Danggui-Shaoyao-San improves cognitive impairment through inhibiting O-GlcNAc-modification of estrogen α receptor in female db/db mice
Graphical abstract
Introduction
Diabetic encephalopathy (DE) is characterized by cognitive impairment caused by diabetes, and is one of the main complications of diabetes (Sima et al., 2004). The main clinical manifestations of DE are learning and memory impairment that seriously develop into dementia (Rui et al., 2018). Epidemiological studies have shown that individuals with diabetes have an increased risk of dementia (Bello-Chavolla et al., 2019). Although the pathological mechanism of DE has not yet been fully understood, the primary has gradually emerged: the brain insulin signaling is inhibited by the increased levels of advanced glycation end products (AGEs), which leads to neuronal oxidative stress (Ahmad et al., 2017), inflammation (Pugazhenthi et al., 2017) and apoptosis (Sadeghi et al., 2016).
Estrogen, a sex hormone, not only promotes the sexual maturity and fertility of female individuals, but also maintains the metabolic homeostasis of the human body, through its effect on intracellular insulin sensitivity (E and Simon, 2010). Epidemiological studies have found that menopausal women are more likely to develop into DE (BRAND et al., 2013; Farrer et al., 1997), and the further mechanism studies have found that the rapid decline of estrogen activity may contribute to the crosstalk between diabetes and dementia (Deborah et al., 2017; Yumi and Masahiro, 2016). There are mainly two homologous estrogen receptors: ERα and ERβ in the brain (Mcewen, 2002). Estrogen can regulate insulin and glucose levels by combining with ERα (Jia M, 2015). When aging, more and more ERα mutants appear in the brain, resulting in a decrease in the efficiency of binding to estradiol, which further leads to the loss of ability of estrogen to maintain metabolic homeostasis and keep normal cognitive ability (Ghosh and Thakur, 2010). Studies showed that insulin resistance and obesity appear in ERα knockout mice, and ovariectomy can also lead to insulin resistance and an increased blood glucose levels in rodents (Barros et al., 2006; Francesc et al., 2006). In addition, many researches showed that the polymorphism of ERα increases the risk of Alzheimer's disease in women (Frick and Behavior, 2009; TANG, 1996; Yaffe et al., 2005).
Danggui-Shaoyao-San (DSS) is a Chinese herbal compound invented by Zhang Zhongjing, which is composed of Angelicae Sinensis (Oliv.) Diels., Paeonia Lactiflora Pall., Atractyolodes macrocephala Koidz., Poria cocos (Schw.) Wolf., Alisma orientale(Sam.) Juzep., and Ligusticum Chuanxiong Hort.. DSS has been used to treat gynecological diseases for thousands years, which is believed that DSS has the functions of promoting blood circulation, nourishing blood, strengthening spleen and promoting dampness. The chemical constituents of DSS are complex, mainly including organic acids, diterpenoids and their derivatives. In the late 1980s, shimajima Xuanzhao, a Japanese scholar, reported for the first time that DSS improved the clinical symptoms of dementia patients, and was widely concerned. Then in the next few decades, some Japanese scholars, Kotanin (Yasuhiro et al., 1997), Inanagak (Inanaga, 2007), Kitabayashiy (Kitabayashi and Neurosciences, 2007) and Matsuokat (Matsuoka et al., 2012) reported the clinical observation of DSS in the treatment of dementia, which further verified its therapeutic effect. Recently, a large number of animal experimental studies have also proved that DSS can improve the cognitive dysfunction of dementia models (Li et al., 2015; Luo et al., 2016). Besides, some studies also have found that DSS may have a certain estrogen-like effect, which can reduce the generation of free radicals and arteriosclerosis caused by high glucose(Yan et al., 2014). However, the specific mechanism of DSS in the treatment of DE is still unknown.
In this study, we used female db/db mice to investigate the pharmacodynamic effect of DSS in treating diabetic encephalopathy. Our results showed that DSS treatment could significantly improve the peripheral insulin resistance in female db/db mice, while improving brain neurotrophin secretion, inflammation and oxidative stress-induced neuronal apoptosis, which may be related to improve the abnormal O-GlcNAc-modification of ERα.
Section snippets
Preparation of DSS
DSS is composed of Angelicae Sinensis(Oliv.) Diels., Paeonia Lactiflora Pall., Atractyolodes macrocephala Koidz., Poria cocos(Schw.) Wolf., Alisma orientale(Sam.) Juzep., and Ligusticum Chuanxiong Hort., in a ratio of 3:16:4:4:8:8. All herbs were supplied by the Kangmei Pharmaceutical Co., Ltd. (GuangZhou, China). All herbs immersed in distilled water (1:8, w/v) for 1 h, and then extracted for 1 h. After the filtrate was collected, distilled water (1:6, w/v) was added to extract again for 1 h.
Identification of major components of DSS extract by HPLC-Q-Exactive Orbitrap HRMS
It is reported that organic acid, diterpenoids and its derivatives were the main compounds of DSS. Based on the optimized elution program, HPLC-Q-Exactive Orbitrap HRMS was conducted to detect the compounds in DSS extract. In the HPLC fingerprint of the DSS extract, tens of compounds were found under the present chromatographic method, and most of compounds were well separated with sharp symmetrical peaks (Fig. 1C). The total ion chromatograms of DSS in negative and positive modes (Fig. 1A and
Discussion
The MWM test was frequently used to measure the ability of spatial learning and memory(Barnhart et al., 2015). The time required for mice to successfully find a platform after entering the water is called the escape latency. The escape latency is used to judge the spatial learning and memory abilities of mice, and the short escape latency indicates that the mice have good learning and memory abilities. Compared with db/m mice, db/db mice presented a significant increase in the escape latency
Conclusions
In summary, our results confirmed that DSS treatment could effectively improve the learning and memory function of db/db mice by inhibiting abnormal O-GlcNAc-modification of ERα in db/db mice. This experiment also proved that DSS is an effective Chinese medicine prescription for the prevention and treatment of DE.
Author contributions
Y.-B.C. and S.-J.Z. designed the study. J.-J.S., H.-F.L and H.T. conducted the experiment. S.-J.Z. and J.-J.S. contributed to initial data analysis and interpretation and drafted the initial manuscript. X.G. and D.T. conducted HPLC-MS/MS experiment. Y.-B.Z. W.-R.L. and Q.W. helped the mouse work and provided the platform. Y.-B.C. and S.-J.Z. supervised all aspects of the study, critically reviewed and revised the manuscript, and approved the final manuscript as submitted.
Funding
This work was supported by National Natural Science Foundation of China (No.81674040, No.82074505, No.82074137), Key laboratory project of colleges and universities in Guangdong province (No.2019KSYS005), Guangdong province science and technology plan international cooperation project (No.2020A0505100052), Guangdong Provincial Science and Technology Project (No. 2017A020213029) and Scientific research project of Traditional Chinese Medicine of Guangdong Province(No.20202046).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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