Hydrogen Sulfide Attenuates the Cognitive Dysfunction in Parkinson's Disease Rats via Promoting Hippocampal Microglia M2 Polarization by Enhancement of Hippocampal Warburg Effect

Identification of innovative therapeutic targets for the treatment of cognitive impairment in Parkinson's disease (PD) is urgently needed. Hydrogen sulfide (H2S) plays an important role in cognitive function. Therefore, this work is aimed at investigating whether H2S attenuates the cognitive impairment in PD and the underlying mechanisms. In the rotenone- (ROT-) established PD rat model, NaHS (a donor of H2S) attenuated the cognitive impairment and promoted microglia polarization from M1 towards M2 in the hippocampus of PD rats. NaHS also dramatically upregulated the Warburg effect in the hippocampus of PD rats. 2-Deoxyglucose (2-DG, an inhibitor of the Warburg effect) abolished NaHS-upregulated Warburg effect in the hippocampus of PD rats. Moreover, the inhibited hippocampal Warburg effect by 2-DG abrogated H2S-excited the enhancement of hippocampal microglia M2 polarization and the improvement of cognitive function in ROT-exposed rats. Our data demonstrated that H2S inhibits the cognitive dysfunction in PD via promoting microglia M2 polarization by enhancement of hippocampal Warburg effect.


Introduction
Parkinson's disease (PD), characterized by the loss of dopaminergic neurons, is a common neurodegenerative disease [1,2]. PD is defined primarily as a movement disorder, with the typical dopamine-related motor symptoms being resting tremor, rigidity, bradykinesia, and postural instability. However, a wide variety of nonmotor symptoms are commonly observed in patients with PD [3]. The cognitive dysfunction is a frequent important nonmotor symptom encountered in PD patients [3]. It has been reported that the lesioned hippocampus, a temporal lobe structure, leads to memory dysfunction [4,5]. Furthermore, aerobic exercise training is effective at reversing hippocampal volume loss, which is accompanied by improved memory function [6]. As reported that verbal memory deficient in recall and recognition is associated with atrophy of the hippocampus in newly diagnosed PD patients [7], recent advancement also observed hippocampal subfield atrophy of CA1 in participants who progressed to PD dementia [8]. All of these reflect an intimate relationship between hippocampal dysfunction and cognitive impairment in PD. However, there is no effective strategy to prevent the cognitive impairment in PD. Hydrogen sulfide (H 2 S), well known as a neuromodulator [9], alleviates cognitive deficits caused by multiple risk factors [10][11][12]. Our previous work demonstrated that the cognitive impairment in formaldehyde-or homocysteine-exposed rats is related to the disturbance of endogenous H 2 S [13,14] and that supplementation of exogenous H 2 S prevents the cognitive impairment in homocysteine- [15,16], chronic restrain stress- [17], and streptozotocin- [18] exposed rats. Collectively, the antagonistic action of H 2 S on cognitive dysfunction is widely recognized. Thus, it seems reasonable to speculate the therapeutic potential of H 2 S for the cognitive deficit in PD.
Microglia is the primary immune cells in the brain, acting as immune surveillance and host defense. Accumulating evidence has reported that microglia regulate neurodevelopment and synaptic plasticity [19,20]. Like macrophages, microglia are designated classically activated (M1) and alternatively activated (M2) phenotypes [21]. M1 microglia release proinflammatory cytokines to destroy neurons, and contrarily, M2 microglia secrete neuroprotective mediators and trophic factors. It has been confirmed that promoting microglia M2 polarization enhances α-synuclein clearance in PD [22]. Furthermore, maintaining the balance of M1 and M2 is crucial to alleviate cognitive disorder in schizophrenia [23] and enhancing M2 polarization mitigates Aβcaused cognitive dysfunction [24]. Additionally, H 2 S exerts a regulatory role in microglia polarization [25,26]. Thus, to understand the mechanism underlying H 2 S-promoted cognitive function in PD, we investigated whether H 2 S skews M1 microglia polarization toward M2 activation.
The Warburg effect, also known as aerobic glycolysis, involves the metabolism of glucose to supply ATP and secrete lactate via glycolysis under aerobic conditions. Otto Warburg's work first reported that cancer cells have a propensity to metabolize glucose via glycolysis despite oxygen availability [27]. Interestingly, the Warburg effect also plays vital regulatory roles in the function of the brain, supporting the molecular demands of neuronal proliferation in embryonic development, promoting the maturational changes of neurons in postnatal development, and is responsible for changes at the synapse in adulthood [28,29]. Moreover, lactate, a metabolite of the Warburg effect, exerts a regulatory role in synaptic plasticity by enhancing the expressions of Egr-1 and c-Fos. A recent study reported that lactate is required for long-term potentiation (LTP) formation [30], providing a new insight into the mechanism of cognition [31,32]. These observations prompted us to raise the question of whether the Warburg effect mediates the antagonistic role of H 2 S in the cognitive deficit in PD.
In this study, we found that H 2 S alleviated the cognitive impairment, promoted hippocampal microglia M2 polarization, and enhanced hippocampal Warburg effect in the rotenone-induced PD rats. Furthermore, inhibition of the hippocampal Warburg effect abrogated H 2 S-exerted protection against the cognitive disorder and promotion of hippocampal microglia M2 polarization in PD rats. Our findings demonstrated that H 2 S enhances hippocampal Warburg effect, leading to promote microglia M2 polarization, which in turn prevents the cognitive disorder in PD.

PD Animal
Model and Animal Dosing. The adult male Sprague-Dawley (SD) rats weighing 260-280 g were obtained from SJA Lab. Each group contained 15 rats and each rat was housed individually with free access to food and water. The room humidity was controlled at 40%-46%, and the temperature was controlled within 22-26°C. SD rats were exposed to ROT (2 mg/kg, s.c.) for 5 w to mimic the PD model [33]. After habituating to this new housing environment for 1 w, rats were administered NaHS (30 and 100 μmol/kg, i.p.) for 1 w. Subsequently, rats were exposed to ROT via subcutaneous injection for 5 w and NaHS for 3 w simultaneously. In the 3 rd week of injecting ROT, rats were cotreated with 2-DG (3 μg/w, i.c.v.) for 3 w. After performing behavioral tests, the brains of rats were collected under euthanasia ( Figure 1). All experiments strictly complied with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Animal Use and Protection Committee of the University of South China.

Intracerebroventricular Injection.
Under deep anesthesia using pentobarbital sodium (45 mg/kg), animals were fixed on the stereotaxic apparatus for the following operation. The hair from the top of the skull was removed, fully exposing the bregma. The position of the right lateral ventricle was determined according to the following coordinates: AP, −1.0 mm; ML, −2.0 mm; DV, −4.0 mm. Then, the aseptic cannula was implanted into this location for drug administration. After surgery, penicillin was administered for 3 consecutive days to prevent infection. Rats were recovered for 1 w. 2-DG was administered into the lateral ventricle along the cannula using a microsyringe. The needle was removed slowly and kept halfway in for an extra 2 min to ensure complete delivery of the drug.

Y-Maze
Test. The Y-maze test was used to evaluate the learning and memory of rats. The experimental apparatus consisted of three identical labeled arms (A, B, and C) at equal angles. At the beginning of the test, the rat was placed in the intersection of the three arms to explore freely for 5 min. The sequence and number of the rat entries into each arm were recorded. The rat entering different arms in turn during three consecutive chances was marked as a correct sequence (for example, ABC, BAC, or ACB, but not ABA 2 Oxidative Medicine and Cellular Longevity and etc.). Once the rat completed the test, the three arms and the bottom of the apparatus were swept and wiped using 70% alcohol to eliminate the potential influence of odors and residues on experimental data. The alternation performance was calculated to evaluate the spatial differentiation memory of rats.

Novel Object Recognition (NOR)
Test. The open-field box that was used in this experiment was 50 cm × 40 cm × 30 cm. To avoid being pushed or bitten by rats, the recognized objects were cylinders and tubes with a certain weight as well as hardness. The whole experiment was divided into three periods: the adaptation period, training period, and testing period. The adaptation period lasted for 2 d. All rats were introduced to the test box from the fixed position and were adapted to this new environment for 5 min without objects. During the training period, the rat entered the box with two identical objects from a fixed position, and the time that the rat spent exploring each of the two objects was recorded. After an hour-long retention interval, the rat was reintroduced to the test box, with one familiar object replaced by one novel object, and the time that the rat spent exploring each of the two objects over the course of 5 min was recorded. Touching, sniffing, and direct attention to an object rather than climbing or chewing objects were defined as exploratory behaviors. A distance between the nose and object within 2 cm was also regarded as exploratory behavior. The discrimination index was calculated to reflect the cognitive function of the rat, which was defined by the following equation: discrimination index = ½ðtime spent on familiar object − time spent on novel objectÞ/ðtime spent on familiar object + time spent on novel objectÞ × 100.
2.6. Morris Water Maze (MWM) Test. The MWM system mainly includes a circular pool with water and a hidden platform. The pool was divided into four quadrants, and the target quadrant was defined as the platform positioned in the center of this area. In the acquisition phase (days 1-5), rats were randomly introduced into the pool from different quadrants to search for the hidden platform in four trials per day with a 20-minute interval. The maximum swimming time for each rat was 120 s in each trial; otherwise, rats were guided to the hidden platform and allowed to stay there for 20 s. The swimming route and latency to the platform were tracked by the MT-200 Morris Image Motion System. In the probe phase (day 7), rats were reintroduced to the pool without a platform from the opposite position to the target quadrant. The time spent swimming in the target quadrant and the number of crossing the target platform of rats were recorded over the course of 120 s. Following the probe test, rats were subjected to the visible platform test to rule out possible deficits in vision and motor function, which may impact experimental data. The platform was replaced opposite to the target quadrant and was 2 cm higher above the water. Rats were reintroduced to the pool from the side opposite to the platform, and the escape latency and average speed were recorded.

Western Blot Analysis.
After all behavioral tests were completed, rats were sacrificed, and hippocampal tissues of the brain were dissected. Then, hippocampal tissues were homogenized using lysis buffer, and supernatant was collected after centrifugation. The protein content was measured by a BCA protein assay kit. The prepared samples were added into sodium dodecyl sulfate-polyamide gel for electrophoresis and subsequently transferred to the polyvinylidene fluoride membrane. The membranes were blocked using 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). Two hours later, these were incubated with primary antibodies overnight at 4°C and subsequently washed 10 min in TBST followed by incubation with corresponding secondary antibodies for 2 h. Finally, membranes were visualized by an enhanced chemiluminescence system (BeyoECL Plus kit, Beyotime). AlphaImager 2200 software was used to calculate the signal of immunoblot. 3 Oxidative Medicine and Cellular Longevity with the primary antibodies at 4°C overnight. After washing in PBS three times for 5 min, sections were incubated with corresponding secondary antibodies in the dark at 37°C to visualize the fluorescent signal. The nuclei were visualized using DAPI staining solution for 1 h without light. While coverslips were mounted, stained sections were visualized and photographed under a microscope.
2.10. Enzyme-Linked Immunosorbent Assay (ELISA). The contents of TGF-β1, IL-1β, and IL-4 were detected by the corresponding ELISA kits. According to the manufacturer's instructions, supernatants of tissues were collected via lysis and centrifugation. Samples were added into the microplate stripe wells and then mixed with HRP-labeled antibody. Microplate was coated with microplate sealers and incubated at 37°C. After washing, substrate A and substrate B were added separately to each well to incubate in the dark and then supplemented with stop solution. The optical density at 450 nm was measured within 15 min using a microplate reader.
2.11. Griess Assay. The level of NO was detected using a Griess assay kit. Supernatant was collected and added into a 96-well plate. Solution I and solution II were added, mixed, and then incubated for 20 min without light. The optical density at 540 nm was measured using a microplate reader.
2.12. Statistical Analysis. All data in this paper were obtained using SPSS 20 software and described as the mean ± S:E:M: The significance of intergroup differences was determined by one-way ANOVA. The variance and multiple comparisons were evaluated by LSD-t. P < 0:05 was set as the standard for a significant difference.

H 2 S Attenuates the Cognitive Impairment in ROT-
Induced PD Rats. We explored the effect of H 2 S on the cognitive disorder in ROT-induced PD rats using the Y-maze, NOR, and MWM tests.
In the Y-maze test, NaHS (a donor of H 2 S) markedly increased the alternation performance in the ROT-exposed rats ( Figure 2(a)). In addition, there was no significant difference in the number of total entries among experimental groups (Figure 2 In the NOR test, there was no significant difference in the total time spent exploring the two objects among the five groups during the training phase (Figure 3(a)) as well as testing phase (Figure 3(c)). However, the discrimination index in the testing phase was decreased in the ROT-exposed rats and dramatically elevated by treatment with NaHS ( Figure 3(b)).
The MWM test was also used to explore the effect of H 2 S on the declined learning and memory in the ROT-induced PD rats. In the acquisition phase of the MWM test, the swimming routes for searching for the platform were tortuous and complicated for all groups on the first training day, while NaHS significantly simplified the search routes of ROT-exposed rats on the fifth day (Figure 4(a)). Furthermore, ROT treatment lengthened the latency to find the platform compared to the control group (Figure 4(b)), which was significantly reduced by NaHS supplementation (Figure 4(c)).
NaHS treatment alone showed no impact on the normal rats to find the platform (Figure 4(d)). In the probe trial phase of the MWM test, NaHS significantly increased the number across the target platform ( Figure 4(e)) and the time spent in the target quadrant ( Figure 4(f)) among the ROT-exposed rats. In the visible platform phase of the MWM test, no significant differences in the escape latency (Figure 4(g)) and the average speed (Figure 4(h)) were observed among groups.
Together, these findings indicated that H 2 S attenuates the cognitive impairment in the ROT-induced PD rats.

H 2 S Promotes Microglia
Polarization from M1 to M2 in the Hippocampus of ROT-Exposed Rats. Next, we assessed the effect of H 2 S on microglia polarization in the hippocampus of ROTexposed rats. The bright fluorescence of Iba1 in ROT-exposed rats exhibited a reduced state after NaHS treatment (Figures 5(a) and 5(b)), indicating the inhibitory role of H 2 S in microglia activation. The most important characteristic of microglia polarization is the phenotype alteration. We found that H 2 S inhibited M1 polarization of microglia, which was characterized by the decreased expression of iNOS ( Figure 5(c)) and the secretions of NO ( Figure 5(d)) and IL-1β ( Figure 5(e)) in the hippocampus of rats cotreatment with ROT and NaHS. More strikingly, H 2 S accelerated microglia polarization to M2 phenotype, featured with the upregulated protein expressions of Arg-1 ( Figure 5(f)) as well as Ym-1 ( Figure 5(g)) and the increased secretions of TGF-β1 ( Figure 5(h)) as well as IL-4 ( Figure 5(i)) in the hippocampus of rats cotreatment with ROT and NaHS. These data indicated that H 2 S promotes microglia M2 polarization in the hippocampus of ROT-induced PD rats.  Figure 6(e)) in the hippocampus of ROT-exposed rats. Moreover, the content of lactate decreased in the hippocampus of ROT-treated rats, which was increased by treatment with NaHS ( Figure 6(f)). Taken together, these findings indicated that H 2 S enhances the Warburg effect in the hippocampus of ROT-treated rats.

2-DG Prevents the Upregulatory Role of H 2 S in the
Hippocampal Warburg Effect of ROT-Exposed Rats. To investigate whether inhibited the hippocampal Warburg effect reverses the protection of H 2 S against ROT-induced decline in cognitive function, we first investigated whether 2-DG (an inhibitor of the Warburg effect) [34,35] abolished H 2 S-enhanced Warburg effect in the hippocampus of ROTexposed rats. 2-DG dramatically ameliorated NaHS-elicited upregulation in the expressions of HKII (Figure 7 Oxidative Medicine and Cellular Longevity NaHS in enhancing the hippocampal lactate level of ROTexposed rats (Figure 7(f)). These data indicated that 2-DG prevents H 2 S-enhanced Warburg effect in the hippocampus of ROT-induced PD rats.

Inhibited Hippocampal Warburg Effect Reverses H 2 S-Attenuated Cognitive Dysfunction of PD Rats.
To confirm the mediatory role of hippocampal Warburg effect in H 2 S-promoted cognitive function of PD rats, we further explored whether inhibited hippocampal Warburg effect by 2-DG abolishes H 2 S-promoted cognitive function in the ROT-treated rats.
In the Y-maze test, 2-DG dramatically reversed the upregulatory effect of NaHS on the alternative performance of ROT-treated rats (Figure 8(a)). The total entries showed no significant difference among the groups (Figure 8(b)).
In the NOR test, 2-DG greatly decreased the recognition index of rats cotreated with ROT and NaHS (Figure 9(b)). There was no significant difference in the total time spent in exploring objects (Figures 9(a) and 9(c)).
In the acquisition phase of the MWM test, 2-DG significantly complicated the swimming routes ( Figure 10(a)) and extended the latency to find the platform in the rats cotreated  Taken together, suppression in the hippocampal Warburg effect abrogated the protection of H 2 S against the cognitive impairment in ROT-exerted PD rats.

Inhibited Hippocampal Warburg Effect Reverses H 2 S-Promoted Microglia M2
Polarization in the Hippocampus of ROT-Induced PD Rats. Next, we explored whether inhibited hippocampal Warburg effect by 2-DG reverses H 2 S-promoted microglia M2 polarization in the hippocampus of ROTinduced PD rats. Our data showed that 2-DG increased the fluorescence intensity of Iba-1 (Figures 11(a) and 11(b)) in the rats cotreated with ROT and NaHS, indicating that inhibited hippocampal Warburg effect by 2-DG restored the microglia activation. Additionally, 2-DG supplementation led to increases in the expression of iNOS (Figure 11(c)) as well as the contents of NO (Figure 11(d)) and IL-1β (Figure 11(e)) in the rats cotreated with ROT and NaHS, indicating that inhibited hippocampal Warburg effect by 2-DG enhanced M1 polarization. Conversely, 2-DG reduced the expressions of Arg-1 (Figure 11(f)) and Ym-1 (Figure 11(g)) as well as the contents of TGF-β1 (Figure 11(h)) and IL-4 ( Figure 11(i)) in the rats

Discussion
How to effectively ameliorate cognitive impairment is a huge problem that needs to be solved to improve the life quality of PD patients. In this study, we demonstrated that H 2 S   Figure 8: Effect of 2-DG on H 2 S-ameliorated cognitive impairment of ROT-exposed rats in the Y-maze test. After pretreatment with NaHS (100 μmol/kg, i.p.) for 1 w, SD rats were injected with ROT (2 mg/kg, s.c.) for 5 w. In the 3 rd week of ROT injection, rats were cotreated with NaHS (100 μmol/kg, i.p.) and 2-DG (3 μg/w, i.c.v.) for 3 w simultaneously. All rats were subjected to the Y-maze test. The alternation performance (a) and arm entries (b) over the course of 5 min were analyzed. Values are presented as the mean ± S:E:M: (n = 9 -15). * P < 0:05 vs. control group; ## P < 0:01 vs. ROT treatment alone group; && P < 0:01 vs. ROT and NaHS cotreatment groups.  Figure 9: Effect of 2-DG on H 2 S-ameliorated cognitive impairment of ROT-induced PD rats in the NOR test. After pretreatment with NaHS (100 μmol/kg, i.p.) for 1 w, SD rats were injected with ROT (2 mg/kg, s.c.) for 5 w. In the 3 rd week of ROT injection, rats were cotreated with NaHS (100 μmol/kg, i.p.) and 2-DG (3 μg/w, i.c.v.) for 3 w simultaneously. All rats were subjected to the NOR test. The total time in the training period (a) and recognition index (b) as well as the total time (c) in the test period over the course of 5 min were recorded. Values are presented as the mean ± S:E:M: (n = 9 -15). * * P < 0:01 vs. control group; # P < 0:05 vs. ROT treatment alone group; & P < 0:05 vs. ROT and NaHS cotreatment groups. PD is one of the most familiar neurodegenerative diseases in the clinic, featuring motor and nonmotor disorders [36,37]. Dementia, a nonmotor symptom, develops in the early stage of PD. Increasing studies using animal models have verified that H 2 S recovers the number of dopaminergic neurons to normal and mitigates motor disorders [38,39], identifying H 2 S as a new hope for the treatment of PD [40]. As a novel gaseous signaling molecule, H 2 S has the capacity to attenuate deficits in learning and memory induced by risk factors [16,17,41]. Accordingly, our study is aimed at investigating whether H 2 S protects against the cognitive impairment in ROT-induced PD rats. In the Ymaze test, we found that NaHS supplementation markedly increased alternation performance in ROT-exposed rats, suggesting a benefit of H 2 S to the cognitive function of ROT-exposed rats. In the NOR test, the declined recognition index in ROT-treated rats was reversed by the administration of NaHS, which indicated that H 2 S ameliorates deficits in learning and memory in ROT-treated rats. In the MWM test, NaHS greatly shortened the escape latency in the acquisition phase and increased the number of crossing platform as well as the time spent in the target quadrant in the probe trial of ROT-exposed rats, which further suggested that the impairment in spatial learning and memory in ROTtreated rats is reversed by H 2 S. Collectively, our data demonstrate that H 2 S prevents cognitive dysfunction in ROTinduced PD rats. It has demonstrated that H 2 S reduces neurodegeneration in MPP + -, ROT-, and 6-hydroxydopamine (6-OHDA)-caused PD models [42][43][44]. Thus, we believe that H 2 S holds promise for providing therapeutic benefits in the cognitive impairment of PD.
Microglia is the first line of defense of the central nervous system in the resting condition; microglia not only provide surveillance but also respond to danger signals [45]. Activated microglia exhibits two different statuses, M1 and M2. A previous study revealed microglia activation in the hippocampus of PD patients [46]. Furthermore, microglia play a crucial role in neuronal plasticity and neurogenesis [19,47]. The hippocampus, one of limbic system structures, is well known in learning and memory. It also has been reported that hippocampus lesion contributes to memory dysfunction [4,5], and reversing hippocampal volume loss improves memory function [6]. Cognitive impairment and hippocampal atrophy are hallmark features in PD patients, and hippocampal atrophy is reported to predict PD dementia [48,49]. Microglia polarization is known to influence cognitive function and has therefore become a main player in neurodegenerative diseases leading to dementia. It has been reported that young offspring rats with cognitive impairment after maternal sleep deprivation exposure exhibit increase in microglia activation and M1 phenotype of microglia as well as a decrease in M2 phenotype of microglia [50]. Suppressing microglia M1 polarization in the hippocampus is an important mechanism in protecting against chronic unpredictable mild stress-induced cognitive disturbance [51]. Similarly, promoting microglia M2 polarization is able to ameliorate cognitive deficits in AD mice [52]. Inspiringly, inhibition of hippocampal microglial activation alleviates cognitive impairment in a PD model [53]. Therefore, the microglia polarization in the hippocampus was focused in the cognitive decline of PD. There are increased activation and M1 phenotype of microglia and decreased M2 phenotype of microglia in the hippocampus of ROT-treated rats. Therefore, we suggested that the cognitive deficit in PD model is also closely related to increased M1 polarization and reduced M2 polarization of microglia in the hippocampus. Notably, supplementation of NaHS significantly promoted hippocampal microglia M2 polarization in ROT-induced PD rats. Moreover, enhancing endogenous H 2 S via overexpression of cystathionine β-synthase promotes M2 polarization in lipopolysaccharide- [54] or ROT- [26] stimulated microglia. Similarly, exogenous H 2 S application with NaHS evokes the M2 polarization of ROT-exposed primary microglia [26]. Collectively, our data 14 Oxidative Medicine and Cellular Longevity suggest the critical role of promoting hippocampal microglia M2 polarization in H 2 S-exerted protection against the cognitive dysfunction in PD. Microglia polarization occupies an important role in cognitive function and it is worth exploring deeply. Our future studies in this exciting and growing field will continue to reveal the effects of microglia in other brain regions on the cognitive impairment in PD. The Warburg effect, or aerobic glycolysis, is an energy metabolism shift to glycolysis under aerobic conditions. This phenomenon was first discovered in cancer by Otto Warburg [55]. Besides supplying energy quickly, the Warburg effect serves as a vital characteristic of nontumor disease, especially in neurodegenerative disease [56,57]. Increasing the expression of HKII attenuates ROT-induced cell death [58]. Corona et al. confirmed that an elevated Warburg effect in the brain reduces the amount of dopaminergic neuron death and improves PD symptoms [59]. More importantly, the Warburg effect maintains LTP and regulates synaptic reconstruction, resulting in an enhancement in learning and memory [30,32]. Thus, there is a scientific research value to elucidate the mechanism underlying H 2 Sattenuated cognitive impairment in the ROT-exposed rats  Figure 11: Effect of 2-DG on H 2 S-promoted microglia M2 polarization in ROT-exerted PD rats. After pretreatment with NaHS (100 μmol/ kg, i.p.) for 1 w, SD rats were injected with ROT (2 mg/kg, s.c.) for 5 w. On the 3 rd w of injecting ROT, rats were cotreated with NaHS (100 μmol/kg, i.p.) and 2-DG (3 μg/w, i.c.v.) for 3 w simultaneously. Representative images of activated microglia (a). Quantitation of mean fluorescence intensity (MFI) of Iba1 (b). The nuclei were stained by DAPI (blue), and the activated microglia was stained by Iba1 (red). Magnification, 20x. The expressions of iNOS (c), Arg-1 (f), and Ym-1 (g) were measured by western blot. The level of NO (d) was measured by the Griess assay kit. The contents of IL-1β (e), TGF-β1 (h), and IL-4 (i) were detected by ELISA. Values are presented as the mean ± S:E:M: (n = 3). * P < 0:05, * * P < 0:01, and * * * P < 0:001 vs. control group; # P < 0:05, ## P < 0:01, and ### P < 0:001 vs. ROT treatment alone group; & P < 0:05 and && P < 0:01 vs. the ROT and NaHS cotreatment groups.
from the new perspective of the hippocampal Warburg effect. We found that the expressions of HKII, PKM2, PDK-1, and LDHA and the content of lactate in the hippocampus of ROT-treated rats were decreased, while the expression of PDH was increased. All these data indicated the downregulation of the hippocampal Warburg effect in ROT-exposed rats. Recent evidence confirmed that downregulation of Warburg effect via dichloroacetate impairs the learning and memory in mice [60], suggesting a requirement of Warburg effect for spatial memory acquisition. Furthermore, Shannon et al. also support the contribution of brain Warburg effect to learning [61]. In the present work, we demonstrated that H 2 S increased the hippocampal Warburg effect in ROT-exposed rats and that inhibited Warburg effect reversed the protection of H 2 S against cognitive dysfunction in ROT-exposed rats. Collectively, enhancing hippocampal Warburg effect plays a crucial role in H 2 S-provided therapeutic benefits in the cognitive impairment of PD. Immune cells need different amounts of energy to exert various functions, and the metabolic pathways responsible for energy generation are determined by cellular phenotypes [62]. Under the classical inflammatory activation process (M1), microglia often undergo metabolic reprogramming toward aerobic glycolysis. In comparison, microglia with M2 status induced by IL-4 stimulation exhibit decreased lactate release and glucose consumption, suggesting reduced aerobic glycolysis [63,64]. 2-DG, an inhibitor of HKII, is widely used to downregulate the Warburg effect [65], which was also confirmed in our study. More interestingly, 2-DG blunts the release of M1 polarization-associated markers TNF-α and IL-6 in primary microglia [66]. Given the link between microglia polarization and energy metabolism, whether the Warburg effect-mediated M2 microglia polarization contributes to H 2 S-produced neuroprotection in cognitive disorder is worth to research. From our data, 2-DG reversed the reduced M1 markers and the increased M2 markers in the hippocampal microglia by NaHS in ROTinduced PD rats, representing a microglia transition from M2 to M1. Additionally, 2-DG dramatically reversed H 2 S the attenuated cognitive deficits in the ROT-induced PD rats. In summary, our findings verified that H 2 S enhances the hippocampal Warburg effect to accelerate microglia polarization toward M2, which consequently ameliorates cognitive impairment.
Taken together, our findings demonstrated that H 2 S produces a protective response to the cognitive dysfunction of PD as a result of elevated microglia M2 polarization via enhancing the hippocampal Warburg effect. This study elucidates a previously unrecognized mechanism in the antagonistic effect of H 2 S on cognitive deficits and thereby provides a strong basis for the development of therapeutics targeting H 2 S bioavailability or the Warburg effect in the treatment of comorbid cognitive disorder and PD that is refractory to current drugs. Additionally, it is necessary to make effort to clarity the pathologic alteration in Parkinson's cognitive dysfunction. We will pay more attention to the relationship between pathologic alteration and microglia activation in our next step.

Data Availability
The datasets analyzed during the current study are available from the corresponding authors upon reasonable request.

Ethical Approval
All experiments strictly complied with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Animal Use and Protection Committee of the University of South China.

Conflicts of Interest
The authors declare that they have no competing interests.