Penehyclidine hydrochloride alleviates LPS-induced inflammatory responses and oxidative stress via ROS/Nrf2/HO-1 activation in RAW264.7 cells

Background. Inflammation is a biological response of the immune system to harmful stimuli. Penehyclidine hydrochloride (PCH) can alleviate inflammation and oxidative stress by activating reactive oxygen species (ROS), nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase 1 (HO-1) in animal models, but there is a lack of cellular evidence. Objectives. This study investigated the effects of PHC on lipopolysaccharide (LPS)-induced inflammation response and oxidative stress in RAW264.7 cells. Materials and methods. RAW264.7 cells were treated with 1 μg/mL or 5 μg/mL of PHC, with interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-1β, and prostaglandin E2 (PGE2) levels measured with enzyme-linked immunosorbent assay (ELISA) and nitric oxide (NO) measured using the Griess test. Reactive oxygen species were examined with flow cytometry and immunofluorescence, and b-related factor 2 (BRF-2) and NAD(P)H-quinone oxidoreductase 1 (NQO1) using western blot. Results. Penehyclidine hydrochloride partly, but substantially, reversed LPS-related NO and PGE2 production by RAW264.7 cells in a dose-dependent manner and suppressed LPS-induced expression of IL-6, TNF-α and IL-1β messenger ribonucleic acid (mRNA), secretion of IL-6, TNF-α and IL-1β, and ROS production. Lipopolysac-charide stimulation did not affect Nrf2, heme oxygenase 1 (HO-1) or NQO1 protein expression in RWA264.7 cells not treated with PHC. However, PHC treatment significantly elevated Nrf2, HO-1 and NQO1 protein in LPS-treated RWA264.7 cells, an effect that was dose-dependent. The ROS scavenging using N-acetyl-L-cysteine abolished the PHC-induced upregulation of Nrf2 and HO-1. Conclusions. Penehyclidine hydrochloride may alleviate LPS-induced inflammation and oxidative stress by activating Nrf2 signaling in RAW264.7 macrophages. These findings suggest that PHC could alleviate inflammation by targeting activated macrophages.


Background
Inflammation represents a critical biological response of the immune system to harmful stimuli such as pathogenic organisms, injured cells and tissues, toxic molecules, and irradiation. 1Although inflammation is necessary to the normal healing process by promoting the disposal of injured cells and pathogenic organisms, 2,3 uncontrolled or excessive inflammation can trigger acute and/or chronic damage in multiple organs, leading to disease. 1,4Inflammation can even promote the growth of tumor cells and cancers. 5he cytokine storm resulting from acute excessive immune and inflammatory reactions is also involved in the morbidity and mortality of several conditions like sepsis and coronavirus disease-19 (COVID-19). 6,7Low-grade chronic inflammation is involved in conditions such as type 2 diabetes, obesity and hypertension, and contributes to the development of complications and associated diseases. 8,9ncontrolled inflammation is characterized by the overproduction of pro-inflammatory modulators, including nitric oxide (NO), prostaglandin E2 (PGE2) and cytokines, 10 playing a key role in the pathogenetic mechanisms of various chronic disorders, e.g., cardiovascular diseases, type 2 diabetes, rheumatoid arthritis, and cancers. 115][16][17][18] There is a functional interplay between the central and autonomic nervous systems, and prefrontal cortex disruption may contribute to irregular behavioral responses, leading to many cognitive dysfunctions common in neurological diseases. 19In this regard, some anti-inflammatory drugs (e.g., celecoxib and aspirin) or drugs with pleiotropic antiinflammatory effects (e.g., pioglitazone, statins and minocycline) function in bipolar disorder, major depressive disorder and schizophrenia, given that baseline inflammation is present. 20nflammation is a complex process involving several cell types and mechanisms, 1,21 with macrophages critical in its initiation, maintenance and resolution.Indeed, they can be activated and deactivated by different cytokines produced during inflammatory processes.Activating cytokines and signals include interferon gamma (IFNγ) granulocyte-monocyte colony-stimulating factor, tumor necrosis factor alpha (TNF-α), bacterial lipopolysaccharide (LPS), extracellular matrix proteins, and chemical mediators.On the other hand, deactivating cytokines and signals include interleukin (IL)-10, transforming growth factor beta (TGF-β), and the removal or deactivation of mediators. 22,23xidative stress contributes significantly to inflammatory reactions observed in chronic disorders, 24 involves cellular damage that can trigger immune cells and processes, 25,26 and occurs when the reactive oxygen species (ROS) exceed antioxidant capacity. 26Reactive oxygen species are normal by-products of adenosine triphosphate (ATP) production by the mitochondria, but can increase under pathogenic conditions. 27In addition, some immune cells can produce ROS to destroy pathogens and abnormal cells. 28Besides consuming antioxidant compounds, several innate antioxidant mechanisms exist in mammals. 29,30uclear factor erythroid 2-related factor (Nrf2) is a pleiotropic transcription factor that induces defense mechanisms counteracting oxidative stress and inflammation.During oxidative stress, Nrf2 upregulates multiple genes responsible for antioxidant activity, including heme oxygenase 1 (HO-1), which reduces oxidative stress by clearing heme, and NAD(P)H-quinone oxidoreductase 1 (NQO1), that prevents hydroquinone conversion to ROS. 31 Besides its role in oxidative stress, the Nrf2 activation mediates the anti-inflammatory features of some commonly used nonsteroidal anti-inflammatory drugs, 32 suggesting a contribution of Nrf2 in the crosstalk between inflammation and oxidative stress.It can also decrease the transcription of pro-inflammatory cytokines in activated macrophages. 33enehyclidine hydrochloride (C 20 H 29 NO 2 ⋅HCl; PHC) is an anticholinergic drug designed by the Chinese Academy of Military Sciences that exhibits substantial peripheral and central anticholinergic activities by simultaneously binding to the M and N cholinergic receptors. 34t is widely used in China as a reversal drug for treating organophosphorus poisoning and a pre-anesthetic medication to reduce respiratory secretion and vagus nerve reflex. 357][38] Furthermore, PHC can attenuate the Toll-like receptors in inflammatory chronic lung diseases 39 and acute lung injury, 40 and has been shown to decrease the postoperative expression of pro-inflammatory cytokines 41 and reduce cerebral injury in models of cardiopulmonary bypass by decreasing inflammation and neuronal apoptosis. 42Toxicity and adverse events associated with PHC are dry mouth, flushing and dry skin.High doses can cause dizziness, urinary retention, delirium, and elevated body temperature. 35tudies have shown that PHC induces Nrf2/HO-1 signaling in acute lung/kidney damage triggered by ischemia/ reperfusion or rhabdomyolysis in animal models, along with suppressing ROS and pro-inflammatory cytokine production, e.g., TNF-α, IL-6 and IL-1β. 36,43,44However, there is still a lack of cellular evidence of Nrf2/HO-1 pathway involvement in the anti-inflammatory and antioxidant activities of PHC.
Activated macrophages are a major source of pro-inflammatory mediators in the development of inflammation, 45 and LPS-stimulated murine RAW264.7 macrophages are widely used in inflammation research. 46n this study, we performed dose-response experiments on the anti-inflammatory and antioxidant activities of PHC and the PHC-induced alterations in Nrf2 signaling in LPS-treated RAW264.7 cells.The study provides cellular evidence of Nrf2 pathway involvement in the anti-inflammatory and antioxidant activities of PHC, serving as a potential therapeutic target for managing inflammatory diseases.

Objectives
This study aimed to investigate the effects of PHC on LPS-induced inflammatory responses and oxidative stress in RAW264.7 cells.

Cell Counting Kit-8 (CCK-8) assay
Cell viability was assessed using Cell Counting Kit-8 (CCK-8; Solarbio Life Science, Beijing, China) as directed by the manufacturer. 47RAW264.7 cells were cultured in 96-well microplates (Corning Company, Corning, USA) at a density of 5×10 3 /well.Cells were serum starved overnight and administered various PHC doses (0, 1, 5, 10, 20, and 40 μg/mL; Jinzhou Aohong Pharmaceutical Industry Co., Ltd., Jinzhou, China) for 24 h.Doses as high as 40 μg/mL have not been reported in the literature previously, and such a wide range of increasing doses was chosen for this study to verify the toxicity of PHC on RAW264.7 cells and provide a basis for dose selection for future studies.The results of cell viability showed that PHC concentrations ranging from 1 μg/mL to 10 µg/mL had no significant effect on RAW264.7 cell viability.Thus, we treated RAW264.7 cells with 1 μg/mL or 5 μg/mL PHC in the subsequent experiments.Cell Counting Kit-8 solution (10 µL) was added to each well, and absorbance (A) was measured at 450 nm after incubation for 1 h at 37°C using an Epoch microplate spectrophotometer (BioTek, Winooski, USA).Cell viability was calculated as (A-A0)/A0 × 100%.
Nitric oxide measurement using the Griess reagent RAW264.7 cells underwent the treatments outlined above.Secreted NO concentration was measured using an NO assay kit containing Griess reagent (Beyotime Biotechnology, Shanghai, China) as directed by the manufacturer. 48
The 2 -ΔΔCT method was utilized for data analysis.

Statistical analyses
The analysis employed GraphPad Prism 9 (GraphPad Software, San Diego, USA), with Fig. 1,2 using 6 data points and Fig. 3,4 using 3 data points.Differences in characteristics between groups were analyzed using the Kruskal-Wallis test with Dunn's post hoc tests, with p < 0.05 indicating statistical significance.

PHC suppressed LPS-associated NO and PGE2 production in RAW264.7 cells
In order to select the appropriate PHC treatment doses, we examined RAW264.7 cell viability after administering diverse PHC concentrations and found that PHC ranging from 1 μg/mL to 10 µg/mL had no marked effects (Fig. 1A and Supplementary Tables 1,2).Thus, the subsequent assays used 1 μg/mL or 5 µg/mL PHC to remain below the toxicity threshold.For evaluating PHC's effect on LPS-related inflammation, RAW264.7 cells were treated with various PHC concentrations in the presence of LPS.Lipopolysaccharide dramatically enhanced NO and PGE2 production by RAW264.7 cells (Fig. 1B,C and Supplementary Tables 3-6).Penehyclidine hydrochloride partly but substantially reversed LPS-related NO and PGE2 production in a dose-dependent manner, with the same trend observed for iNOS mRNA expression (Fig. 1D and Supplementary Tables 7,8).In addition, compared to untreated round-shaped RAW264.7 cells, LPS-stimulated cells became rod-shaped, bearing pseudopodia.Of note, the morphological alterations were alleviated by PHC treatment dose-dependently (Fig. 1E).The above findings indicated that PHC inhibited LPS-related inflammation in macrophages.

PHC suppressed LPS-induced expression and secretion of pro-inflammatory cytokines in RAW264.7 cells
To assess PHC's effect on LPS-associated inflammation, we determined the expression and secretion of proinflammatory cytokines in RAW264.7 cells administered LPS and PHC.As depicted in Fig. 2A-F and Supplementary Tables 9-20, LPS dramatically elevated IL-6, TNF-α and IL-1β mRNA amounts and secretion, and these effects were partially but significantly attenuated by PHC dose-dependently.The above data suggested that PHC suppressed LPS-related inflammation in macrophages.

PHC abrogated LPS-induced ROS production in RAW264.7 cells
In order to examine PHC's effect on LPS-induced oxidative stress, we determined ROS production in RAW264.7 cells administered with LPS and PHC.DCFH-DA-based flow cytometry analysis (Fig. 3A,B and Supplementary Tables 21-23) and immunofluorescence (Fig. 3C,D and Supplementary Tables 24-26) consistently showed that PHC dose-dependently and remarkably abrogated LPS-stimulated ROS production.These data suggested that PHC alleviated LPS-induced oxidative stress in macrophages.

PHC activated Nrf2 signaling in RAW264.7 cells
In order to investigate Nrf2 pathway involvement in PHC's effect in RWA264.7 cells, we determined the protein amounts of Nrf2 and its target genes HO-1 and NQO1 in RWA264.7 cells.As depicted in Fig. 4A,B and Supplementary Tables 27-34, LPS stimulation did not affect the protein expression of Nrf2, HO-1 and NQO1 in cells without PHC.However, PHC treatment significantly elevated Nrf2, HO-1 and NQO1 protein in LPS-treated cells dose-dependently.
We also focused on the effects of the ROS scavenger n-acetyl-l-cysteine (NAC) on PHC-induced Nrf2 and HO-1.Penehyclidine hydrochloride at a concentration of 5 μg/mL significantly upregulated Nrf2 expression, but this was significantly inhibited by NAC.Moreover, PHC at a concentration of 5 μg/mL showed a similar trend in nuclear HO-1 expression, although the difference was not statistically significant, possibly due to limitations in sample size.These findings suggest that the activation of Nrf2/HO-1 in macrophages by PHC is associated with ROS (Fig. 4C and Supplementary Tables 35-40).

Discussion
This study showed that PHC could reverse LPS-related NO and PGE2 production in RAW264.7 cells.PHC also suppressed LPS-induced IL-6, TNF-α and IL-1β mRNA expression, IL-6, TNF-α and IL-1β secretion, and ROS production.LPS stimulation did not affect Nrf2, HO-1 and NQO1 protein expression in RWA264.7 cells without PHC, but PHC treatment elevated Nrf2, HO-1 and NQO1 protein after LPS treatment.Reactive oxygen species scavenging using NAC abolished the PHC-induced upregulation of Nrf2 and HO-1, highlighting the role of ROS in the process.Therefore, PHC partially abrogated LPSrelated overproduction of pro-inflammatory mediators and ROS in RAW264.7 cells, and, in the presence of LPS, PHC enhanced Nrf2, HO-1 and NQO1 protein expression.These findings suggest that PHC may enhance Nrf2 signaling in activated macrophages, and could serve as an antiinflammatory and antioxidant compound for managing inflammatory disorders.
Penehyclidine hydrochloride toxicity on RAW264.7 cells was examined.Weng et al. demonstrated that PHC ranging from 1 μg/mL to 5 μg/mL did not change the activity of cultured alveolar macrophages but dose-dependently reduced LPS-mediated apoptosis. 51Penehyclidine hydrochloride can also decrease neuronal apoptosis in the context of inflammation caused by cardiopulmonary bypass. 42Our results showed that PHC concentrations ranging from 1 μg/mL to 10 µg/mL had no significant effect on RAW264.7 cell viability.Thus, we treated RAW264.7 cells with 1 μg/mL or 5 μg/mL PHC in the subsequent experiments.Selecting the lowest doses could ensure the avoidance of possible PHC toxicity.
Lipopolysaccharide promoted the release of pro-inflammatory factors such as NO, PGE2, IL-6, IL-1β, and TNF-α, consistent with the known effects of LPS. 10,52,53Meanwhile, LPS-induced ROS production by RAW264.7 cells was similar to previous reports. 54,55These data suggest that an in vitro inflammation and oxidative stress model was successfully established.Macrophages are among the first immune cells to encounter pathogenic organisms and other stimuli, and activated macrophages are important sources of pro-inflammatory cytokines. 22,23Polarized macrophages are important in the pathogenetic mechanisms of inflammatory diseases by inducing the production of pro-inflammatory mediators. 56As shown above, PHC effectively reversed LPS-associated excessive synthesis of pro-inflammatory factors and ROS, suggesting that PHC may alleviate the inflammatory response of macrophages.
Oxidative stress is critical in the etiology and perpetuation of inflammation. 57Recently, Liu et al. reported that PHC administration enhanced Nrf2 expression while suppressing mouse lung inflammation resulting from renal ischemia/reperfusion. 43 Similarly, Yang et al. found that PHC upregulated Nrf2 and HO-1 while suppressing the production of pro-inflammatory cytokines and ROS in renal ischemia/reperfusion-induced injured lungs.Furthermore, PHC-induced protection is lost in Nrf2 -/-mice, suggesting that Nrf2 is essential for the protective role of PHC against inflammation and oxidative stress. 44However, whether PHC affects Nrf2 signaling in macrophages remains unknown.
A protective role for PHC in LPS-associated inflammation and oxidative stress has been extensively demonstrated Fig. 3. Penehyclidine hydrochloride (PHC) abrogated lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) in RAW264.7 cells.RAW264.7 cells were administered 1 μg/mL LPS alone or in combination with 1 μg/mL or 5 μg/mL PHC for 24 h. A. Flow cytometry analysis was carried out to determine cellular ROS production; B. Quantification of (A); C. Dichlorofluorescein diacetate (DCFH-DA) immunofluorescent staining was performed to detect ROS production in cells; D. Quantification of the fluorescence intensity of ROS (C) in animal models.Indeed, Guo et al. found that PHC alleviated LPS-associated acute lung injury and lung inflammation in a rat model, accompanied by considerable reductions in TNF-α, IL-8 and IL-6 in bronchoalveolar lavage fluid and downregulation of iNOS expression in the lung tissue. 58e et al. revealed that PHC inhibits LPS-associated lung myeloperoxidase expression and ROS production in rats. 59urthermore, PHC can attenuate the impact of Toll-like receptors in models of inflammatory chronic lung diseases 39 and acute lung injury. 40Similarly, PHC decreases serum TNF-α and IL-1β while improving renal function indicators in rats with LPS-induced acute kidney injury. 60n humans, PHC decreased the expression of pro-inflammatory cytokines after surgery. 41At the cellular level, PHC inhibited NO, PGE2, IL-1β, and TNF-α release in LPS-activated microglia, 61 and reduced the inflammatory response in cultured human pulmonary microvascular endothelial cells after LPS stimulation, reflected by lower secretion of lactate dehydrogenase, TNF-α and IL-6, and attenuated expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1). 62-acetyl-l-cysteine has been shown to downregulate Nrf2/HO-1 signaling in the presence of hydrogen peroxide (H 2 O 2 ) in MC3T3-E1 preosteoblasts 63 and attenuate kaempferol-induced Nrf2 interaction with the HO-1 promoter and subsequent HO-1 expression.64 Our findings suggest that Nrf2/HO-1 signaling induction by PHC in macrophages is ROS-dependent.The findings are consistent with Zhu et al., who demonstrated that Nrf2 decreased the transcription of pro-inflammatory cytokines in activated macrophages.33 Therefore, it is our opinion that PHC could have a role in managing disorders involving inflammation and oxidative stress.Of course, such management is outside the approved indications, and studies are necessary to examine the possibility, efficacy and safety of using PHC for acute or chronic inflammatory diseases.Acute and chronic inflammatory diseases carry high morbidity and mortality worldwide, and the available treatments have limited efficacy.As such, exploring novel strategies warrants attention.
Several points require further elucidation.For example, in Fig. 3C, only a select number of cells showed increased ROS levels instead of a universal increase across all treated cells.Unfortunately, the present study cannot provide the answer.In order to understand what is happening in those cells, microdissection and single-cell omics will be necessary but were not performed in the present study.Compared with the control group, the cells in the LPS group showed a general increase in ROS levels, and this difference was dosedependent.In addition, the western blots appeared to show secondary bands, and since there are different post-translational modifications and different forms of proteins, multiple bands can be recognized by the same antibody.Still, the target bands were analyzed according to the molecular weight of the corresponding marker, and the trend of the bands was consistent.Nonetheless, the nature of the protein modifications and isoforms could be investigated.Furthermore, LPS stimulation did not affect Nrf2, HO-1 and NQO1 protein expression in RWA264.7 cells without PHC.It could be hypothesized that the effect of PHC is specific to Nrf2, HO-1 and NQO1 elevation instead of a reversal of the effects of LPS, but the present study was not designed to examine such mechanisms, and additional studies are necessary.

Limitations
This study failed to verify that PHC can relieve LPSstimulated macrophage inflammation and oxidative stress through the Nrf2 signaling pathway by silencing the expression of Nrf2.Inflammation is a complex process involving several effectors, pathways and cells.Future studies should use agonists, antagonists, and knockout/silenced cell and animal models to examine the involvement of these effectors, pathways and cells in the mechanisms involved in PHC modulation of inflammation and oxidative stress.
Only 1 μg and 5 µg doses were explored and were selected because they were far below the toxic dose, avoiding any risk of interference from PHC toxicity.In addition, starting with a small dose that works is consistent with clinical dosing principles, where anticholinergics have been found to increase the incidence of postoperative cognitive impairment in the elderly. 41Still, future studies should include doses up to 20 µg to determine the exact dosedependency of the effects of PHC.Therefore, the results of this study need further verification.

Conclusions
In conclusion, PHC may alleviate LPS-induced inflammation and oxidative stress while activating Nrf2 signaling in RAW264.7 macrophages.These findings suggest that PHC could be used for inflammation therapy by targeting activated macrophages.