Effects and Mechanisms of Five Psoralea Prenylflavonoids on Aging-Related Diseases

During the aging process, senescent cells gradually accumulate in the organs; they secrete proinflammatory cytokines and other factors, collectively known as the senescence-associated secretory phenotype (SASP). SASP secretions contribute to “inflammaging,” which is a state of chronic, systemic, sterility, low-grade inflammatory microenvironment and a key risk factor in the development of aging-related diseases. Fructus psoraleae is a traditional Chinese medical herb best known for delaying aging and treating osteoporosis. Prenylflavonoids from fructus psoraleae are the main bioactive compounds responsible for its pharmacological applications, such as beaching, bavachinin, bavachalcone, isobavachalcone, and neobavaisoflavone. In previous decades, there have been some promising studies on the pharmacology of fructus psoraleae. Here, we focus on the anti-inflammatory and antiaging diseases of five psoralea prenylflavonoids, such as cardiovascular protection, diabetes and obesity intervention, neuroprotection, and osteoporosis, and discuss the mechanism of these active ingredients for better understanding the material basis and drug application of fructus psoraleae in Chinese medicine.


Introduction
Fructus psoraleae (补骨脂), a traditional Chinese medical herb, is the dried and mature fruit of Psoralea corylifolia Linn., which is an annual herb of Leguminosae (Figures 1(a)-1(c)). After drying, the psoraleae fruits are mixed with salt water and gently stir-baked until the fruits are slightly swollen. Then, the salt-processed fructus psoraleae could be used as a traditional Chinese medicine in the clinic. Fructus psoraleae is considered to have the efficacy of "relieving chronic morning diarrhea, warming and invigorating the Kidney-Yang, gathering the spirit, and enriching the bone marrow" in the Compendium of Materia Medica. According to the traditional Chinese medicine theories, fructus psoraleae has the function of delaying aging. The chemical constituents of fructus psoraleae mainly include coumarin, terpenoid phenols, and prenylflavonoids, which are the therapeutic material basis of fructus psoraleae. The structure of prenylflavonoids is characterized by the presence of isopentenyl side chains on the flavonoid skeletons. The structural types of isopentenyl are mainly 5 kinds of side chains: isopentenyl, hydroxyisopentenyl, pyran ring isopentenyl, lavender, and hydroxylavender. Most of psoralea prenylflavonoids are contained in ethyl acetate extract active fractions from Psoralea corylifolia [1,2]. A recent study has shown that all the tested compounds in Psoralea corylifolia were absorbed into the blood plasma of rats rapidly, almost evenly distributed in the brain parenchyma, and the overall trend in the brain was basically similar to the results of plasma concentration time. Moreover, in terms of the ratio of total brain to plasma, isoprene flavonoids were easier to enter the brain than coumarin [3]. Another pharmacokinetic report also showed that rats taking oral administration and intraperitoneal injection with 20 mg/kg bavachalcone were detected with maximum plasma concentrations of 165 and 740 μg/L, with a drug half-life of 1 and 1.25 h, respectively [4]. Fructus psoraleae has certain hepatotoxicity mainly caused by its constituents, such as bakuchiol, psoralen, and isopsoralen [5,6]. Some psoralea prenylflavonoids exhibited inhibitory effects on microsomal enzymes in in vitro experiments. For example, acyl CoA:cholesterol acyltransferase (ACAT), an enzyme that catalyzes the esterification of cholesterol in the intestine and the production of lipoproteins in the liver, was inhibited by bavachin and isobavachalcone [7]. The following are some similar reports: (1) neobavaisoflavone, isobavachalcone, bavachinin, corylifol A, and bakuchiol blocked human carboxylesterase 2 [8]; (2) bavachalcone, bavachin, and corylifol A strongly and neobavaisoflavone, isobavachalcone, and bavachinin moderately inhibiting UGT1A1 [9,10]; and (3) psoralidin, isobavachalcone, and neobavaisoflavone inhibiting CYP2E1 mRNA expression. Meanwhile, isobavachalcone showed a weak competitive inhibition on CYP3A4, whereas psoralidin and neobavaisoflavone exhibited significant induction of CYP3A4 mRNA expression [11].
With the development of aging, senescent cells gradually accumulate in tissues, which produce proinflammatory factors and form a low-degree of sterility and potential systemic inflammation to result in aging-related diseases. This negatively affected microenvironment is called "inflammaging," which is the key factor connecting cell aging and the development of aging-related diseases. Modern pharmacological studies showed that bavachin, bavachinin, bavachalcone, isobavachalcone, and neobavaisoflavone ( Figure 1(d)) had various pharmacological actions and could improve aging-related diseases. In this paper, we will review the effects and mechanisms of these five psoralea prenylflavonoids on ameliorating aging-related diseases, such as aginginduced chronic low-grade inflammation, cardiovascular dysfunction, diabetes, and obesity, and improving neurodegenerative diseases and osteoporosis.  Oxidative Medicine and Cellular Longevity

Anti-Inflammatory Action
The accumulation of senescent cells with age causes a series of pathological manifestations. Nonproliferating cells occupy key cell niches and produce proinflammatory factors, which form a negatively affected microenvironment and present a senescence-associated secretory phenotype (SASP). SASP contributes to a state of sterile, systemic, low-grade inflammation, also known as "inflammaging," which precedes many age-related diseases [12]. Considering the antiaging activity, a variety of synthetic and natural compounds has got keen interest in senolytic discovery (drugs selectively eliminating senescent cells) [13]. Fructus psoraleae is a traditional antiaging Chinese medical herb, and its resistance to "inflammaging" has become one of research concerns in antiaging field. Psoraleae prenylflavonoids have been shown to suppress Toll-like receptor-(TLR-) or IL-1β-induced inflammation response. Multiple reports indicated that isobavachalcone, bavachinin, and bavachin inhibit the expression of iNOS, COX-2, and mPGES-1 and the production of nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) in microglia, macrophages, and chondrocytes [14][15][16][17][18]. Bavachin also inhibited IκBα degradation and increased the nuclear translocation of p65 and p50 proteins in the inflammatory chondrocytes and endothelial cells [19,20]. In addition, bavachin significantly inhibited the activity and expression of matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS), while upregulating tissue inhibitors of metalloproteinases (TIMPs) in mouse chondrocytes [17]. These findings showed that bavachin has an anti-inflammatory effect on chondrocytes and could be used to treat articular cartilage degeneration [17,19]. Furthermore, bavachin has shown to attenuate LPS-induced inflammation and inhibit macrophage NLRP3 inflammasome activation. Bavachin suppressed caspase-1 activation, IL-1β secretion, and inflammasome complex formation [18]. Isobavachalcone can significantly downregulate the levels of intercellular adhesion molecule-1 (ICAM-1) and interferon-β (IFN-β) and inhibit leukocyte adhesion to brain endothelial cells through inhibiting the TLR4/MyD88 signaling pathway [20]. Studies have determined that neobavaisoflavone significantly inhibits the production of reactive oxygen species, reactive nitrogen species, and cytokines in LPS+IFN-γ-or PMA-stimulated RAW264.7 macrophages, such as IL-1β, IL-6, and tumor necrosis factor-(TNF-) α [21]. A previous study has shown that isobavachalcone, bavachin, bavachinin, and neobavaisoflavone also have inhibitory effects on IL-6-induced STAT3 promoter activity and phosphorylation in Hep3B cells [22].
Bavachinin attenuated HIF-1α activity under hypoxia in a concentration-dependent manner and reduced HIF-1regulated transcription of genes related to energy metabolism, such as glucose transporter type 1 (Glut1) and hexokinase 2 [23]. HIF-1α is the main oxygen sensor within cells and is essential to the regulation of cell responses to varying oxygen levels. The transcription factor HIF-1α forms a complex relationship with inflammation and mitochondrial metabolism [24][25][26][27]. Recent studies have elucidated a new mechanism for mitochondrial metabolism to promote IL-1β expression and activity via HIF-1α regulation [25,28]. Activated PKM2 inhibited the LPS-induced expression of HIF-1α and IL-1β, as well as a series of other HIF-1α-dependent genes. PKM2 induced by LPS forms a complex with HIF-1α and directly binds to the IL-1β promoter. Researchers also observed that activated PKM2 inhibited LPS-induced glycolytic reprogramming and succinate production [28]. LPS-induced succinate stabilizes HIF-1α which leads to activation of the proinflammatory cytokine IL-1β [25]. In obesity-related heart failure with preserved ejection fraction (HFpEF), HIF-1α is responsible for recruiting M1 macrophages that mediate obesity-associated inflammation. M1 macrophages produce proinflammatory cytokines IL-6, monocyte chemoattractant protein-1 (MCP-1), TNF-α, and IL-1β and increase the expression of thrombospondin, pro α2 (I) collagen, transforming growth factor (TGF)-β, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and connective tissue growth factor (CTGF) [29]. Therefore, the inhibition of HIF-1α activity by bavachinin antagonized the inflammatory response under ischemia and hypoxia ( Figure 2).
Prenylflavonoids also induced the expression and activity of transcription factors. For example, isobavachalcone antagonized lung injury and myotube atrophy by increasing the activity and expression of nuclear factor erythroid 2-related factor 2 (Nrf2) [30,31]. Nrf2 is highly sensitive to oxidative stress, which is closely related to inflammatory response [32] and aging-related diseases, cardiovascular diseases, Alzheimer's and Parkinson's diseases, and rheumatic diseases [33][34][35][36][37][38]. Researchers demonstrated that Nrf2 inhibition of ROS-induced NLRP3 priming required the participation of NAD(P)H quinone dehydrogenase 1 (NQO1) [39]. Furthermore, the inhibition of Nrf2 also regulated the NLPR3 inflammasome formulation, assembly-mediated cleaved caspase-1, and IL-1β secretion through the Trx1/TXNIP complex [40]. In addition, Nrf2 binding to antioxidant responsive element (ARE) site can trigger the transcription of more than 200 endogenous protective genes, such as antioxidant, anti-inflammatory, and antiapoptotic genes [33,41]. Therefore, the moderate expression and balance of Nrf2 promoted by prenylflavonoids are beneficial to the regulation of antioxidant, anti-inflammatory, and mitochondrial homeostasis ( Figure 3).
Our latest study has shown that 4'-O-methybavachalcone significantly reduced cerebral infarction and edema, improved neurobehavioral indexes, inhibited the production of IL-1β, TNF-α, and IL-6 in ischemic brain, and reduced the level of NLRP3, cleaved caspase1, and GSDMD-N (gasdermin D N-terminal domain) in the mouse stroke model and in oxygen glucose deprivation-(OGD-) RAW264.7 cells [42]. Recently, gasdermin D and NLRP3 have been identified as programmed death patterns of stroke cell inflammation and pyrolysis [43]. Moreover, a specific pyroptosis inhibitor Vx765 has a potential therapeutic value for cerebral ischemia by inhibiting the canonical inflammasome pathway of pyroptosis [44]. In brief, bavachalcone has the ability to inhibit the inflammatome NLRP3 activity and cell pyrolysis ( Figure 3).
It has been reported that the bavachin activated gene expression of peroxisome proliferator-activated receptor gamma (PPARγ) [45]. As we all know, PPARγ can transform proinflammatory cytokines produced by neutrophils, platelets, and macrophages into anti-inflammatory mediators. PPARγ and its ligands further regulate platelet and neutrophil function, reduce trafficking, promote neutrophil apoptosis, and prevent platelet-leukocyte interactions. PPARγ alters macrophage transport, increases efferocytosis and phagocytosis, and promotes macrophages to switch to M2-type polarity [46]. What is more, endothelial PPARγ is an indispensable factor to prevent endothelial dysfunction with aging [47], and interference with PPARγ function can accelerate vascular aging and atherosclerosis [48,49]. Conversely, cell senescence may also alter the PPARγ-dependent fatty acid handling in human microvascular endothelial cells and contribute to inflammation [50].
In the following summary (Table 1), the cell or animal models, inflammation induction methods, active recipients, experimental doses, and pharmacological effects in more detail are provided. In general, prenylflavonoids inhibited JNK1/2, ERK1/2, NF-κB, STAT3, inflammatome NLRP3 activity, iNOS and mPGES-1 expression, secretion of many cytokines, and cell pyroptosis. These findings indicate that psoralea prenylflavonoids have the potential effects on improving "inflammaging" environment and aging-related diseases.

Cardiovascular Function Regulation
The age-related status changes lead to low-degree inflammation and increase of oxidative stress in the cardiovascular system. The phenotypes of these changes include rigidity of HO-1, NQO1 Chronic low-degree inflammation Cell pyroptosis Aging-related diseases Isobavachalcone ROS Bavachalcone Figure 3: The schematic diagram of the Nrf2 signal transduction pathway activated by isobavachalcone and of NLRP3 inflammasome inhibited by bavachalcone in the anti-inflammation.  Oxidative Medicine and Cellular Longevity (i) Concentration-dependent inhibitory effects on NO and PGE 2 production with an IC 50 for inhibiting NO production: 1:6 ± 0:11 μmol/L (ii) Blocked the I-κBα degradation and downregulated NF-κB activity (iii) Inhibited the iNOS and COX-2 mRNA and protein expression [14] RAW264 5 Oxidative Medicine and Cellular Longevity elastic arteries, endothelial dysfunction, hypoperfusion cerebral ischemia, stroke, and cardiac diastolic dysfunction. This involves the Klotho longevity gene, the AMPK energysensitive pathway, and the activity and expression of eNOS, MnSOD, and PGC-1α genes.
The cardiovascular protective effects of prenylflavonoid derivatives from fructus psoraleae have been reported ( Table 2). Kassahun et al. [51] reported that isobavachalcone inhibited hTRPC3 current, which significantly relaxes the contraction of the endothelium-intact aortic rings induced by phenylephrine. In addition, a previous study has shown that isobavachalcone inhibits platelet aggregation induced by arachidonic acid, collagen, and platelet activating factor in rabbits [52]. As we all know, platelet aggregation is closely related to myocardial infarction and stroke [53,54].
Acyl-CoA:cholesterol acyltransferase (ACAT), a key enzyme regulating cholesterol esterification, induced the accumulation of cholesterol esters in the endoplasmic reticulum of macrophages and smooth muscle cells and promoted the formation of foam cells [55,56]. However, the enzyme was inhibited by isobavachalcone and bavachin in an ACAT noncompetitive inhibition type [7]. In in vitro studies performed by our team in human endothelial cells, bavachalcone suppressed cell senescence by decreasing the activity of a senescence-associated β-galactosidase (a senescent markers) and downregulating mRNA expression of p16 (ink4a) (a marker of replicative senescence) and IL-1α (a proinflammatory cytokine of SASP) [57]. Bavachalcone also activated retinoid-related orphan receptor alpha 1 (RORα1) reporter gene activity and induced RORα1 mRNA expression in a dose-dependent manner, resulting in the enhancement of the amplitude of circadian rhythm of Bmal1 mRNA expression after serum shock [57]. Another study has shown that bavachalcone protected endothelial cells by increasing the activity of adenosine 5′-monophosphate-activated protein kinase (AMPK) and the expression of manganese-superoxide dismutase (MnSOD) and PGC-1α, inhibiting mitochondrial oxidative stress and improving mitochondrial biogenesis [58]. We also have found that bavachalcone promoted endothelial progenitor cell (EPC) differentiation and neovascularization through the AMPK pathway. Oral administration of bavachalcone for 14 days stimulated neovascularization and reestablishment of microcirculation in a rat ischemic hind limb model. The number of EPCs and the level of erythropoietin (EPO) in the circulation were increased in rats with bavachalcone treatment. Furthermore, bavachalcone enhanced the activity of RORα1 and EPO reporter genes [59]. Bahlmann et al. [60] reported that the treatment of recombinant human EPO caused a significant mobilization of circulating CD34(+)/CD45(+) EPC in the peripheral blood and increased the number of functionally active EPCs in patients and healthy subjects. Previous clinical studies and animal experiments also have shown that EPO therapy increased the number of circulating EPCs and promoted homing and improved acute myocardial infarction, heart failure, stroke, and cerebral aneurysm [61][62][63][64]. Our multiple results indicate (Figure 4) that AMPK-mediated cardiovascular protection of bavachalcone can include regulating mitochondrial biogenesis [65], delaying cell senescence [66], activating eNOS, MnSOD and PGC-1α activity and expression [67,68], and promoting EPC differentiation [69], which presents consistency with the results of other research groups.

Diabetes and Obese Intervention
Aging, diabetes, and obesity had a synergistic effect on inflammation and oxidative stress among the elderly, which in turn increased the risk of diabetes and obesity. Early interventions to reduce inflammation, oxidative stress, and insulin resistance may improve age-related diseases [70][71][72][73]. Fructus psoraleae, as an option for seeking treatment and prevention of diabetes and obesity, has the potential to improve the active life expectancy of the elderly.
A recent report revealed that psoralea prenylflavonoid extract (PFE) significantly reduced body weight and fat mass in a dose-dependent manner, increased energy expenditure, improved insulin sensitivity, and prevented hepatic steatosis by increasing lipid oxidation and secretion in high-fat dietfed mice. The study also found that PFE induced clear browning in subcutaneous white adipose tissue, increased brown adipose tissue activity, and thermogenic genes [74]. Another research group also reported that the extract of Psoralea corylifolia seeds attenuated methylglyoxal-induced insulin resistance and significantly increased the phosphorylation of Akt, IRS-1/2, and glucose uptake [75]. Moreover, this extract reduced nonalcoholic fatty liver disease, the expression of lipogenic, and inflammatory genes, such as TNF-α, IL-1β, and iNOS in high-fat diet-induced obese mice [76]. In addition, Lee et al. [45] showed that bavachin (2 μM) increased the expression and secretion of adiponectin and (i) Improved the area of cerebral infarction, brain edema, and neurobehavioral indexes 48 hours after MCAO/R (ii) Lowered active-PARP and cleaved-caspase-3 levels (iii) Inhibited IL-1β, TNF-α, and IL-6 production in ischemic cerebral homogenate (iv) Reduced NLRP3/GSDMD-mediated pyroptosis of brain tissue and cells [43] 6 Oxidative Medicine and Cellular Longevity  (i) Inhibited acidic betagalactosidase activity (ii) Induced RORα1 expression in RORα reporter luciferase activity, mRNA, and protein levels in a dose-dependent manner and enhanced the circadian amplitude of Bmal1 mRNA expression after serum shock (iii) Suppressed senescence in human endothelial cells and mRNA expression of p16 (ink4a) (a marker of replicative senescence) and IL-1α (a proinflammatory cytokine of the senescence- [57] 7 Oxidative Medicine and Cellular Longevity  Oxidative Medicine and Cellular Longevity   10 Oxidative Medicine and Cellular Longevity enhanced glucose uptake via GLUT4 translocation by activating the protein kinase B (Akt) and AMPK pathway in the presence or absence of insulin. Targeting the AMPK signaling pathway is one of the target pathways to control diabetes. Some studies have shown that diabetic patients have impaired AMPK activity, and metformin, which is used to treat diabetes, works by regulating AMPK. Natural medicines of plant origin showed great potential in regulating and activating the AMPK pathway and can be used as drug candidates for the treatment of diabetes and its complications [77,78].
Moreover, bavachin promoted lipid accumulation in 3T3-L1 cells at differentiation day 8 by activating gene expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα) [45], suggesting that bavachin might have therapeutic potential for type 2 diabetes. Like bavachin, bavachinin is a PPARγ agonist. Isolated bavachinin is a mixture of S and R configurations. A time-resolved fluorescence resonance energy transfer-based competitive binding assay showed that (S)-and (R)-bavachinin had similar PPARγ

11
Oxidative Medicine and Cellular Longevity agonist activities with IC 50 616.7 nM and 471.2 nM, respectively [79]. Subsequent in vivo studies have shown that bavachinin exhibited blood glucose-lowering properties without causing weight gain and liver toxicity. Importantly, bavachinin had synergistic effects with the synthetic PPARγ agonist thiazolidinediones and PPARα agonist fibrates, which induced PPAR transcriptional activity, as well as lowered glucose and triacylglycerol levels in db/db mice [80]. Further research found that bavachinin bound not only to the canonical site of PPAR agonists but also to a novel alternative binding site. Moreover, rosiglitazone pretreatment prevented bavachinin from binding to the canonical site instead of a novel alternative binding site [80]. The sketch of bavachinin activating PPARs to improve insulin resistance and obesity is shown in Figure 5.
Isobavachalcone caused 3T3-L1 cell cycle arrest at G0/G1 and dose-dependently inhibited the protein expression levels of cyclin D1, cdk4, and cd6 within 48 h after adipocyte differentiation medium MDI stimulation. Moreover, while isobavachalcone inhibited adipocyte fat accumulation, it also reduced intrahepatic fat deposition and hepatic steatosis in zebrafish fed high fat cholesterol diets [81]. Table 2 summarizes the experimental data related to diabetes and obesity.

Neuroprotection
Aging is the most important independent risk factor for neurodegenerative diseases. Millions of people suffer from Alzheimer's disease (AD), Parkinson's disease (PD), vascular dementia (VD), and ischemic stroke around the world. Fructus psoraleae is a promising antiaging Chinese herb which is introduced for the therapy of neurodegenerative diseases. Several studies have demonstrated that Psoralea corylifolia seed extracts can exert neuroprotection in vivo and in vitro. Alzheimer's disease (AD) is an age-related neurodegenerative disease. Evidence showed that four components of Psoralea corylifolia, named bavachin, bavachinin, bavachalcone, and isobavachalcone, differentially inhibited neuroinflammation, oxidative damage, and key AD-related protein targets, such as amyloid β-peptide 42, β-secretase, glycogen synthase kinase 3β, and acetylcholinesterase in vivo [82]. The researchers further revealed that isobavachalcone and bavachinin regulate the aggregation of aβ42 through different mechanisms. Isobavachalcone significantly inhibited both oligomerization and fibrillization of Aβ42, whereas bavachinin inhibited fibrillization and led to off-pathway aggregation. Both of the compounds attenuated Aβ42induced toxicity in an SH-SY5Y cell model [83]. Meanwhile, isobavachalcone can also effectively relieve Parkinson's disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), prolong the residence time of mice on Rota-rod, and alleviate the neuronal necrosis [84]. Isobavachalcone also inhibited the activation of microglia and decreased the expression of IL-6 and IL-1β in the brain of PD mice [80]. In vitro, isobavachalcone significantly suppressed BV-2 microglia activation and the NO production induced by LPS [84], while neobavaisoflavone had a strong inhibitory activity on H 2 O 2 -induced cell death in HT22 hippocampal cells [85]. Monoamine oxidase B (MAO-B) is an enzyme that breaks down dopamine in the brain. MAO-B inhibitors block the action of the enzyme and therefore improve the symptoms in early PD. A molecular docking test found that the C7 methoxy group of bavachinin has a high affinity for MAO-B, suggesting that bavachinin might be a selective and competitive human MAO-B inhibitor and could be used in the PD treatment [86]. The experimental details are summarized in Table 2.
Recently, bavachinin was identified as a novel agonist that activates all three PPAR isoforms, so-called pan-PPAR agonist [80], which indicated its potentially powerful application of prenylflavonoids in neuroprotection. PPARγ agonists have been demonstrated as neuroprotective agents. Pioglitazone and 15d-prostaglandin J2 significantly reduced the infarct volume, attenuated the aggregation of parenchymal neutrophils, decreased the release of TNF-α, IL-1β, and IL-6, and improved neurological function in the cerebral ischemia injury mice [87,88]; reduced β-amyloid levels effectively reversed cerebrovascular damage, improved energy metabolism and antioxidant capacity of model mice's cortex, and inhibited β-amyloid-stimulated COX-2, TNF-α, and IL-6 expression in Alzheimer's disease models [89][90][91]. Pioglitazone and MDG548 also increased the phagocytosis of microglia on necrotic cells, reduced the death of dopaminergic neurons and the production of cellular reactive oxygen species, upregulated the expression of MRC1, CD68, and IL-10, elevated the activity of glutathione-S-transferase, and blocked the activity of monoamine oxygenase B in the Parkinson's disease MPTP-mice [92][93][94].
Endogenous Nrf2 is the main regulator of the antioxidant response and an indicator of oxidative stress. Numerous research data indicated that Nrf2 mediated the neuroprotective effect of active ingredients of Chinese herbal medicine on Parkinson's disease [95,96], Alzheimer's disease [97,98], vascular dementia [99,100], and cerebral ischemiareperfusion injury [101][102][103]. In vivo and in vitro studies suggested that isobavachalcone may act as a potential neuroprotective agent by increasing Nrf2 activity and expression [30,31]. The therapeutic potential of EPO in brain plays a neuroprotective role against Alzheimer's disease [104,105], Parkinson's disease [106][107][108], chronic cerebral hypoperfusion-induced vascular dementia [109,110], and cerebral ischemia-reperfusion injury [111][112][113]. The elevation of expression and circulating concentration of EPO improved the therapeutic effects of bavachalcone and other flavonoids of Chinese herbal medicine in brain diseases [59,[114][115][116]. The neuroprotective effects of psoralea prenylflavonoids are summarized in Figure 6.

Improve Osteoporosis
Senile osteoporosis is caused by aging and affects both men and women. The evidences showed that (1) senescence of bone marrow stromal cells (BMSCs) was more likely to differentiate into adipocytes rather than osteoblasts [117,118]; (2) age-dependent bone loss is associated with increased bone cell DNA damage, cell aging, SASP, and elevated RANKL levels [119][120][121]; and (3) excessive osteoclast activity induced by aging is closely related to inflammation, which 12 Oxidative Medicine and Cellular Longevity induced osteoporosis, while anti-inflammatory drugs inhibit osteoclast formation and bone resorption [122][123][124].
In China, fructus psoraleae is often used to treat fractures, osteomalacia, and osteoporosis. A phytoestrogen effect was considered to play an important role in studies on the material basis of fructus psoraleae and its pharmacological action ( Table 2). Xin and colleagues [125] found that bavachin, isobavachalcone, and neobavaisoflavone activate estrogen receptor ERα and ERβ, while pure estrogen antagonists ICI-182,780 completely block the activation of estrogen receptors. Bavachin, isobavachalcone, and neobavaisoflavone also significantly promoted osteoblast proliferation [126]. In a model of bone loss caused by ovariectomy, in vivo experi-ments showed that bavachin prevents bone loss by upregulating the Wnt signaling pathway [127]. Wnt/β-catenin signaling pathway regulated cell fate and promoted BMSCs transfer to osteoblasts rather than adipocytes [128,129]. Neobavaisoflavone stimulated osteogenesis of MC3T3-E1 osteoblasts in a dose-dependent manner, which is manifested by notable enhancement of ALP activity, significantly increased bone-specific matrix proteins including type I collagen (Col-I), osteocalcin (OCN), and bone sialoprotein (BSP) and formation of bone nodules. Neobavaisoflavone also upregulated the mRNA expression of transcription factors Runx2 and Osterix (Osx) through a p38-dependent signaling pathway then exerted osteogenic activity [130].  13 Oxidative Medicine and Cellular Longevity Runx2, ALP, and OCN are integral factors for the control of osteogenesis. Many research reported that Runx2 can promote osteogenic differentiation and inhibit fat differentiation [131,132]. As an osteoblast-specific transcription factor, Osx is also required for osteoblast differentiation. Osx showed a transcriptional regulatory mechanism for BSP gene expression, which is related to osteoblast differentiation and the onset of mineralization [133]. Interestingly, aiming at 162 potential bone-promoting target proteins, Ge et al. [134] revealed that 8 ingredients of 23 known psoralen components, such as bavachalcone, bavachinin, and neobavaisoflavone, may be the main activities of psoralea-promoting bone formation in MC3T3-E1 cells. In summary, psoralea prenylflavonoids promoted bone formation in many ways (Figure 7).
Contrary to osteoblasts, osteoclast is a kind of cell that has a special effect on bone absorption. Disorder of bone absorption is the main culprit of many bone diseases such as osteoporosis. Bavachalcone inhibited the differentiation of precursor cells into osteoclasts by interfering with the extracellular regulated protein kinases (ERK) and Akt pathways and suppressed the expression of c-Fos and NFATc1 via receptor activator of nuclear factor κB ligand (RANKL) [135]. NFATc1 induced by the cytokine RANKL is a major regulator of the osteoclast transcriptome. NFATc1 promoted the expression of many genes, such as Itgb3, Oscar, Acp5, and Ctsk promoters, which were required for osteoclastogenesis and bone resorption [136]. NFATc1 protein expression and transcriptional activity also played important roles in inflammatory osteoclastogenesis and pathological bone loss [137,138]. An earlier study reported that bavachalcone can intervene with osteoclastogenesis by inhibiting the induction of c-Fos and NFATc1 [135]. AMPK, a negative regulator of  14 Oxidative Medicine and Cellular Longevity NFATc1 activation, antagonized osteoclastogenesis [139,140]. Estrogen and phytoestrogens also negatively regulated the RANKL-induced osteoclastic differentiation [141][142][143]. Many studies revealed that psoralea prenylflavonoids activate the AMPK pathway and present estrogen-like effects, which explained their inhibitory mechanism on osteoclastogenesis ( Figure 8).
In conclusion, this article summarizes the numerous pharmacological effects of psoralea prenylflavonoids on aging-related diseases, including anti-inflammatory action, cardiovascular protection, diabetes and obese intervention, neurodegenerative diseases improvement, and osteoporosis alleviation. This article also discusses the regulation of these active ingredients on the key pathways, such as AMPK, EPO, HIF-1α, NFATc1, NF-κB, Nrf2, Osx, PPARγ, Runx2, and Wnt/β-catenin, as well as inflammatome NLRP3. However, there is insufficient evidence to prove that these five psoralea prenylflavonoids can be used as senolytics (eliminating senescent cells) or as senomorphics (modifying SASP components) drugs and can only be considered to prevent certain aging-related signaling pathways by entering cells. The summary and discussion of the pharmacological effects and regulatory mechanisms of psoralea prenylflavonoids will help to improve the recognition of the material basis of the pharmaceutical function of fructus psoraleae and will have a positive significance for the interpretation of its pharmacological effect.

Conflicts of Interest
The authors declare that they have no competing interests.

Authors' Contributions
Yi-Ting Zhou and Lin Zhu contributed equally to this work.