Lingguizhugan decoction protects PC12 cells against Aβ25-35-induced oxidative stress and neuroinflammation by modulating NF-κB/MAPK signaling pathways

https://doi.org/10.1016/j.jep.2022.115194Get rights and content

Abstract

Ethnopharmacological relevance

Alzheimer's disease (AD) is recognized as one of the most prevalent neurodegenerative diseases. Lingguizhugan decoction (LGZGD) is a classical traditional Chinese medicine (TCM). Many studies have shown that LGZGD can alleviate the symptoms of AD.

Aim of the study

The aim of this study was to assess the neuroprotective effects of LGZGD and elucidate its molecular mechanism on Aβ25-35-induced PC12 cells.

Materials and methods

PC12 cells were used MTT assays, ELISA, fluorescence probe analyses, Hoechst 33342 staining, immunofluorescent staining and western blot analyses were systematically conducted to evaluate the underlying mechanisms of LGZGD.

Results

In Aβ25-35-induced PC12 cells, LGZGD remarkably increased cell viability, reduced the generation of TNF-α, IL-1β, IL-6, MDA and ROS, increased the activity of GSH-Px, inhibited cell apoptosis, downregulated the expression of Bax and cleaved caspase-3, and upregulated the expression of Bcl-2. Moreover, LGZGD modulated the NF-κB/MAPK signaling pathways by upregulating the levels of IκBα and phospho-ERK, while downregulating the levels of phospho-p65, phospho-IκBα, and phospho-p38. Furthermore, LGZGD repressed the nuclear translocation activity of NF-κB p65. Meanwhile, LGZGD increased the expression of phospho-GSK-3β and reversed the hyperphosphorylation of Tau proteins by inhibiting the activation of the ERK MAPK pathway.

Conclusions

Taken together, the present study suggested that LGZGD may be a valuable drug candidate that can attenuate the neurotoxicity induced by Aβ25-35 by modulating the NF-κB/MAPK signaling pathways in PC12 cells.

Introduction

Alzheimer's disease (AD) is the most common, irreversible neural disorder associated with dementia and is clinically characterized by memory decline, cognitive dysfunction and behavioral disorders. The incidence of AD has been increasing, and the total prevalence could reach 13.8 million by 2050 (Alzheimer’s Association, 2015). Despite decades of research efforts, the pathogenesis of AD remains controversial. To date, the extracellular amyloid beta (Aβ) peptide plaques along with intraneuronal tau lesions are predominantly considered to be the pathological features of AD (Bloom, 2014; Goedert, 2015). At present, drugs approved by the US Food and Drug Administration (FDA) for the clinical treatment of AD include cholinesterase inhibitors (ChEIs) and N-Methyl-D-Aspartate (NMDA) receptor antagonists (Auld et al., 2002). However, they are all single-target drugs that neither prevent nor reverse the disease and can only partially alleviate the symptoms with the risk of side effects (Fang et al., 2020). Therefore, it is important to identify more efficient drugs for AD treatment.

With the characteristics of multiple ingredients, focusing on multiple targets, and modulating multiple signaling pathways, traditional Chinese medicines (TCMs) have been used to treat and prevent neurodegenerative diseases in China for thousands of years (Ji et al., 2015). It has been proven that TCMs might slow down the cognitive decline in AD patients by regulating the Aβ generation and aggregation, tau phosphorylation, and gut microbiota-brain axis among others (Zhang et al., 2020). In recent years, many researchers have found that TCMs exhibit excellent efficacy in treating AD with fewer side effects, such as Ginkgo biloba extract and Kai-Xin-San (Guo et al., 2019; Zeng et al., 2018). To date, the advantages of many TCMs over single-target medicines in the treatment of AD have been reported (Anekonda and Reddy, 2005).

Lingguizhugan decoction (LGZGD) consists of the four herbs Poria cocos Schw. Wolf (Poria), Cinnamomum cassia Presl (Cinnamomi ramulus), Atractylodes macrocephala Koidz. (Atractylodis macrocephalae rhizoma) and Glycyrrhiza uralensis Fisch. (Glycyrrhizae radix et rhizoma) with a ratio of 4:3:3:2 (w/w/w/w). The classic formula was originally recorded in “Treatise on Febrile Diseases” and “Synopsis of Golden Chamber” with a long history of application, both of which were written by Zhong-Jing Zhang in the Han Dynasty (Song et al., 2003). As a typical formula, it's traditionally applied to warm yang for phlegm and retained fluid, strengthen the spleen and clear away dampness (Lv and Song, 2017). Previous studies have shown that LGZGD possesses the ability to treat multiple diseases, such as hypertension (Xie et al., 2021), Meniere's disease (Lv and Song, 2017), obesity (Yang et al., 2017), type 2 diabetes (Chen et al., 2012), arrhythmia, thrombus, angina pectoris and other cardiovascular diseases (Zhu et al., 2018), also for the treatment of AD (Xi et al., 2012). Wang et al. used LGZGD as an antioxidant alternative drug model to against complex pathophysiologies diseases related to oxidative stress (Wang et al., 2006). Recently, Xi et al. have found that LGZGD can increase the survival rate and cell viability of SH-SY5Y cells treated with Aβ25-35, and inhibit the secretion of pro-inflammatory mediators and nitric oxide in BV-2 microglia (Xi et al., 2012). In addition, Yu et al. have reported that LGZGD exerted positive effects in AD by regulating Aβ transportation and inhibiting RAGE/MAPK and NF-κB signaling pathways (Yu et al., 2014). Furthermore, we also elucidated the multi-target mechanisms of LGZGD on AD using a network pharmacology-based strategy (Wang et al., 2019). However, the underlying mechanisms of LGZGD in AD still require further confirmation.

In the present study, the neuroprotective effects of LGZGD on Aβ25-35-induced AD-like PC12 cells were evaluated. Furthermore, this system was employed to study the biological mechanisms by which the NF-κB/MAPK pathway is modulated to play a neuroprotective role in PC12 cells. This study will endeavor to provide insights into the pharmacological mechanism of AD treatment with LGZGD.

Section snippets

Reagents and antibodies

All herbs were purchased from Shenyang GuoDa Pharmacy (Shenyang, China) and genetically identified by Qingdao Science Innovation Quality Testing Co., Ltd. (Qingdao, China). Aβ25-35 and donepezil hydrochloride (DHCL) were purchased from Aladdin Biochemical Technology (Shanghai, China). PD98059 was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). RPMI 1640 medium was supplied by Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Fetal bovine serum (FBS)

Effects of Lingguizhugan decoction on cells viability

First of all, we studied the concentration and time of Aβ25-35 used to establish AD cell models, as shown in Fig. 1A, Aβ25-35 (10–30 μM) led to reduction in the viability of PC12 cells at 12, 24, 36 h compared to the respective control group (P < 0.05, P < 0.01 and P < 0.001). The results showed that cell viability is dose-dependent and time-dependent. The Aβ25-35 (30 μM) application for 24 h was used in further experiments as model group (P < 0.001). As shown in Fig. 1B, exposure to Aβ25-35

Discussion

Aβ accumulation leads to oxidative stress and inflammation, which ultimately causes neuronal death and tissue damage (Lobello et al., 2012). Therefore, the prevention of oxidative stress and inflammation is considered as an effective therapeutic approach for AD. At present, TCMs are highly popular owing to their high rates of compliance and lower toxicity. LGZGD is a classic Chinese medicine prescription with various pharmacological effects (Yang et al., 2017). Several reports of evidence have

Conclusion

In conclusion, we demonstrated that LGZGD has a neuroprotective effect on the Aβ25-35-induced neurotoxicity by modulating NF-κB/MAPK pathways in PC12 cells. Our results suggest that LGZGD may be a useful anti-neurotoxicity agent that can be applied to the treatment of neurodegenerative diseases.

Authors’ contributions

Jing Han ([email protected]) and Haotian Zhang ([email protected]) were responsible for the execution of the experiments, the analysis of data and the writing of the manuscript. Yu Zhang ([email protected]), Zan Zhang ([email protected]), Maomao Yu ([email protected]) and Sijie Wang ([email protected]) were involved in the performance of the cell experiments, the immunohistochemistry analysis, the ELISA and western blotting test in this study. Fei Han ([email protected]) designed and managed

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the Natural Science Foundation of Liaoning Province (No. 2021-MS-220).

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