Effects of Baicalin on the ATP Levels in the Prefrontal Cortex in Mice Exposed to Chronic Unpredictable Mild Stress

Shuai-fei Lu Henan University of Traditional Chinese Medicine: Henan University of Chinese Medicine Xiao-hui Jin Henan University of Traditional Chinese Medicine: Henan University of Chinese Medicine Lei-lei Zhu Henan University of Traditional Chinese Medicine: Henan University of Chinese Medicine Ji-duo Shen Henan University of Traditional Chinese Medicine: Henan University of Chinese Medicine Ming Bai Henan University of Traditional Chinese Medicine: Henan University of Chinese Medicine Yu-cheng Li (  Liyucheng@hactcm.edu.cn ) Henan University of Traditional Chinese Medicine Er-ping Xu Henan University of Traditional Chinese Medicine: Henan University of Chinese Medicine


Introduction
Depression is mainly manifested as persistent low mood, anhedonia, energy loss, and sleep disturbance (Belmaker and Agam 2008;Willner, Scheel-Kruger, and Belzung 2013). Chronic external stresses have become the leading cause of depression in humans. The increasing number of patients with depression will inevitably become a mountain burden on the family and society (Secretariat, T 2012). Although the rst-line clinical antidepressants are widely used, they have the disadvantages of slow onset, poor curative effect, low cure rate, and serious adverse reactions (Westergren, Narum, and Klemp 2019).
Therefore, it is urgent to develop new effective antidepressants.
Increasing evidence showed that the pathogenesis of depression was extremely complicated (Malhi and Mann 2018). Previous studies mainly focused on the hypothesis of "monoamines de ciency", "hyperactivation of hypothalamic-pituitary-adrenal (HPA) axis", "in ammatory response", and "neurotrophic factor de ciency" (Schildkraut and Kety 1967;Barden 2004;Koo et al. 2010;Duman and Monteggia 2006). In recent years, the correlation between energy metabolism disorder and depression has been widely studied (Cao et al. 2013). In addition, increasing studies have shown that depression is closely related to mitochondrial dysfunction (Bansal and Kuhad 2016;Sharma and Akundi 2019). Recent research reported that the adenosine triphosphate (ATP) content in the brain was reduced dramatically in an animal model of depression (Detka et al. 2015;Guo et al. 2021). And the lower energy levels in the brain of depression patients and animal models could be signi cantly reversed by antidepressants (Glombik et al. 2020;Park et al. 2020;Weckmann et al. 2017;Lam et al. 2017). Moreover, direct intra-cerebroventricular injection of ATP into the brain could signi cantly improve the depression-like behavior of chronic unpredictable mild stress (CUMS)-exposed mice (Cao et al. 2013). These ndings suggested that improving the energy level in the brain may be one of the new strategies to treat depression.
Baicalin, the main active ingredient in Scutellaria baicalensis Georgi, possess multiple biological functions, including neuroprotective effects, anti-apoptotic and anti-in ammatory (Shen et al. 2016;Sowndhararajan et al. 2018;Li, Cheng, and Liu 2020). In addition, our previous studies have shown that baicalin can improve depression-like behaviors caused by chronic stress and repeated corticosterone injection Li et al. 2015). However, whether the antidepressant effects of baicalin are related to the regulation of energy metabolism in the brain is still unclear. Therefore, the present study was planned to investigate the effects of baicalin on the levels of ATP, glycolysis, tricarboxylic acid (TCA) cycle, and mitochondrial electron transport chain (ETC) in the prefrontal cortex and to explore the possible antidepressant mechanism of baicalin in CUMS-exposed mice.

Animals
Male C57BL/6N mice, 16-20 g, were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). Mice were housed under a 12 h light/dark cycle with 23-25°C and 40-60 % humidity. Mice were acclimatized for one week with free access to water and food before the experiments. All animal experiments were performed following the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the Committee of Animal Care of Henan University of Chinese Medicine (DWLL16020024).

Drug administration
The mice were randomly divided into 2 groups: the control group (n = 9) and the stress group (n = 27). The stress group received CUMS for 6 weeks, and the control group was kept in a separate room without any stimulation. After week 6, the stress group was subdivided into 3 groups based on the equal sucrose preference: CUMS group, baicalin group (10 mg/kg), and uoxetine group (10 mg/kg). The dose of baicalin was selected from our previous study, which found that 10 mg/kg of baicalin can produce an antidepressant effect in mice (Li et al. 2015). All drugs were suspended in saline and were administered by oral gavage daily in a volume of 10 mL/kg for 4 weeks. CUMS group received an equal volume of saline. The CUMS procedure still lasted during the period of drug administration.

CUMS procedure
The CUMS procedure was slightly modi ed from that previously described by Willner et al. (Willner et al. 1987). Brie y, stress group mice were individually fed in a cage and received 1-2 different stimuli every day. Stressors including day and night reversal for 24 h, food deprivation for 12 h, 45° tilting cage for 24 h, cold (4°C) or hot (45°C) water swimming for 4 min, white noise (90dB) for 12 h, soiled cage for 24 h, restraining for 3 h, ashlight (150 ashes/min) for 6 h, pair-housing for 2 h.

Sucrose preference test
The sucrose preference test (SPT) was carried out according to the protocol described in Liu et al. (Liu et al. 2018). Before the test, all mice were trained to consume two bottles of 1% sucrose solution for 24 h. Then, one bottle of sucrose solution and one bottle of freshwater were given to the mice for another 24 h. The SPT was performed after 12 h of fasting. A bottle of sucrose solution and a bottle of fresh water were weighed and given to the mice for 24 h. To avoid the position preference, two bottles were replaced every 12 h. At the end of the test, all bottles were reweighed. Sucrose preference was calculated as following: sucrose preference (%) = sucrose intake / (sucrose intake + water intake) ×100%.

Forced swimming test
The forced swimming test (FST) was carried out according to the protocol described in Porsolt et al. (Porsolt et al. 2001). The mice were placed in a 13 cm×25 cm (diameter × height) glass cylinder lled with 10 cm deep water (23-25°C). The mice were forced to swim in the water for 6 minutes, and the camera recorded the immobility time of the mice in the last 4 minutes.

Brain tissues collection
Thirty minutes after the last administration, all mice were sacri ced by decapitation. The prefrontal cortex was collected immediately and frozen by liquid nitrogen and stored at -80°C until analysis.

The extraction of the mitochondrion
The prefrontal cortex tissues were homogenized in a 10-fold volume lysis buffer (SM0020, Solarbio, Beijing, China). The homogenate was centrifuged at 1000×g for 5 min, and the supernatants were recentrifuged at 1000×g for 5 min. The supernatants were centrifuged at 12000×g for 10 min, and the supernatants were collected, which is the cytoplasmic extract. Then the precipitate was resuspended with wash buffer and centrifuged at 1000×g for 5 min. The supernatant was recentrifuged at 12000×g for 10 min, and the precipitate was resuspended with store buffer, which is the high-purity mitochondrial extract.

Biochemical analysis
The level of ATP in the prefrontal cortex was detected by ATP kit (S0027, Beyotime Biotechnology, Shanghai, China). The mitochondrial membrane potential (MMP) was detected by JC-10 kit (CA1310, Solarbio, Beijing, China), and the activity of mitochondria complex I-V were detected by kits (ab109721, ab109908, Abcam, Cambridge, UK; BC3245, BC0945, BC1445, Solarbio, Beijing, China). All procedure was carried out following the manufacturer's instructions.

Quantitative real-time polymerase chain reaction
The extraction of total RNA in the prefrontal cortex and cDNA synthesis were using commercial kits following the manufacturer's instructions (DP451, Tiangen, Beijing, China). Real-time PCR reactions were performed in the ABI-Q6 system (Applied Biosystems, USA) by SYBR Green kit (208054, Qiagen, Germany). The primers were synthesized by Invitrogen (Shanghai, China). The sequences of primers were listed in Table 1. GAPDH was used to normalize the gene expression of SIRT1 and PGC1α. Because GAPDH is one of the enzymes in glycolysis, β-actin were used to normalize the related gene expression involved in the glycolysis and tricarboxylic acid (TCA) cycle.

Western blot
The mitochondrial and cytoplasmic proteins in the prefrontal cortex were extracted by the kit (SM0020, Solarbio, Beijing, China) and quanti ed by the BCA kit (CW0014S, CWBIO, Beijing, China). The protein samples were mixed with loading buffer and separated by SDS-PAGE. Then the proteins were transferred onto polyvinylidene di uoride (PVDF) membranes (IPVH00010, Millipore, Billerica, MA, USA) for blocking in 5% skim milk (BD Biosciences, Franklin Lakes, NJ, USA). The membranes were incubated with polyclonal rabbit primary antibodies in blocking solution at 4°C overnight, including anti-GAPDH (KC-5G5, Kangchen, Shanghai, China) and anti-COXIV (4850, Cell Signaling Technology, Danvers, MA, USA). The membranes were washed 3 times with PBST and incubated with goat anti-rabbit secondary antibody (KC-RB-025, Kangchen, Shanghai, China) for 30 min at room temperature. The immunoblotting bands were visualized by incubation with ECL reagent (WBLUF0100, Merck Millipore, Darmstadt, Germany) and exposed to X-ray lm (6535876, Kodak, Rochester, NY, USA).

Statistical Analysis
The SPSS 24.0 software was used for statistical analysis. The Shapiro-Wilk test was used to analyze the normality of the data. Data were expressed as mean ± SD or median and interquartile range. After evaluating homogeneity of variance, the data were analyzed by one-way ANOVA followed by Turkey post hoc test. And the non-parametric data were analyzed by Kruskal-Wallis test. The p < 0.05 was considered statistically signi cant. The statistical analysis was performed by Ming Bai. She did not participate in the relevant experiment and was blinded to the experimental design.

Baicalin reversed the depression-like behaviors in mice
To investigate the effects of baicalin on CUMS-induced depression-like behaviors in mice, the SPT and FST were performed. In the SPT (Fig. 1a)

Baicalin increased the ATP levels in the prefrontal cortex of mice
To investigate whether the antidepressant effects of baicalin were related to the regulation of the energy level in the brain, the ATP level in the prefrontal cortex was detected. Figure 2 showed that the level of ATP was signi cantly reduced in the prefrontal cortex in CUMS group [t (8) = 4.148, p = 0.003]. The reduced ATP level was signi cantly reversed by baicalin treatment [F (2, 12) = 12.705, p = 0.007], but not uoxetine [F (2, 12) = 12.705, p = 0.604]. These results indicated that baicalin could increase the level of ATP and then elevate the energy level in the prefrontal cortex of CUMS-induced mice.

Baicalin increased the level of glycolysis in the prefrontal cortex of mice
To investigate whether baicalin can affect the glycolysis level in the prefrontal cortex, we detected the mRNA expression of three key rate-limiting enzymes of glycolysis. As shown in Fig. 3, The mRNA expression of HK and PK were signi cantly down-regulated in the CUMS group [t (8) = 7.744, p < 0.001; t (4.304) = 9.478, p < 0.001] compared with the control group. And the mRNA expression of HK was signi cantly increased in the baicalin group and uoxetine group [F (2, 12) = 13.961, p = 0.048, p < 0.001].
And the mRNA expression of PK was signi cantly decreased in the uoxetine group [F (2, 12) = 11.157, p = 0.042]. Both baicalin and uoxetine had no effects on the PFK mRNA levels. This result suggested that baicalin might accelerate the glycolysis process by upregulating the expression of HK to produce ATP.

Baicalin increased the TCA levels in the prefrontal cortex of mice
To investigate whether baicalin can affect the TCA level in the prefrontal cortex, the mRNA expression of four key rate-limiting enzymes in TCA was detected. The results were shown in Fig. 4. There were no signi cant changes between CUMS and control groups. However, the mRNA expression of PDHα and IDH were signi cantly up-regulated in the baicalin group compared with CUMS group {[Chi]2 (2, N = 15) = 10.820, p = 0.040; [Chi]2 (2, N = 15) = 9.420, p = 0.033}. Although there was no statistical difference in the mRNA expression of CS and OGDH in the baicalin group, there was still a slight up-regulated in the mRNA expression level. The results indicated that baicalin could enhance the TCA level and thus promote the production of ATP.

Baicalin activated the electron transport chain in the prefrontal cortex of mice
To investigate whether the reduced energy levels resulted from the impairment of the mitochondrial electron transport chain, the activity of ve mitochondrial respiratory chain complexes was detected. The results were shown in Fig. 5a-e. The activity of complexes II and V were signi cantly decreased in the CUMS group compared with the control group [t (7.963) = 8.005, p < 0.001; t (6) = 15.528, p < 0.001].
Interestingly, the activity of complex I was signi cantly increased by baicalin treatment [F (2, 15) = 7.732, p = 0.013], but the activity of complex II was further reduced in the baicalin and uoxetine group {[Chi]2 (2, N = 18) = 12.028, p = 0.003, p = 0.032}. In addition, the activity of complex V was signi cantly increased in the baicalin and uoxetine group {[Chi]2 (2, 12) = 7.385, p = 0.019, p = 0.019}. These results suggested that the effect of baicalin on complex I may be responsible for the increase of ATP level. The purity of the extracted mitochondrion was determined by western blotting (Fig. 5f, supplementary gure S2).
3.6 Baicalin increased the level of mitochondrial membrane potential in the prefrontal cortex of mice To further investigate the effects of baicalin on mitochondrial function, the level of mitochondrial membrane potential (MMP) was detected. The result was shown in Fig. 6. The level of MMP was reduced in the CUMS-exposed mice [t (8)

Discussion
The antidepressant effect of baicalin has been widely demonstrated in our and other previous studies and suggested that the mechanism may be related to brain-derived neurotrophic factor (BDNF), in ammation, and HPA axis (Li, Cheng, and Liu 2020;Li et al. 2013;Li et al. 2015). In the present study, the antidepressant effect of baicalin was con rmed again in CUMS-exposed mice, which was exhibited by the increased sucrose preference and the reduced immobility time in FST. Moreover, we found that baicalin signi cantly reversed the reduction of ATP in the prefrontal cortex induced by CUMS, which may be related to its antidepressant effect.
In recent years, the relationship between abnormal energy metabolism and depression has received increasing attention. Imaging studies have found that there were obvious disorders of energy metabolism in the brain of patients with depression (Kahl et al. 2020;Ernst et al. 2017), which were also con rmed in the brain of chronic mild stress (CMS)-exposed rats (Khan et al. 2018).
ATP is the most important energy source, and its content directly re ects the level of energy status. Previous studies have shown that the ATP content is lower in the brain of depression patients and animal models (Karabatsiakis and Schonfeldt-Lecuona 2020;Kolar et al. 2021). And Jun et al. (Jun et al. 2018) reported that calcium homeostasis modulator protein 2 (Calhm2) knockout mice have a low content of ATP in the brain and exhibit depression-like behavior. Cao et al. (Cao et al. 2013) found that lateral intracerebroventricular and intraperitoneal injection of ATP, or increase of endogenous ATP released by astrocytes, could signi cantly improve the depression-like behavior of mice induced by chronic social defeat stress (CSDS). In addition, the increasing ATP can alleviate synaptic damages, increase spine density and neural activity in the hippocampus region and improve the depression-like behavior of CUMSexposed mice (Cao et al. 2013;Jun et al. 2018).
Together, these studies showed that increasing ATP might be a new strategy for treating depression. In the present study, we found that the ATP content in the prefrontal cortex was signi cantly reduced in CUMS group and was reversed by baicalin. Therefore, our results suggested that the increased ATP content by baicalin will be bene cial for maintaining the function of neurons, which may partly explain its antidepressant mechanisms.
Glycolysis is an important pathway for ATP production in organisms (Gu et al. 2021). A lower level of glycolysis was found in various neurodegenerative diseases, such as depression, Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (Xie et al. 2020;Bell et al. 2020). HK, PFK, and PK are three key rate-limiting enzymes in the glycolytic pathway (Hipkiss 2019). To observe the effect of baicalin on the glycolytic level, we detected the mRNA expression of these enzymes in the prefrontal cortex. We found that baicalin and uoxetine signi cantly up-regulated the mRNA level of HK in the CUMS group, but not PK and PFK. This result indicated that the baicalin can up-regulate the expression of HK to accelerate glycolysis. Similarly, previous reports showed that the level of glycolysis was elevated by paroxetine in depressed rats (Park et al. 2020). Our results suggested that baicalin could increase the production of ATP by improving the level of glycolysis in the prefrontal cortex of mice in the CUMS group.
Under aerobic conditions, the tricarboxylic acid cycle and the coupled oxidative phosphorylation are the main ways to produce ATP in the mitochondrion. Studies have shown that mitochondrial function is closely related to depression (Streck et al. 2014). And other researchers found that chronic stress can inhibit the oxidative phosphorylation of mitochondria, destroy the mitochondrial membrane potential, and damage the mitochondrial structure of different brain regions such as the hippocampus and prefrontal cortex of mice (Gong et al. 2011;Ling-Hu et al. 2021). In addition, a study reported that disruption of the TCA cycle was shown to reduce ATP production in a rat model of depression induced by CMS (Shao et al. 2015). In this study, we found that the mRNA expression of PDHα and IDH, two key rate-limiting enzymes of the TCA cycle, were upregulated by baicalin, which suggested that the level of the TCA cycle might be increased by baicalin.
In addition, the electrons generated during the TCA cycle, which were carried by nicotinamide adenine dinucleotide (NADH) and (nicotinamide adenine dinucleotide phosphate) NAPDH, were delivered to the mitochondrial membrane space by the "proton pump" in ETC, and then this proton gradient was used by ATP synthase to generate large amounts of ATP (Sousa, D'Imprima, and Vonck 2018). The dysfunction of mitochondrial ETC is closely related to depression (Rezin et al. 2009;Villa et al. 2017). In the present study, the activity of complex I and V was upregulated by baicalin, while complex II was the opposite.
Mitochondrial respiratory chain complexes I and II together provide the electrons needed for the reaction of the electron transport chain, which is the rst in the energy production of mitochondria, but complex I is often the starting point of energy production (Willems et al. 2015).
Therefore, this result suggests that baicalin may increase the level of TCA and activate the activity of mitochondrial complex I, then increase the content of ATP. It is well known that the mitochondrial membrane potential (MMP) was formed by the electrons transferred in the respiratory chain. Then, the potential will be used by complex V to produce ATP. And a study reported that the MMP in the prefrontal cortex of CUMS-induced mice was signi cantly reduced (Gong et al. 2011). In the present study, we found that the level of MMP was increased by baicalin and uoxetine in the CUMS group. These results suggest that the antidepressant effect of baicalin may be achieved by activating complex I and V to increase MMP and then increase ATP content.
SIRT1 and PGC1α are two key genes closely related to mitochondrial function, biogenesis, and oxidative stress damage (Tang 2016). In previous studies, depression was associated with decreased expression of SIRT1 (Lo Iacono et al. 2015). PGC1α is a downstream target gene of SIRT1, which is closely linked to the biogenesis of mitochondria (Li, Hou, and Hao 2017). Increasing the expression of PGC1α can signi cantly improve mitochondrial dysfunction and reduce neuronal damage (Jia et al. 2020). In addition, studies have found that the activation of the SITR1/PGC1α pathway plays a major role in mitochondrial function and neuroprotection (Fanibunda et al. 2019). Our data showed that the baicalin could signi cantly increase the mRNA expression of SIRT1 and PGC1α. This result suggested that the baicalin could activate the SIRT1/PGC pathway to increase the level of energy metabolism.

Conclusions
Taken together, the present study found that baicalin could improve the depression-like behavior of mice caused by CUMS and increase the ATP level in the prefrontal cortex. Moreover, baicalin could promote the glycolysis and TCA cycle by upregulating the mRNA expression of HK, PDHα, and IDH. Baicalin also upregulated the mRNA expression of PGC1α and SIRT1, improved mitochondrial function, activated Complex I and V, and increased MMP. These ndings may clarify the possible antidepressant mechanism of baicalin by increasing ATP levels through multiple pathways and provide a new strategy for the development of new antidepressant drugs.

Declarations
Author's contribution SFL designed and performed experiments, collected data, and wrote the manuscript. XHJ and LLZ performed experiments. JDS contributed to the data collection. MB contributed to the data analysis. YCL designed the experiments and revised the manuscript. EPX supervised and managed the experiments.

Data availability
The data are available upon request.

Figure 1
Baicalin reversed the depression-like behaviors in mice. All values are presented as means ± SEM (n = 9).
(A) The sucrose preference in the SPT. (B) The immobility time in the FST. ###p < 0.001 and #p < 0.05 as compared with the control group; ***p < 0.001 and *p < 0.05 as compared with the CUMS group.

Figure 2
Baicalin increased the ATP levels in the prefrontal cortex of mice. All values are presented as means ± SEM (n = 5). ##p < 0.01 as compared with the control group; **p < 0.01 as compared with the CUMS group.

Figure 3
Baicalin increased the level of glycolysis in the prefrontal cortex of mice. The mRNA levels were normalized to β-actin, and subsequently represents as fold change relative to control group. All values are presented as means ± SEM (n = 5). ###p < 0.001 as compared with the control group; ***p < 0.001 and *p < 0.05 as compared with the CUMS group. HK: Hexokinase; PFK: Phosphofructokinase; PK: Pyruvate kinase.

Figure 4
Baicalin increased the TCA levels in the prefrontal cortex of mice. The mRNA levels were normalized to βactin, and subsequently represents as fold change relative to control group. All values are presented as means ± SEM (n = 5). *p < 0.05 as compared with the CUMS group. PDHα: Pyruvate dehydrogenase alpha; CS: Citrate synthase; IDH: Isocitrate dehydrogenase; OGDH: Oxoglutarate dehydrogenase.

Figure 5
Baicalin activated the electron transport chain in the prefrontal cortex of mice. All values are presented as means ± SEM (n = 4-6). (A-E) Mitochondria electron transport chain complex I-V. (F) Purity of mitochondria. 1,2 Mitochondrial. 3,4 Cytoplasmic. ###p < 0.001 as compared with the control group; **p < 0.01 and *p < 0.05 as compared with the CUMS group.

Figure 6
Baicalin increased the level of mitochondrial membrane potential in the prefrontal cortex of mice. All values are presented as means ± SEM (n = 5). ##p 0.01 as compared with the control group; ***p 0.001 and **p 0.01 as compared with the CUMS group.