitophagy.2.In vitro studies The study was carried out on neuronal cells of rats（PC 12 cells and primary rat cerebral cortical neurons）.Neuronal cells of rats were treated with different concentrations of cadmium chloride（0 μM,2.5 μM,5 μM,10 μM and 20 μM）for 6 h or 12 h,10 μM cadmium chloride for different times（0 h,3 h,6 h,9 h,12 h and 24 h）,different concentrations of puerarin（50 μM,100 μM and 200 μM）alone or with 10 pM cadmium chloride for 12 h,100 μM puerarin or different concentrations of Srt1720（0.05 μM,0.5 μM,1 pM,3 μM and 5 μM）with 10 μM cadmium chloride for 12 h.A series of experiments were carried out.The cell viability was detected by CCK-8 assay.The ultrastructure of mitochondria was observed by transmission electron microscope.The level of ATP was detected by luminometer,the activity of MnSOD was detected by colorimetry.The level of MitoSOX was detected by flow cytometry.The fluorescence accumulation of MitoSOX was observed by fluorescence microscope.The transcription levels of mitochondrial oxidative stress-related gene were detected by qRT-PCR.The expression levels of mitochondrial damage-related proteins were detected by Western blot.The results show that:①Compared with the control group,the cell viability of PC 12 cells decreased significantly after treatment with 10,20 μM cadmium for 6 h,or treatment with 5,10,20 μM cadmium for 12 h（P<0.05）.Compared with the cadmium group,the viability of PC 12 cells in 100 μM puerarin and cadmium co-treated group increased significantly（P<0.05）.②Compared with the control group,there were disruption and shadow of mitochondrial cristae and vacuoles in the mitochondrial matrix in neuronal cells of rats in the cadmium group.Compared with the cadmium group,the arrangement of the mitochondrial cristae was more regular,and the degree of vacuolization in the mitochondrial matrix was low in neuronal cells of rats in the cadmium and puerarin cotreated group.③Compared with the control group,the protein expression levels of Sirtl in neuronal cells of rats and PGC-1α in primary rat cerebral cortical neurons decreased significantly after treatment with 10 μM cadmium for 12 and 24 h（P<0.05）,the protein expression level of PGC-1α in PC 12 cells decreased significantly after treatment with 10 μM cadmium for 9,12 and 24 h（P<0.05）,the protein expression levels of PGC-1α in neuronal cells of rats and Sirtl in the primary rat cerebral cortical neurons decreased significantly after treatment with 10 μM and 20 μM cadmium for 12 h（P<0.05）,the protein expression level of Sirtl in PC 12 cells decreased significantly after treatment with 5 μM,10 μM and 20 μM cadmium for 12 h（P<0.05）.④Compared with the control group,the protein expression levels of Sirtl and PGC-1α in PC12 cells,Sirtl in primary rat cerebral cortical neurons in the cadmium group decreased significantly（P<0.05）.Compared with the cadmium group,the protein expression levels of Sirtl in neuronal cells of rats,PGC-1α in PC 12 cells increased significantly（P<0.05）in 1,3,5 μM Srt1720 and cadmium co-treated group,the protein expression level of PGC-1α in primary rat cerebral cortical neurons increased significantly（P<0.05）in 3,5 μM Srt1720 and cadmium co-treated group.⑤Compared with the control group,the cell viability in neuronal cells of rats in the cadmium group decreased significantly（P<0.05）.Compared with cadmium group,the cell viability in neuronal cells of rats increased significantly（P<0.05）in 1,3,5 μM Srt1720 and cadmium co-treated group.⑥Compared with the control group,the protein expression levels of Sirtl and PGC-1α in neuronal cells of rats in the cadmium group decreased significantly（P<0.05）.Compared with the cadmium group,the protein expression levels of Sirtl and PGC-1α in neuronal cells of rats in cadmium and puerarin co-treated group increased significantly（P<0.05）.⑦Compared with the control group,the ATP level in neuronal cells of rats in the cadmium group decreased significantly（P<0.05）.Compared with the cadmium group,the ATP levels in 3,5 μM Srt1720 and cadmium co-treated group of PC 12 cells and 5 μM Srt1720 and cadmium co-treated group of primary rat cerebral cortical neurons increased significantly（P<0.05）.⑧Compared with the control group,the ATP level in neuronal cells of rats in the cadmium group decreased significantly（P<0.05）.Compared with the cadmium group,the ATP level in neuronal cells of rats in the cadmium and puerarin co-treated group increased significantly（P<0.05）.⑨Compared with the control group,the activity of MnSOD and the level of MitoSOX in neuronal cells of rats in the cadmium group increased significantly（P<0.05）.Compared with the cadmium group,the activity of MnSOD and the level of MitoSOX in neuronal cells of rats decreased significantly（P<0.05）in 3,5 μM Srt1720 and cadmium co-treated group.⑩Compared with the control group,the activity of MnSOD and the level of MitoSOX in neuronal cells of rats,the transcription levels of UCP1,UCP2,UCP3,GST and CAT mRNA in PC12 cells in the cadmium group increased significantly（P<0.05）.Compared with the cadmium group,the activity of MnSOD and the level of MitoSOX in neuronal cells of rats,the transcription levels of UCP3,GST and CAT mRNA in PC 12 cells in cadmium and puerarin co-treated group decreased significantly（P<0.05）.11Compared with the control group,the total protein expression level of Drpl in PC 12 cells and the protein expression level of Drpl in mitochondria in neuronal cells of rats increased significantly（P<0.05）,the total protein expression level of Mfn2 in neuronal cells of rats decreased significantly（P<0.05）in the cadmium group.Compared with the cadmium group,the protein expression level of Drpl in mitochondria in neuronal cells of rats in cadmium and puerarin co-treated group decreased significantly（P<0.05）.12Compared with the control group,the protein expression levels of Pink1,Parkin and LC3II in mitochondria in neuronal cells of rats in the cadmium group increased significantly（P<0.05）.Compared with the cadmium group,the protein expression levels of Pink1 and Parkin in mitochondria in neuronal cells of rats,LC3II in mitochondria in the primary rat cerebral cortical neurons decreased significantly（P<0.05）in the cadmium and puerarin co-treated group.These results suggest that the inhibition of Sirtl/PGC-1αpathway plays an important role in cadmium-induced mitochondrial damage in neuronal cells of rats,and puerarin alleviates the inhibition of Sirtl/PGC-1α pathway and mitochondrial damage induced by cadmium in neuronal cells of rats,indicating that puerarin may alleviate the mitochondrial damage induced by cadmium through Sirtl/PGC-1α pathway.To sum up,this study indicates that the inhibition of Sirt1/PGC-1α pathway plays an important role in cadmium-induced mitochondrial damage in neuronal cells of rats,and puerarin alleviates cadmium-induced mitochondrial damage mediated by Sirtl/PGC-1αpathway in neuronal cells of rats. |
PDF Full Text Request