The Regulatory Effect And Mechanism Of Baicalin Magnesium On Lippolysaccharide-induced Depression Model In Rats

Depression is a mental disease caused by various genetic,biological,psychological and environmental factors,with persistent depressed mood,slowed thinking and reduced volitional activity as the main symptoms.The current traditional antidepressants have certain efficacy,but the drugs take a long time to work and need to be taken continuously,with greater side effects.Therefore,the search for safe and effective antidepressants has become a hot topic in the treatment of depression.Scutellaria baicalensis Georgi is a commonly used Chinese medicine in clinic,which has anti-inflammatory,antioxidant,immunomodulatory and neuroprotective effects.Baicalin is the main active ingredient of Scutellaria baicalensis Georgi.A large number of studies have confirmed that baicalin has obvious neuroprotective effects on central nervous system disease models,like Alzheimer’s disease,Parkinson’s disease,epilepsy and depression.Our previous studies have found that baicalin exists in the form of baicalin magnesium in medicinal materials,but it is still unclear whether baicalin magnesium has neuroprotective effect,so this study aims to explore the regulatory effect and mechanism of baicalin magnesium on lipopolysaccharide（LPS）-induced depression model in rats.Objective:In this study,rat models of depression were induced and established by intraperitoneal injection of LPS.The behavioral,pathological and molecular biological techniques were applied to study the effects of baicalin magnesium on the LPS-induced depression rats,providing theoretical and experimental basis for the research of baicalin magnesium as an antidepressant.Methods:1.A rat depression model was induced and established using LPS induction.Male Sprague-Dawley（SD）rats were divided into 6 groups:control group,model group,baicalin magnesium low-dose group(50 mg·kg^-1),and baicalin magnesium high-dose group(150 mg·kg^-1),baicalin group(146.4 mg·kg^-1)and magnesium sulfate（Mg SO₄）group(19.7 mg·kg^-1),with 8 rats in each group.The dose of baicalin group was equal to that of the parent nucleus of baicalin in the high-dose group of baicalin magnesium,and the dose of Mg SO₄ group was equal to that of magnesium ion in the high-dose group of baicalin magnesium.The drug was given by tail vein injection once a day for 7 days.On the 5th day of administration,LPS(1 mg·kg^-1)was injected intraperitoneally for 3 days,once a day.Behavioral experiments were performed before（0 day）and at the end of 7 day of experiment,including the open field test（OFT）,forced swimming test（FST）and sugar water preference test（SPT）.During the experiment,the body weight of rats was recorded at the same time every day,and the mental status of rats,such as coat color,body shape,exercise vigor and approach responsiveness were observed.On the 7th day after the behavioral experiment,the specimens were collected and processed.2.Hematoxylin-eosin（HE）staining and Nissl staining were used to observe the morphological changes of hippocampal neurons.The level of malondialdehyde（MDA）and the activity of superoxide dismutase（SOD）in hippocampal and cortical tissues,and the levels of interleukin（IL）-6,IL-10,IL-1β,and tumor necrosis factor（TNF）-αin serum were determined by the kit.The protein expressions of nucleotide binding oligomerization domain like receptor protein 3（NLRP3）,toll like receptor 4（TLR4）,nuclear transcription factorκB（NF-κB）p65 and phosphorylated NF-κB（p-NF-κB）p65 in hippocampus were detected by western blot.The gene expressions of IL-1βand TNF-αin brain tissue were condueted by real-time polymerase chain reaction（PCR）.Results:1.On day 0 of experiment,there were no distinct differences in weight and behavioral experiments of rats（P>0.05）.After continuous intraperitoneal injection of LPS for 3 days,compared to the control group,the rats in the model group lost a lot of weight,and prolonged the immobility time of FST,the stay time of OFT center and the number of cleaning actions decreased.Sugar water consumption was markedly reduced,the SPT was decreased,and depression behavior was dramatically decreased（P<0.01）.Compared to the model group,baicalin magnesium in high dose group notably improve the depressive behavior of rats,shorten the immobile time of FST,increase the stay time of OFT center and the SPT rate,improve the general condition and weight of rats（P<0.01）.2.The results of HE and Nissl staining figured that the neurons in the CA1region of the hippocampus in the model group were disordered and irregular,some neurons shrank,and the number of nissl vesicles decreased.The number of neurons in the hippocampal CA1 region of rats in baicalin groups and Mg SO₄groups increased to a certain extent,with more orderly arrangement and clear staining,and the number of nissl vesicles increased.In the low dose group of baicalin magnesium,the degree of injury was not significantly relieved（P>0.05）,while the number of neurons in the hippocampal CA1 region of the rats in the high dose group of baicalin magnesium increased virtually,with neat arrangement,clear staining,and more nissl vesicles.3.Oxidative stress and inflammatory factors results showed that compared to the control group,the levels of IL-6,IL-1βand TNF-αin serum of rats in the model group were increased,while the level of IL-10 was decreased（P<0.01）.The level of MDA in hippocampus and cortex was increased,and the activity of SOD was decreased（P<0.01）.Compared to model group,IL-6,IL-1βand TNF-αin serum were decreased,while IL-10 was increased in baicalin magnesium,prominently（P<0.01）.The level of MDA was decreased,and the activity of SOD was increased（P<0.01）.4.The results of western blot indicated that the protein expression levels of NLRP3 and TLR4 in hippocampus of the model group were up-regulated,and the p-p65/p65 ratio was increased.High-dose baicalin magnesium markedly down-regulated the expressions of NLRP3 and TLR4 protein,and decreased the p-p65/p65 ratio（P<0.01）,while the effect of baicalin magnesium in low dose group was not obvious（P>0.05）.5.The results of real-time PCR showed that the m RNA expressions of IL-1βand TNF-αin hippocampus and cortex of the model group were observably up-regulated（P<0.01）.Compared to the model group,high-dose baicalin magnesium strikingly decreased the m RNA expressions of IL-1βand TNF-α（P<0.01）,while the effect of baicalin magnesium in low dose group was not obvious（P>0.05）.Conclusions:1.The rat model of acute depression was induced and established by intraperitoneal injection of LPS.The body weight of rats in the model group was decreased,the immobility time of FST was increased,the stay time of OFT center was decreased,and the SPT rate was decreased,which were similar to the clinical symptoms of depression（loss of appetite,weight and interest）,which proved that the acute depression rat models was successfully reproduced.The behavior of the rats improved after the drug intervention,with the most significant effect in the high dose group of baicalin magnesium.2.Baicalin magnesium can significantly improve LPS-induced depressive behavior and neuroinflammation in rats through anti-inflammatory and antioxidant effects.The mechanism may be related to the inhibition of the activation of TLR4/NF-κB/NLRP3 inflammatory signaling pathway.