| Objectives:Aging is a progressive deterioration process of the cells and organisms after they have become mature. Along with the growth of the age and/or the increasing stimulation of exogenous adverse factors (such as UV radiation, photolytic, chemicals, etc), a series of changes in morphological and functional characteristics of the organism’s take place gradually, accompanied by the corresponding compensatory responses. The study of Traditional Chinese Medicine (TMC) on aging mechanism has a long history; from the period of Inner Canon of Huangdi to modern time, there were always a lot of records about aging. For hundreds of years, many scientists in Chinese medicine and pharmacy have verified that blood stasis is closely associated with aging. As Hua Tuo put it,"the valley of blood circulation, the disease may not be born"; as Yan De-Xin said,"the essence of organism aging is the dysfunction of qi and blood as well as qi deficiency and blood stasis", which means that "blood stasis is the reason, and asthenia is the result". Moreover, Han Xiang-Ming clearly put forward the theory of "organism senescence caused by blood stasis", who believed that failing to nourish of brain and other body organs was the key factor for aging, and pointed out that the characteristics of organism senescence (such as dementia, schizophrenia, dim complexion, skin pigmentation, thick long sublingual vein, scleral opacity, knotted intermittent pulse, etc) were always the signs of blood stasis. In recent years, anti-aging researchers of Blood-Activiating and Stasis-Dissolving Drugs (BASDs) have made a great progress. Meanwhile, a large number of data have confirmed that the BASDs, especially their active components, have obvious advantages in adjusting and improving the adverse trends in physiology, pathology, biochemistry, and/or immunity of organism aging. Consequently, they are expected to become important candidate drugs in the field of anti-aging. Flos Carthami (FC) is the dry tubiform floret of Carthamus tinctorius L., the1annual herbaceous plants of Compositae, the major producing area of which is Xinjiang. The traditional effect of FC is activiating blood and dissolving stasis, which has been confirmed by clinical validation, and it has been used to treat coronary heart disease, cerebral thrombosis and other cardiovascular and cerebrovascular diseases for thousands of years. It has been shown that FC mainly contains pigments, phenolic acids, flavonoids and fatty oil, and that hydroxysafflor yellow A (HSYA) is the main water-soluble active ingredient. At the moment, HSYA is the principal efficiency ingredient of Safflor Yellow Injection (approval number:Z20050582). Numerous prior research studies have shown that HSYA, mainly used in the clinical treatment of cardiovascular and cerebrovascular diseases, exhibits many pharmacological properties such as scavenging oxygen-free radicals, lowering blood pressure and heart rate, anti-platelet aggregation, and anti-inflammatory action. However, little experimental data of this compound extend to other typical aging-related diseases, for example, Alzheimer’s disease (AD) and skin photoaging. Therefore, to explore the anti-aging role of HSYA has great research and development value.In order to open up new areas for the clinical applications of FC and/or HSYA, as well as its related Chinese patent medicines, the present study was therefore undertaken to evaluate HSYA for its possible activities and underlying mechanisms in prevent age-related diseases, including AD and photoaging, which has won more and more attention in recent decades. Methods:1. The in vitro anti-Alzheimer’s disease effect of HSYAThe anti-Alzheimer’s disease effect of HSYA was investigated in β-Amyloid (Aβ)-stimulated PC12cells, which had been differentiated into neuron-like cells by nerve growth factor (NGF). This is a classical in vitro model of the AD, and it can simulate similar pathological changes in the brain cells of Alzheimer’s patient. The PC12cells were pretreated with or without HSYA (at different concentrations) and then further treated with Aβ.To test whether HSYA has potential neuroprotective effect and to observe its optimal concentrations, we used a series of tests, including the morphological observation, MTT method, LDH method and Hoechst staining method.To elucidate the underlying mechanism of action associated with this protective effect of HSYA, mitochondrial membrane potential (MMP), the ratio of Bcl-2/Bax protein expression, mRNA expression of the pro-apoptotic protein PUMA, the formation of DNA fragmentation, and the levels of intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and glutathione (GSH) were studied in Aβ-stimulated PC12cells.2. The anti-photoaging effect of HSYAThe aging of skin is usually divided into two types:inherent aging and exogenous aing. Inherent aging, also called endogenous aging or intrinsic aging, is an irreversible process tightly regulated by the passage of time and the genetic information. Exogenous aing, also called phtoaging, is a preventable and treatable disease caused by the external environment, especially the ultraviolet (UV) ray in many environmental factors. Therefore, study in the field of anti skin aging research essentially refers to anti photoaging.In this part, simulated solar irradiation was provided by the long-wave ultraviolet (UVA) combined with medium wave ultraviolet (UVB). To test whether HSYA has an anti skin photoaging activity, we investigated the effect of topical HSYA application on a UV (UVA+UVB) induced photoaging mouse model. The effectiveness of this natural compound was tested by macroscopic evaluation of the skin, the pinch test, and the levels of skin moisture.In order to further study the underlying mechanism of action associated with this protective effect of HSYA, activity analysis of the main antioxidant enzymes (including SOD, GSH-Px, CAT), content determination of MDA and hydroxyproline (Hyp), and the activity evaluation of matrix metalloproteinases (MMP)(including MMP-1and MMP-3) of skin were held in UV-induced photoaging mice model. Furthermore, H&E staining and Gomori’s aldehyde fuchsin staining of the skin tissues were carried out to evaluate the extent of epidermal hyperproliferation, and to detect elastic and collagenous fibres (mainly assessing their distribution, integrity and structure).Results:1. The in vitro anti-Alzheimer’s disease effect of HSYAThe results of efficacy evaluation showed that HSYA (at the concentrations of20,40or80μM) could maintain the normal morphology and structure of the cell as well as its synapses, significantly increased cell viability and reduced the rate of LDH release, which were changed by Aβ. These results indicated that HSYA exerted a dose-dependent protective effect against Ap-induced neurotoxicity in PC12cells. Our data on the mechanisms of anti-Alzheimer’s disease exhibited that Ap observably decreased GSH level, MMP and the ratio of Bcl-2/Bax protein expression, while elevated the mRNA level of PUMA, the formation of DNA fragmentation, the levels of MDA and intracellular ROS in PC12cells. However, pretreatment with HSYA (20,40,80μM) could effectively reverse the above changes induced by Aβ in PC12cells. These experimental results demonstrated that HSYA exerted a protective effect against Aβ-induced neurotoxicity in PC12cells, and the neuroprotective effect of this natural compound is, at least partially, associated with inhibiting oxidative stress, stabilizing mitochondrial function, preventing the release of apoptosis factors, and finally reducing neuronal apoptosis. Base on these findings, HSYA appears to protect the nerve cells through the mitochondrial pathway of apoptosis.2. The anti-photoaging effect of HSYAThe results of efficacy evaluation demonstrated for the first time that HSYA (especially100-200μg/mouse) could significantly reverse the UV-induced mice skin dehydration, relaxation, rough deep wrinkles, nodules, increased pigmentation, leather like appearance and other undesirable state. Specifically, after HSYA treatment, the ability of mice skin to regain its initial shape was markedly promoted after deformation, and the level of mouse skin moisture was significantly increase. These data revealed that topical HSYA application could protect mouse skin from photoaging induced by UV irradiation.The current research results on the mechanisms of anti-photoaging revealed that topical HSYA (especially100-200μg/mouse) treatment significantly elevated the activities of these antioxidant enzymes and the content of Hyp, suppressed the abnormal expression of MMPs, decreased the content of MDA, and maintained the normal structure and distribution of elastic and collagenous fibres. Our results demonstrated for the first time that topical HSYA treatment could protect mouse skin from photoaging induced by UV irradiation, and revealed that this effect might be attributable to its free radical scavenging property, which in turn influenced the expression of MMPs.Conclusion:In this study, a classical in vitro model of the AD was used to investigate the potential neuroprotective effect of HSYA. The results indicated that HSYA exerted a dose-dependent protective effect against Aβ-induced neurotoxicity in neuron-like cells, and that HSYA appeared to protect the nerve cells through the mitochondrial pathway of apoptosis. Meanwhile, a UV-induced photoaging mouse model was used to test whether HSYA possessed anti skin photoaging activity. Our results demonstrated for the first time that topical HSYA treatment could protect mouse skin from photoaging induced by UV, and revealed that this effect might be attributable to its free radical scavengin property, which in turn influenced the abnormal expression of MMPs, and ultimately inhibited the degradation of collagen. In brief, this study is helpful to pen up new areas, such as preventing or treating age-related diseases, for the clinical application of HSYA, and provides a new application area for the active ingredients of Blood-Activiating and Stasis-Dissolving Drugs. |
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