Synergistic effects of lipopolysaccharide and rotenone on dopamine neuronal damage in rats

Abstract Introduction The etiology of Parkinson's disease (PD) is still unknown. Until now, oxidative stress and neuroinflammation play a crucial role in the pathogenesis of PD. However, the specific synergistic role of oxidative stress and neuroinflammation in the occurrence and development of PD remains unclear. Methods The changes in motor behavior, dopamine (DA) neurons quantification and their mitochondrial respiratory chain, glial cells activation and secreted cytokines, Nrf2 signaling pathway, and redox balance in the brain of rats were evaluated. Results Lipopolysaccharide (LPS)‐induced neuroinflammation and rotenone (ROT)‐induced oxidative stress synergistically aggravated motor dysfunction, DA neuron damage, activation of glial cells, and release of related mediators, activation of Nrf2 signaling and destruction of oxidative balance. In addition, further studies indicated that after ROT‐induced oxidative stress caused direct damage to DA neurons, LPS‐induced inflammatory effects had stronger promoting neurotoxic effects on the above aspects. Conclusions Neuroinflammation and oxidative stress synergistically aggravated DA neuronal loss. Furtherly, oxidative stress followed by neuroinflammation caused more DA neuronal loss than neuroinflammation followed by oxidative stress.

the occurrence of various biological functions. 3 When the redox balance in the body is destroyed, an imbalance in the biochemical processes that produce and remove reactive oxygen species (ROS), known as antioxidant cascade reactions, occurs. When excess ROS accumulates, the oxidative stress response in cells is activated. 4 As an important organelle to maintain the normal function of cells, the mitochondria are injured by oxygen toxicity by a large amount of ROS in the state of oxidative stress, resulting in respiratory chain dysfunction. 5 Mitochondrial respiratory chains are located on the mitochondrial inner membrane and consist of five complexes, in which electron leakage at mitochondrial complex I releases individual electrons to oxygen and further produces superoxide radicals, a process exacerbated by damage to mitochondrial function. 6,7 Inhibition of mitochondrial complex I activity in DA neurons leads to the increased ROS production and subsequent oxidative damage to proteins, lipids, and DNA. 8 NADH: Ubiquitin oxidoreductase core subunit S3 (NDUFS3), a subunit of mitochondrial complex I, is introduced into the mitochondria after nuclear transcription, and this process is down-regulated in dysfunctional mitochondria. 9 Rotenone (ROT) is an inhibitor of mitochondrial NADH dehydrogenase, a commonly used natural insecticide that is highly lipophilic and easily penetrates the blood-brain barrier (BBB) into the brain. It has been confirmed that rats exposed to ROT for a long time showed the main characteristics of PD, including loss of DA neurons and motor dysfunction, which is now widely used to induce mammalian PD models. 10,11 Due to the inherent threat of oxidative stress, mitochondria are often associated with antioxidant defense mechanisms to protect their redox environment. This task is performed by superoxide dismutase (SOD), an antioxidant function that prevents neurodegenerative changes in animal models of PD. 12,13 Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that is sensitive to redox states and maintains cellular redox homeostasis by regulating transcription of antioxidant response elements and expression of antioxidant defense enzymes. 14,15 Under normal circumstances, Nrf2 binds to Kelch-like ECH-Associated Protein 1(Keap1) in the cytoplasm and is not actively transported into the nucleus. However, Nrf2 is stimulated with elevated ROS levels and the stability of binding to Keap1 is disrupted, causing its release and transfer to the nucleus of the cell. 16 Antioxidant response element (ARE) is a cis-acting element. 17 After entering the nucleus, Nrf2 binds to ARE and transcribes downstream genes through relevant activation procedures, thereby activating the expression of heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1), generating antioxidant activity. 18 Therefore, Nrf2 pathway is considered to be an effective way to regulate ROS. Currently, neuroprotective pathways regulated by Nrf2 in astrocytes of the brain have been reported to prevent neuronal oxidative stress. 19 Astrocyte is the ubiquitous glial cell in the brain that regulates water transport and blood flow in the brain.
In addition, various neurotrophic molecules, such as glial cell linederived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), are also produced, which are particularly important for the development and survival of DA neurons. 20 Furthermore, BDNF is also essential for neuronal development, survival, and synaptic plasticity. 21 Lipocalin 2 (LCN2) is considered to be a chemokine inducer of astrocytes and a classical pro-inflammatory activation autocrine promoter. 22 Compared with the A2 phenotype, which polarizes to protect neurons during hypoxia, A1-phenotype astrocytes not only lose the ability to promote neuronal survival, growth, synaptic formation, and phagocytosis but also directly kill DA neurons by secreting neurotoxic complement 3d (C3d). 23,24 Neuroinflammation is initially a protective response of the brain.
It is regulated by immune cells, cytokines, and chemokines. However, excessive neuroinflammation is toxic and could inhibit the regeneration of DA neurons. 25 Lipopolysaccharide (LPS), as a bacterial endotoxin and glial cell activator, not only promoted the aggregation of DA neuronal loss, but also stimulated the activation and release of ROS by glial cells to mediate neuroinflammation. [26][27][28] Microglia is the resident immune cell in CNS. Normally, microglia play a key role in maintaining tissue homeostasis and promoting brain development, while activated microglia and subsequent release of pro-inflammatory factors are closely related to death of DA neurons in the brain of PD patients. 29 The activated microglia consist of two groups of cells with different or even opposite functions. 30 The designation of microglia anti/pro-inflammatory status was originally developed according to the two main differentiation subtypes of T cells (Th1/Th2), based on which activated microglia could form more anti/pro-inflammatory phenotypes, referred to as M1 or M2, respectively. 23 Among them, M1 is a classically activated pro-inflammatory phenotype, secreting pro-inflammatory factors, such as tumor necrosis factorα (TNFα) and interleukin-1β (IL-1β). The accumulation of a large number of proinflammatory factors would increase the damage of DA neurons. M2 is an alternately activated anti-inflammatory phenotype, secreting anti-inflammatory-related factors, such as interleukin-10 (IL-10) and arginase-1 (Arg1). The production of anti-inflammatory factors could reduce the damage of DA neurons. 31 To sum up, oxidative stress and neuroinflammation play a crucial role in the pathogenesis of PD, and inducible factors of these two events are also widely present in daily life. Our previous studies revealed that peripheral inflammatory stimulation induced by LPS and neurotoxicity caused by ROT co-aggravated the damage of DA neurons. 32

| Animals and treatment
Adult male Sprague-Dawley rats (220-260 g) were purchased from the Liaoning Long Life Biotechnology Co., Ltd. LPS-or ROT-induced rat PD model was applied in this study. 33,34 All animal experiments were performed in accordance with Chinese Guidelines of Animal Care and Welfare and the present study was approved by the Animal Care and Use Committee of Zunyi Medical University. All animals were housed in a temperature (19-25°C) and humidity (40%-70%) environment and given access to food and water ad libitum. The experimental animals were randomly divided into 6 groups: control, LPS (1 mg/kg/d), ROT (0.5 mg/kg/d), LPS + ROT (simultaneous administration), LPS→ROT, and ROT→LPS (sequent administration) groups. LPS (1 mg/kg/d) was injected intraperitoneally for 4 consecutive days. ROT (0.5 mg/kg/d) was subcutaneously injected six times a week for consecutive 4 weeks. The detail of specific grouping method is shown in Figure 1. After the end of the administration, behavioral tests were performed uniformly and then animals were sacrificed.

| Rotarod test
Rotarod test is a common test used to evaluate the effects of drugs on animal behavior dysfunction. Before the experiment, all the rats were trained on the rotating rod until they remained on the rod for at least the specified time. The starting speed of the test was 10 rpm, increasing by 5 rpm every 30 s until rats fell off the rotating rod. The behavior changes in rats were analyzed by recording the time that each rat stayed on the rod.

| Open field test
Open field test is a sensorimotor test used to determine the general activity level, total motor activity and exploratory habits of rodent models of neurological disorders. In this study, each rat was placed in a separate open field area, and rat behavioral parameters were recorded within 10 min. Before each round of test, the equipment was cleaned with 75% alcohol solution to avoid odor interference. After the experiment, the total moving distance of rats was calculated.

| Tissue preparation and immunofluorescence staining
After the behavioral tests, anesthetized rats were perfused with PBS before fixation with 4% paraformaldehyde. The brain was peeled off and fixed with 4% paraformaldehyde for 1 week. Within dehydration and embedding into paraffin blocks for subsequent dyeing, the wax block was placed on a paraffin microtome (Leica) and the brain was cut into a six microns cross-section attached to a glass slide.
Brain slices were separately treated with 3% hydrogen peroxide and 0.1 M citrate buffer and blocked with goat serum. These slices were F I G U R E 1 Grouping and time schedule of LPS and ROT administration.

| Immunohistochemical staining
The slices attached to the brain tissue were dried and treated with 3% hydrogen peroxide and 0.1 M citrate buffer, and sealed with goat serum in an oven at 37°C. The slices were incubated overnight at 4°C with the following primary antibodies: GFAP (1:300, the marker of astrocytes) and Iba-1 (1:200, the marker of microglia). Brain sections were incubated with secondary antibody working solution at 37°C for 20 min followed by incubation with biotin for 18 min and developed with DAB developer. Five rats in each group were used for detection, and 2-3 brain slices were taken from each rat for measurement and the mean value was calculated. The images of GFAPpositive astrocytes and Iba1-positive microglia were presented by an Olympus microscope, and then the density was analyzed by ImageJ software and the average value was taken.

| Western blot analysis
The total protein was extracted from rat midbrain tissue with lysate containing protease inhibitor. Nucleoplasmic protein extraction kit (Solabio) was used to extract nuclear and cytoplasmic parts. The pro-

| Real-time RT-PCR assay
Total RNA was extracted with Trizol reagent and purified by RNeasy kit. Nrf2, NQO1, Keap1, HO1, and β-actin genes were amplified using forward and reverse primers. SYBR Green Supermix was used to perform real-time RT-PCR in accordance with the instructions, and then the CFX96 real-time RT-PCR detection system (Bio-Rad) was applied. The target gene expression level was normalized withβactin expression level. The control group was set to 100%.

| Malondialdehyde (MDA) and SOD assays
The content of MDA was determined with a lipid peroxidation MDA detection kit (Beyotime) and calculated with a standard curve according to the manufacturer's instructions in kit. Total SOD detection kit WST-8 (Beyotime) was used to detect SOD activity and the calculation was performed according to the manufacturer's instructions. All experiments were repeated three times independently.

| Statistical analysis
Data were presented as mean ± SEM. The control group was set as 100% and the differences between the other groups and control group were compared. Statistical comparison used SPSS statistical software for one-way analysis of variance (ANOVA) and normal distribution test. When analysis of variance showed significant differences, all pairwise comparisons among means were accessed by Bonferroni's post hoc test with correction. All data were tested for normality. Data that did not exhibit a normal/Gaussian distribution were analyzed by non-parametric equivalents. A value of p < 0.05 was considered statistically significant.

| Effects of different sequential administration of LPS and ROT on motor dysfunction and DA neuronal damage in rats
To investigate whether LPS and ROT had initiating or promoting factors in the pathogenesis of PD, effects of different sequential administration of LPS and ROT on rat behavior changes and DA neuronal damage were observed. First, as shown in Figure 2A, the time of rats stayed on rod in LPS alone and ROT alone groups was shorter than that in control group. Compared with LPS group, time of rats stayed on rod in LPS + ROT group was shorter than that in LPS group. In addition, compared with LPS + ROT group, time of rats stayed on rod in ROT→LPS and LPS→ROT groups were shortened, in which time in ROT→LPS group was much shorter than that in LPS→ROT group.
However, in open field test ( Figure 2B), an obvious decrease was just in ROT→LPS group.
To further investigate the effects of different sequential administration of LPS and ROT on DA neuronal loss in rats, the quantification of TH-positive DA neurons in rat SN and the expression of TH protein were detected. As shown in Figure 2C, compared with single LPS or ROT group, the number of DA neurons in rat midbrain of LPS + ROT group was reduced. Compared with LPS + ROT group, DA neuronal number in ROT→LPS group was apparently decreased, whereas no significant difference of DA neurons between LPS + ROT and LPS→ROT groups was discerned. Similar results were exhibited in TH protein expression detection ( Figure 2D, Figure S1).
These results indicated that ROT and LPS synergistically aggravated DA neuronal damage of rats, and the later application of LPS could more effectively promote the damage of ROT to DA neurons.

| Effects of different sequential administration of LPS and ROT on mitochondrial respiratory chain of DA neurons in rats
Next, we verified the effects of different sequential administration of LPS and ROT on the mitochondrial respiratory chain of DA neurons in rat SN. First, TH and NDUFS3 protein expressions in rat midbrain were double-stained by double-labeled immunofluorescence. Compared with control group, the expression of NDUFS3 protein in ROT group was decreased. Compared with the single LPS or ROT administration group, the expression of NDUFS3 protein in LPS + ROT group was down-regulated. Furthermore, compared with LPS + ROT group, NDUFS3 protein expression in ROT→LPS group was reduced, while no significant difference was shown in LPS→ROT group ( Figure 3A). In addition, the protein expression levels of NDUFS3 and SDHA in rat midbrain were determined. Similar phenomenon was discerned in NDUFS3 protein detection but not in SDHA ( Figure 3B, Figure S1).

| Effects of different sequential administration of LPS and ROT on microglia activation and secretion of cytokines in rat midbrain
Since glial cells were involved in neuroinflammatory response, microglia activation and the production of various cytokines were assessed. As shown in Figure 4A of Iba-1 immunohistochemical staining analysis, compared with control group, single LPS-or ROTinduced microglia activation. However, no obvious difference of microglia activation between LPS /ROT and LPS + ROT groups was indicated. Furthermore, compared with LPS + ROT group, apparent microglia activation was demonstrated in ROT→LPS and LPS→ROT groups, where ROT→LPS induced more microglia activation than LPS→ROT. In addition, similar results were present in Iba-1 protein level detection ( Figure 4B, Figure S1).
Furtherly, the protein levels of M1-type microglia proinflammatory factors, such as IL-1β and TNFα, and M2-type microglia anti-inflammatory factors, such as IL-10 and Arg1, were tested. As shown in Figure 4C and Figure S1, the increase in pro-inflammatory factors (IL-1β and TNFα) in ROT→LPS group was the most obvious. However, there was no significant difference in increase of

F I G U R E 2 Effects of different sequential administration of LPS and ROT on motor dysfunction and DA neuronal damage in rats. Rotating rod test (A) and open field test (B) were performed on rats after LPS and ROT administration. (C) TH-positive neurons number was counted after rat midbrain sections immunofluorescence staining (Scale bar = 200 μm). (D) TH protein expression was detected by Western blot
assay. Data were represented as mean ± SEM from five rats. *p < 0.05 compared with control group, # p < 0.05 compared with LPS group, ω p < 0.05 compared with LPS + ROT, Ψ p < 0.05 compared with LPS→ROT group. anti-inflammatory factors (IL-10 and Arg1) among different groups.
These results indicated that ROT combined with LPS to aggravate microglia activation and pro-inflammatory cytokine secretion, and after ROT destroyed the redox balance, LPS provided inflammatory stimulation to further promote microglia activation and proinflammatory cytokine secretion.

| Effects of different sequential administration of LPS and ROT on astrocytes activation and release of cytokines in rat midbrain
In addition to microglia, astrocytes participated in DA neurodegeneration. Immunohistochemical staining analysis ( Figure 5A) showed that, compared with control group, single LPS or ROT did not induce the activation of astrocytes but LPS + ROT elicited astrocytes activation. Compared with LPS + ROT group, both LPS→ROT and ROT→LPS administration induced obvious astrocytes activation, where ROT→LPS induced more astrocytes activation than LPS→ROT. GFAP protein level measurement ( Figure 5B, Figure   S1) was consistent with results of immunohistochemical staining.
Moreover, compared with LPS→ROT group, ROT→LPS caused more A1-type astrocytes neurotoxic factors, such as LCN2 and C3 release, and less A2-type astrocytes neurotrophic factors, such as BDNF and GDNF release ( Figure 5C, Figure S1).

| Effects of different sequences of administration of LPS and ROT on brain oxidation balance and activation of Nrf2 signaling in rat midbrain
From the above results, we found that ROT→LPS caused more DA neuronal damage than LPS→ROT. It is well-known that ROT is closely associated with redox balance. Next, the effects of different sequences of administration of LPS and ROT on brain oxidation balance and activation of Nrf2 signaling in rat midbrain were explored. First, as shown in Figure 6A, MDA and SOD levels detection indicated that compared with control group, LPS + ROT and ROT alone not LPS alone caused increase in MDA and decrease in SOD.
Compared with LPS + ROT group, more increase in MDA and more decrease of SOD were displayed in ROT→LPS group than those in LPS→ROT group. Second, activation of Nrf2 signaling in rat midbrain was analyzed. As shown in Figure 6B Figure S1) and also more protein expressions of HO-1, Keap1, and NQO1 ( Figure 6D, Figure S1).

| DISCUSS ION
In the present study, the changes in motor behavior, DA neurons quantification, and their mitochondrial respiratory chain, glial cells activation and secreted cytokines, Nrf2 pathway, and redox balance in the brain of rats were evaluated. It was found that LPS- Furthermore, the progressive loss of DA neurons in SN is the major feature of PD, and the cellular mitochondrial complex I NDUFS3 was introduced into the mitochondria after nuclear transcription, and this process was down-regulated in dysfunctional mitochondria. 9 The present study found that the expression of NDUFS3 was decreased along with the aggravation of midbrain DA neuron injury.
Since the pathogenesis of PD is very complex and has not yet been clarified, oxidative stress and neuroinflammation, as the important factors that induce PD development, have attracted extensive attention. ROT is a highly lipopilic pesticide, which plays its function as a mitochondrial complex I inhibitor in brain after long-term exposure, including damaging the normal function of DA neurons,

F I G U R E 4 Effects of different sequential administration of LPS and ROT on microglia activation and secretion of cytokines in rat midbrain. (A)
The activated microglia in the substantia nigra of rat midbrain were detected by immunocytochemical staining with anti-Iba-1 antibody. The density of activated microglia was recorded. (B) Iba-1 protein expression was tested by Western blot assay. (C) IL-1β, TNFα, IL-10, and Arg1 protein expressions was determined by Western blotting. Data were represented as mean ± SEM from 5 rats. *p < 0.05 compared with control group, # p < 0.05 compared with LPS group, △ p < 0.05 compared with ROT group, ω p < 0.05 compared with LPS + ROT group, Ψ p < 0.05 compared with LPS→ROT group. increasing the production of ROS, causing imbalance of redox in brain, and further inducing oxidative stress damage to neurons. 36 ROT is a typical example of exogenous toxins simulating the clinical and pathological characteristics of PD in animal models and is widely used in the construction of PD animal model to explore the therapeutic effects of drugs on the oxidative stress pathway of PD. [37][38][39] LPS is an endotoxin of gram-negative bacteria, which is widely used in investigating the role of the inflammatory process in PD and the anti-inflammatory treatment of PD. [40][41][42] Therefore, this study induced oxidative stress and inflammatory state in rat brain by introducing ROT and LPS, respectively. Furthermore, M2-type microglia could play an important role in brain environment monitoring, cell maintenance and innate immunity. Alternately, activated M1-type microglia release a large number of neurotoxic and pro-inflammatory factors to damage DA neurons, which in turn damaged DA neurons generated neurotoxic factors, such as damage-associated molecular patterns (DAMPs), further resulting in microglia activation and forming a self-amplification cycle of neuronal damage and microglia activation and thus leading to more DA neuronal death. 43 Studies have shown that inhibiting the polarization of microglia to M1 phenotype could alleviate neuroinflammation. 44,45 However, this study found that with the aggravation of ROT combined with LPS-caused damage, the secretion of anti-inflammatory factors of microglia in the midbrain of rats was increased similar to that of pro-inflammatory factors, indicating that the activation states of microglia were more dynamic and varied between the two extreme states of M1 and M2-type microglia. 46 Although the mechanisms of oxidative stress-and neuroinflammation-induced PD were different, it was not comprehensive to discuss these two events separately.
Recent studies revealed that over-structured ROS promoted the polarization of microglia to M1 phenotype. [47][48][49][50] Here and GDNF protein expressions were tested by Western blot assay. Data were represented as mean ± SEM from 5 rats. *p < 0.05 compared with control group, # p < 0.05 compared with LPS group, △ p < 0.05 compared with ROT group, ω p < 0.05 compared with LPS + ROT group, Ψ p < 0.05 compared with LPS→ROT group. are widely used determine the redox state in brain. 51,52 This study found that ROT and LPS synergistically increased MDA level, especially first stimulation of ROT followed by LPS-induced apparent increase in MDA level, while SOD level detection were opposite. In addition, as a major regulator of oxidative stress, Nrf2 could regulate the redox state of cells in response to oxidative stress. 53 Normally, Nrf2 is located in the cytoplasm of DA neurons in SN. 54 In patients with PD, Nrf2 is activated and enters the nucleus, and the expressions of NQO1 and HO-1 in Nrf2 downstream pathway are subsequently up-regulated. 55   are several limitations in this study. First, the time-course study on both LPS or ROT alone administration and LPS combined with ROT application would provide more detail information. Second, why oxidative stress followed by neuroinflammation damaged more DA neurons than neuroinflammation followed by oxidative stress was unrevealed. Thus, the underlying mechanisms warrant further exploration. This study provides a baseline for understanding the crosstalk between neuroinflammation and oxidative stress on PD pathogenesis.

| CON CLUS IONS
This study demonstrated that neuroinflammation combined with oxidative stress aggravated DA neuronal loss. Furtherly, oxidative stress followed by neuroinflammation caused more DA neuronal loss than neuroinflammation followed by oxidative stress.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data that support the findings of this study were available from the corresponding author upon reasonable request.