Exploring the mechanism of Huangqi San in treating Alzheimer’s disease based on network pharmacology and experimental verification
Alzheimer’s disease (AD) is a central nervous system degenerative disease characterized by progressive cognitive impairment and behavioral impairment. With the acceleration of the aging process of the population, the incidence rate of AD in China is increasing year by year, which seriously threatens the health of the middle-aged and elderly, and creates a heavy burden on society and families. At present, the anti AD drugs approved by the US FDA are mainly N-methyl-D-aspartate (NMDA) receptor blockers and acetylcholinesterase (AchE) inhibitors. Although they can moderately improve patients’ symptoms, they still cannot cure or reverse the development of AD. Traditional Chinese medicine formulas have the synergistic effects of multiple components, targets, and pathways, and have significant therapeutic effects in improving cognitive function, daily living ability, and quality of life in AD patients. They also have minimal toxic side effects and are not easily resistant to long-term use.
Some scholars call AD type 3 “diabetes” in the brain and point out that the core of AD may be caused by the obstacle of brain insulin and insulin-like growth factor signal transduction pathway. Research has confirmed that diabetes is a very important high-risk factor of AD, and diabetes patients have a very high risk of AD, and many AD patients have diabetes at the same time, which greatly increases the pain of patients and brings greater difficulty to clinical treatment. Huangqi San originated from the “Shengji Zonglu” and is a classic formula for treating thirst. It is composed of kudzu root, Huangqi root, and mulberry bark. The study found that Huangqi Powder can reduce the fasting blood glucose, glucose tolerance and adrenaline induced hyperglycemia in diabetes mice. Experimental results have shown that Huangqi San can improve learning and memory, spatial cognitive dysfunction, and AD like pathology in 5 × FAD mice. Traditional Chinese medicine formulas have the characteristic of synergistic effects of multiple components, targets, and pathways, which are difficult to systematically and comprehensively explain from the whole to the cellular and molecular levels. Network pharmacology is an emerging discipline that combines computer technology, systems biology, and bioinformatics to systematically analyze the interrelationships between drugs, targets, pathways, and diseases. It can elucidate the pharmacological substance basis and mechanism of action of traditional Chinese medicine formulas from a molecular and holistic perspective, providing technical support for the screening of effective ingredients in traditional Chinese medicine formulas and the study of their mechanisms of action. It is highly compatible with the overall principles of traditional Chinese medicine treatment and the synergistic effects of multiple components, targets, and pathways in traditional Chinese medicine formulas.
Huangqi San has numerous active ingredients and rich targets, but the effective ingredients and mechanism of action of Huangqi San in treating AD are still unclear. Therefore, this study adopts the method of network pharmacology to predict and analyze the effective ingredients, targets, and signaling pathways of Huangqi San, and verifies them through cell experiments, attempting to explore the mechanism of Huangqi San in treating AD at the molecular level, in order to provide reference for further basic experimental research and clinical rational application of Huangqi San.

 

 

AD has been called “type 3 diabetes” in academic circles because of its similar pathogenesis with type 1 and 2 diabetes, which is mainly reflected in brain insulin deficiency and insulin resistance. Diabetes belongs to the traditional chinese medicine, which is mainly characterized by deficiency of both qi and yin and excess of dryness and heat. Therefore, nourishing qi, nourishing yin, and clearing heat are the basic principles for treating diabetes. Huangqi San is a classic formula for treating thirst. In the formula, kudzu root produces saliva to quench thirst and invigorate the spleen to clear yang. The body fluid is infused to irrigate the five organs, nourishing yin and clearing heat; Huangqi (Astragalus membranaceus) has the meaning of nourishing qi and strengthening the spleen, taking its qi to restore fluids, and applying it to the clouds and rain; Mulberry white skin is sweet, cold, and can relieve fire, nourish yin and moisten dryness. Its sweet and cold nature is similar to the slightly hot nature of Huangqi; The combination of three medicines has the effects of tonifying qi and spleen, nourishing yin and clearing heat, generating fluids and quenching thirst, and treating the symptoms in a balanced manner.

The active ingredient target network shows that Huangqi San has the characteristics of multi-component and multi-target treatment for AD. The results show that quercetin, puerarin, astragaloside IV, and resveratrol are its key active ingredients. Research has shown that quercetin can increase the Bcl-2/Bax expression ratio and decrease the expression of Caspase-3 protein through the classical estrogen receptor pathway and MAPK pathway, thereby reducing the occurrence of apoptosis. Research has shown that puerarin can effectively slow down the increase in tau protein phosphorylation levels in the olfactory bulb of AD model rats, and its mechanism of action may be related to its reduction of GSK-3 β activity levels. Research has shown that the MEK5/ERK5 signaling pathway has an inhibitory effect on the activity of microglia in AD rats and can antagonize neuronal apoptosis in AD rats. Research has found that resveratrol may alleviate inflammatory damage and improve cognitive impairment in AD mice by promoting M2 polarization of hippocampal microglia.

Mapping the active ingredients to the common target of AD resulted in 102 drug disease common action targets. The top three key targets of Huangqi San are PINK1, NLRP3, and BDNF. PINK1 is a major member of the PINK1/Parkin signaling pathway, and research has found that activating PINK1 Parkin mediated mitochondrial autophagy affects AD pathology and improves cognitive and memory impairment in AD model mice. Research has shown that NLRP3 inflammasome may play a crucial role in the occurrence and development of Alzheimer’s disease by regulating the inflammatory response. The two key markers of NLRP3 inflammasome activation, IL-1 β and IL-18, are at high levels in the brains of AD patients. BDNF, as an important neurotrophic factor, plays a crucial role in synaptic plasticity, neurogenesis, and neuronal survival.

GO enrichment analysis showed that the gene functions of Huangqi San in treating AD mainly include autophagy, inflammatory response, regulation of synaptic plasticity, response to insulin, neuronal apoptosis, oxidative stress response, and other processes. Among the signal pathways screened in this study, the mitochondrial autophagy signaling pathway has the highest correlation with the effective targets of Huangqi San, followed by insulin resistance. In this study, combined with the pathogenesis of AD, the regulatory mechanisms of mitochondrial autophagy, inflammatory response, and synaptic plasticity predicted in the results were screened as possible mechanisms for Huangqi San’s intervention in AD through in vitro cell experiments. The PINK1/Parkin signaling pathway is a key pathway regulating mitochondrial autophagy, which activates autophagy through ubiquitination of parkin. PINK1 is the main detector of mitochondrial damage. When mitochondria are damaged, PINK1 phosphorylates parkin on the outer membrane of mitochondria to recruit them from the cytoplasm to the damaged mitochondria, and then phosphorylates E3 ubiquitin protein ligase parkin in the cytoplasm to induce its recruitment from the cytoplasm to the damaged outer membrane of mitochondria. Activated parkin connects ubiquitin to the basement protein to form a ubiquitin chain, which binds with p62 and other molecules to recruit autophagy marker LC3 in the cytoplasm, forming autophagosomes with a bilayer membrane structure. Autophagosomes ultimately fuse with lysosomes to completely degrade damaged mitochondria, completing the process of mitochondrial autophagy. The higher the degree of transformation of LC3-II, the more autophagosomes there are, and the level of autophagy can be determined by LC3-II/LC3-I. P62 can directly bind to specific regions of LC3 protein and ultimately be selectively degraded by autophagosomes. During autophagy, p62 protein is degraded within autophagosomes, making its degradation one of the markers reflecting autophagy levels. Research has shown that impaired mitochondrial autophagy may lead to the accumulation of A β and tau proteins, exacerbating oxidative damage, resulting in synaptic dysfunction and cognitive impairment. Therefore, mitochondrial autophagy plays a crucial role in Alzheimer’s disease. Research has shown that activating the PINK1/Parkin pathway to promote mitochondrial autophagy can improve cognitive dysfunction, enhance mitochondrial function, and provide neuroprotective effects in AD model mice. The results of this study indicate that Huangqi San can increase the number of autophagosomes, promote autophagosome encapsulation of damaged mitochondria, increase the LC3 Ⅱ/Ⅰ ratio, upregulate the expression of PINK1 and parkin proteins, and downregulate the expression of p62 protein, suggesting that Huangqi San may promote mitochondrial autophagy by activating the PINK1/Parkin pathway. Research has shown that ROS is a key signal that promotes the activation of NLRP3 bodies and the expression of downstream inflammatory molecules such as IL1 β, IL-18, and TNF – α. Experiments have shown that activating the PINK1/Parkin pathway to promote mitochondrial autophagy can reduce ROS levels, inhibit NLRP3 inflammasome activity, and decrease downstream levels of inflammatory factors such as IL-1 β and IL-18, playing a protective role in AD. The results of this study indicate that Huangqi San can significantly reduce ROS levels, inhibit NLRP3 inflammasome activity, and lower the levels of inflammatory factors IL-1 β, IL-18, and TNF – α, which may be related to Huangqi San activating the PINK1/Parkin pathway to promote mitochondrial autophagy levels. Neurons are the structural units of the nervous system, in which neurons are closely connected through synapses. The outward growth of neural processes in neurons is a sign of neurodegeneration and neuroprotection. The growth of neural processes is an important prerequisite for neuronal development, synapse formation, and nerve regeneration. Therefore, promoting the growth of neural processes and repairing damaged nerve cells is particularly important in the treatment of central nervous system diseases. BDNF, as an important neurotrophic factor, plays a crucial role in synaptic plasticity, neurogenesis, and neuronal survival. Research has shown that activating the PINK1/Parkin pathway to promote mitochondrial autophagy can improve learning, memory, cognitive impairment, synaptic and dendritic spine structure and function damage in 3xTg AD mice. In our study, we found that Huangqi San can increase the survival rate of PC12 cells induced by A β 25-35, increase the length of cell processes, and enhance BDNF protein expression, which may be related to Huangqi San activating the PINK1/Parkin pathway to promote mitochondrial autophagy.

In summary, this study analyzed the mechanism of Huangqi San in treating AD using network pharmacology and experimental verification methods. The conclusion drawn is that Huangqi San may exert its therapeutic effect on AD by activating the PINK1/Parkin pathway to promote mitochondrial autophagy and eliminate ROS, thereby inhibiting the activation of NLRP3 inflammasome and improving synaptic plasticity. Elaborate on the molecular and cellular levels, providing reference for further basic experimental research and rational clinical application of Huangqi powder.