Construct Phenylethanoid Glycosides Harnessing Biosynthetic Networks, Protein Engineering and One-Pot Multienzyme Cascades.

Phenylethanoid glycosides (PhGs) exhibit a multitude of structural variations linked to diverse pharmacological activities. Assembling various PhGs via multienzyme cascades represents a concise strategy over traditional synthetic methods. However, the challenge lies in identifying a comprehensive set of catalytic enzymes. This study explores biosynthetic PhG reconstruction from natural precursors, aiming to replicate and amplify their structural diversity. We discovered 12 catalytic enzymes, including four novel 6'-OH glycosyltransferases and three new polyphenol oxidases, revealing the intricate network in PhG biosynthesis. Subsequently, the crystal structure of CmGT3 (2.62 Å) was obtained, guiding the identification of conserved residue 144# as a critical determinant for sugar donor specificity. Engineering this residue in PhG glycosyltransferases (FsGT61, CmGT3, and FsGT6) altered their sugar donor recognition. Finally, a one-pot multienzyme cascade was established, where the combined action of glycosyltransferases and acyltransferases boosted conversion rates by up to 12.6-fold. This cascade facilitated the reconstruction of 26 PhGs with conversion rates ranging from 5-100 %, and 20 additional PhGs detectable by mass spectrometry. PhGs with extra glycosyl and hydroxyl modules demonstrated notable liver cell protection. This work not only provides catalytic tools for PhG biosynthesis, but also serves as a proof-of-concept for cell-free enzymatic construction of diverse natural products.

Identification of Potential Neddylation-related Key Genes in Ischemic Stroke based on Machine Learning Methods.

Ischemic stroke (IS) is a complex neurological disease that can lead to severe disability or even death. Understanding the molecular mechanisms involved in the occurrence and progression of IS is of great significance for developing effective treatment strategies. In this context, the role of neddylation refers to the potential impact of neddylation on various cellular processes, which may contribute to the pathogenesis and outcome of IS. First, differential analysis was conducted on the GSE16561 dataset from the GEO database to identify 350 differentially expressed genes (DEGs) between the IS and Control groups. By intersecting the differential genes with neddylation-related genes, 11 neddylation-related DEGs were obtained. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses showed that the DEGs were mainly enriched in hematopoietic cell lineage and neutrophil degranulation, while the neddylation-related DEGs were mainly enriched in apoptosis and post-translational protein modification. Further Lasso-Cox and random forest analyses were performed on the 11 neddylation-related DEGs, identifying key genes SRPK1, BIRC2, and KLHL3. Additionally, validation of the key genes was carried out using the GSE58294 dataset and clinical patients. Finally, the correlation between the key genes and ferroptosis and cuproptosis was analyzed, and a ceRNA network was constructed. Our study helps to elucidate the complex role of neddylation in the mechanism of ischemic stroke, providing potential opportunities for the development of therapeutic interventions.

Diet and exercise interventions reduce serum asprosin and the corresponding hypothalamic- pituitary-gonad-axis dysfunction in obese men.

Background: Asprosin (ASP) is a recently discovered adipocyte factor that participates in glucose metabolism and inflammatory reactions. Recent findings suggest that it may be involved in the regulation of sex hormone secretion in the hypothalamic-pituitary-gonad (HPG) axis, but no studies have been reported in related populations. The purpose of this study was to evaluate the changes in serum ASP levels in healthy men and obese men, as well as before and after exercise weight loss, and to investigate male hypogonadism, insulin resistance, inflammatory response, and relationships induced by ASP and obesity. Methods: Thirty-eight young male volunteers were recruited and divided into a normal group (n = 20) and an obese group (n = 18) according to their body mass index. Fourteen of the obese men underwent a 14-week exercise and diet intervention (first 8 weeks of aerobic exercise at 60%-70% HRmax for 30-50 min/4 days a week). Beginning at week 9, the intensity was increased to 75% HRmax. Participants in the obese groups maintained a calorie-restricted diet throughout the study period. Results: Serum ASP levels in the obese group were significantly higher than those in the normal group, and serum gonadotropin-releasing hormone (GnRh), luteinizing hormone (LH), and testosterone (T) levels were decreased. After 14 weeks of exercise and diet intervention, serum ASP decreased significantly, the levels of body weight, lean body weight, body fat rate, fasting insulin (FINS), homeostatic model assessment for insulin resistance, TNF-α, IL-6, and IL-1ß decreased significantly, and the serum GnRH, LH, and T levels increased significantly. ASP was positively correlated with body weight, body fat percentage, FINS, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß and negatively correlated with relative lean body weight and serum LH and T levels. Conclusion: The serum ASP levels were increased in obese men compared with those of normal weight individuals, resulting in a chronic inflammatory reaction, high serum insulin, and HPG axis injury. Fourteen weeks of exercise and diet intervention effectively alleviated this phenomenon. It has been speculated that ASP might regulate male reproductive function by regulating the inflammatory response and insulin sensitivity.

Single-cell transcriptomics reveals the natural product Shi-Bi-Man promotes hair regeneration by activating the FGF pathway in dermal papilla cells.

BACKGROUND: Finasteride and minoxidil are two commonly used drugs for the treatment of hair loss. However, these two drugs have certain side effects. Thus, the further elucidation of treatments for hair loss, including those using Chinese herbal medicine, remains important clinically. Shi-Bi-Man (SBM) is a hair health supplement that darkens hair and contains ginseng radix, tea polyphenols, polygonum multiflorum, radix angelicae sinensis, aloe, linseed, and green tea extract. PURPOSE: This study aimed to find potential effective monomer components to promote hair regeneration from SBM and to explore the mechanism of SBM to promote hair regeneration. METHODS: Supplementation with the intragastric administration or smear administration of SBM in artificially shaved C57BL/6 mice, observe its hair growth. UPLC/MS and UPLC/LTQ-Orbitrap-MS detect the main components in SBM and the main monomers contained in the skin after smearing, respectively. A network pharmacology study on the main components of SBM and single-cell RNA sequencing was performed to explore the role of SBM for hair regeneration. RESULTS: SBM significantly induced hair growth compared with a control treatment. TSG and EGCG were the main monomers in the skin after SBM smearing. The results of single-cell sequencing revealed that after SBM treatment, the number of hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs) increased significantly. Cell interactions and volcano dots show that the interaction of the FGF signaling pathway was significantly enhanced, in which Fgf7 expression was especially upregulated in DPCs. In addition, the Wnt signaling pathway also had a partially enhanced effect on the interactions between various cells in the skin. The network pharmacology study showed that the promotion of the FGF and Wnt pathways by SBM was also enriched in alopecia diseases. CONCLUSION: We report that SBM has a potential effect on the promotion of hair growth by mainly activating the FGF signaling pathway. The use of SBM may be a novel therapeutic option for hair loss.

Re-Du-Ning injection ameliorates radiation-induced pneumonitis and fibrosis by inhibiting AIM2 inflammasome and epithelial-mesenchymal transition.

BACKGROUND: Radiation-induced lung injury (RILI) is a common side effect in chest radiotherapy patients, and there is no good medicine to treat it. Re-Du-Ning (RDN) injection is a traditional Chinese medicine that is clinically used to treat upper respiratory tract infections and acute bronchitis. RDN has the advantage of high safety and mild side effects. The mechanism of most traditional Chinese medicine preparations is unknown. PURPOSE: To illustrate the mechanisms of RDN for the treatment of RILI. METHODS: Female C57BL/6 mice were used to establish a RILI model via irradiation, and RDN injection was intraperitoneally administered at doses of 5, 10, and 20 ml/kg. The cytokines were measured by ELISA and qPCR. The data related to Absent in melanoma 2 (AIM2) inflammasome were analyzed via ELISA and a network pharmacological approach. In addition, the data related to epithelial-mesenchymal transition (EMT) were analyzed via immunofluorescence, Western blotting, and a network pharmacological approach. RESULTS: RDN robustly alleviated RILI. Meanwhile, RDN downregulated inflammatory cells' infiltration and the expression of pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α. Next, the potential molecular mechanisms of RDN were predicted through network pharmacology analysis. RDN may ameliorate radiation pneumonitis (RP) by inhibiting AIM2-mediated pyroptosis. Moreover, RDN treatment inhibited EMT and phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway. The active compounds from Lonicera japonica Thunb. decreased the phosphorylation of Akt. CONCLUSION: These findings demonstrate that RDN, as a traditional Chinese medicine preparation, will be a candidate drug for treating RILI.

Tai Chi for spatiotemporal gait features and dynamic balancing capacity in elderly female patients with non-specific low back pain: A six-week randomized controlled trial.

BACKGROUND: Non-specific low back pain (NS-LBP) is a serious public health problem. Tai Chi is promising in reducing the risk of falls and alleviating symptoms in this population. OBJECTIVE: To investigate the effect of Tai Chi on gait and dynamic balance in elderly women with NS-LBP. METHODS: 20 women (age > 65 yr.) with NS-LBP were randomly assigned to a Tai Chi group (n= 10) or a control group (n= 10). The Tai Chi group practiced Tai Chi exercise 3 times a week for 6 weeks. Each session lasted 60 minutes. Pain, spatiotemporal gait features and dynamic balancing capacity were assessed at 0 and 6 weeks. RESULTS: Compared to the control group at 6 weeks, the Tai Chi group had a significant decrease in VAS (p= 0.027) and stride width (p= 0.019), significant improvement in gait velocity, stride length (p< 0.001). Regarding dynamic balance capacity, the Tai Chi group had significant improvements in anterior (Left: p= 0.001; Right: p= 0.038), postero-lateral (Left: p< 0.001; Right: p= 0.038), and postero-medial (Left: p= 0.015; Right: p= 0.018). CONCLUSION: 6-week Tai Chi can relieve pain and improve gait and dynamic balance in elderly women with NS-LBP, which suggests Tai Chi could be a promising rehabilitation intervention to reduce the risk of falls in this population.

A cross-sectional comparative study on the effects of body mass index and exercise/sedentary on serum asprosin in male college students.

Adipocytes regulate the body's metabolism by secreting adipokines to maintain energy homeostasis. Asprosin is a new type of adipokine, and its relationship with obesity remains controversial. There are a few reports on the effect of long-term exercise on serum asprosin level. This study aimed to investigate the effects of body mass index (BMI) and exercise/sedentary habit on serum asprosin in male college students as well as the relationship between serum asprosin and body composition and related metabolic indicators and provided a basis for further exploration of the biological function of asprosin. Ninety-six male college students were classified into the sedentary habit group (SD; 48) and the special training experience group (ET; 48). Both groups included three subgroups of normal BMI, overweight, and obesity, with 16 people in each subgroup. One-way analysis of variance, analysis of covariance, and Pearson correlation analysis were performed. The results showed that serum asprosin levels in the obesity subgroup were significantly higher than those in the normal and overweight subgroups. Excluding BMI interference, there were no significant differences in serum asprosin between the SD and ET groups; however, there were significant differences in body composition, tumor necrosis factor-α, interleukin-6, and interleukin-10. Asprosin was positively correlated with BMI, body fat percentage, visceral fat area, fasting insulin, insulin resistance homeostasis model, total cholesterol, low-density lipoprotein, tumor necrosis factor-α, interleukin-6, and leptin levels and was negatively correlated with relative lean body mass, relative skeletal muscle mass, high-density lipoprotein, and interleukin-10, and adiponectin levels. In conclusion, serum asprosin is closely related to body weight, body composition, glucose and lipid metabolism, inflammatory response, and fat hormones. Long-term exercise training cannot prevent BMI increase from increasing serum asprosin level. If the influence of BMI is excluded, long-term exercise training does not affect serum asprosin.

SHP2 allosteric inhibitor TK-453 alleviates psoriasis-like skin inflammation in mice via inhibition of IL-23/Th17 axis.

SHP2 is the first oncogenic tyrosine phosphatase encoded by PTPN11, which plays a significant regulatory role in cancer and inflammation-related diseases. Although SHP2 allosteric inhibitors have been used in phase I/II clinical trials for solid tumors, whether SHP2 inhibition alleviates psoriasis remains unclear. Here we expressed and purified SHP2 related proteins, and established an enzyme activity screening system for different conformations of SHP2. We launched an iterative medicinal chemistry program and identified the lead compound, TK-453. Importantly, TK-453 possessed stronger affinity with SHP2 than SHP099, evidenced by the cocrystal structure of SHP2/TK-453, revealing that the additional aryl-S-aryl bridge in TK-453 induces a 1.8 Å shift of the dichlorophenyl ring and an approximate 20° deviation of the pyrazine ring plane relative to SHP099. Furthermore, TK-453 significantly ameliorated imiquimod-triggered skin inflammation in mice via inhibition of the IL-23/Th17 axis, proving that SHP2 is a potential therapeutic target for psoriasis.

Re-Du-Ning injection ameliorates LPS-induced lung injury through inhibiting neutrophil extracellular traps formation.

BACKGROUND: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening diseases and could occur in severe COVID-19 patients. Re-Du-Ning injection (RDN) is a tradition Chinese medicine preparation which has been clinically used for treatment of respiratory diseases including COVID-19. PURPOSE: To elucidate the potential mechanisms of RDN for the treatment of ALI. METHODS: Female C57BL/6J mice were used to establish ALI model by intraperitoneal injection 10 mg/kg LPS, and RDN injection was intraperitoneally administered with the dose of 5 and 10 ml/kg. The cytokines were measured by ELISA and qPCR. The data related to NETs were analyzed by ELISA, immunofluorescence, Western blotting and network pharmacological approach. RESULTS: RDN robustly alleviated LPS-induced ALI. Meanwhile, RDN downregulated the expression of pro-inflammatory cytokines, such as IL-1ß, IL-6 and TNF-α. Specifically, RDN treatment inhibited the formation of neutrophil extracellular traps (NETs) and remarkably suppressed the protein of PAD4. The active compound from RDN decreased the phosphorylation of ERK1/2. CONCLUSION: These findings demonstrate that RDN ameliorates LPS-induced ALI through suppressing MAPK pathway to inhibit the formation of NETs.

HSCs transdifferentiate primarily to pneumonocytes in radiation-induced lung damage repair.

Accumulative radiation exposure leads to hematopoietic or tissue aging. Whether hematopoietic stem cells (HSCs) are involved in lung damage repair in response to radiation remains controversial. The aim of this study is to identify if HSC can transdifferentiate to pneumonocytes for radiation-induced damage repair. To this end, HSCs from male RosamT/mG mice were isolated by fluorescence-activated cell sorting (FACS) and transplanted into lethally irradiated female CD45.1 mice. 4 months after transplantation, transplanted HSC was shown to repair the radiation-induced tissue damage, and donor-derived tdTomato (phycoerythrin, PE) red fluorescence cells and Ddx3y representing Y chromosome were detected exclusively in female recipient lung epithelial and endothelial cells. Co-localization of donor-derived cells and recipient lung tissue cells were observed by laser confocal microscopy and image flow cytometry. Furthermore, the results showed HSC transplantation replenished radiation-induced lung HSC depletion and the PE positive repaired lung epithelial cells were identified as donor HSC origin. The above data suggest that donor HSC may migrate to the injured lung of the recipient and some of them can be transdifferentiated to pneumonocytes to repair the injury caused by radiation.

Guizhi Fuling Capsule ameliorates endometrial hyperplasia through promoting p62-Keap1-NRF2-mediated ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Guizhi Fuling Capsule (GFC) is a classical traditional Chinese medicine officially recorded in Synopsis of the Golden Chamber and has long been used to treat gynecological diseases in China. However, scientific evidence for the anti-endometrial hyperplasia potential of GFC used in traditional medicine is lacking. AIM OF THE STUDY: This study evaluated whether GFC protects against endometrial hyperplasia and its potential mechanism in mice. METHODS AND MATERIALS: We used estrogen (estradiol) to induce endometrial hyperplasia in mice. C57BL/6 mice were treated with estradiol subcutaneously for 21 days, and GFC (75 mg/kg and 150 mg/kg) was given intragastric administration from the first day of the modeling. H&E staining is used to evaluate endometrial tissue structure change. Malondialdehyde was measured to explore lipid peroxidation. Western blot, immunohistochemistry and immunofluorescence were performed to observe the expressions of GPX4, p62, Keap1 and NRF2. RESULTS: The degree of ferroptosis in endometrial tissue of patients with endometrial hyperplasia was lower than normal endometrial tissue. In addition, ferroptosis inducer imidazole ketone erastin could improve endometrial hyperplasia in mice. Interestingly, GFC significantly alleviated endometrial hyperplasia through triggering ferroptosis. Furthermore, GFC inhibited p62-Keap1-NRF2 pathway in estradiol-induced endometrial hyperplasia model. CONCLUSIONS: GFC may attenuate estrogen-induced endometrial hyperplasia in mice through triggering ferroptosis via inhibiting p62-Keap1-NRF2 pathway. These findings suggest that GFC might act as a promising traditional Chinese medicine to treat endometrial hyperplasia.

Leptin and inflammatory factors play a synergistic role in the regulation of reproduction in male mice through hypothalamic kisspeptin-mediated energy balance.

BACKGROUND: Energy balance is closely related to reproductive function, wherein hypothalamic kisspeptin mediates regulation of the energy balance. However, the central mechanism of kisspeptin in the regulation of male reproductive function under different energy balance states is unclear. Here, high-fat diet (HFD) and exercise were used to change the energy balance to explore the role of leptin and inflammation in the regulation of kisspeptin and the hypothalamic-pituitary-testis (HPT) axis. METHODS: Four-week-old male C57BL/6 J mice were randomly assigned to a normal control group (n = 16) or an HFD (n = 49) group. After 10 weeks of HFD feeding, obese mice were randomly divided into obesity control (n = 16), obesity moderate-load exercise (n = 16), or obesity high-load exercise (n = 17) groups. The obesity moderate-load exercise and obesity high-load exercise groups performed exercise (swimming) for 120 min/day and 120 min × 2 times/day (6 h interval), 5 days/week for 8 weeks, respectively. RESULTS: Compared to the mice in the normal group, in obese mice, the mRNA and protein expression of the leptin receptor, kiss, interleukin-10 (IL-10), and gonadotropin-releasing hormone (GnRH) decreased in the hypothalamus; serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone levels and sperm quality decreased; and serum leptin, estradiol, and tumor necrosis factor-α (TNF-α) levels and sperm apoptosis increased. Moderate- and high-load exercise effectively reduced body fat and serum leptin levels but had the opposite effects on the hypothalamus and serum IL-10 and TNF-α levels. Moderate-load exercise had anti-inflammatory effects accompanied by increased mRNA and protein expression of kiss and GnRH in the hypothalamus and increased serum FSH, LH, and testosterone levels and improved sperm quality. High-load exercise also promoted inflammation, with no significant effect on the mRNA and protein expression of kiss and GnRH in the hypothalamus, serum sex hormone level, or sperm quality. Moderate-load exercise improved leptin resistance and inflammation and reduced the inhibition of kisspeptin and the HPT axis in obese mice. The inflammatory response induced by high-load exercise may counteract the positive effect of improving leptin resistance on kisspeptin and HPT. CONCLUSION: During changes in energy balance, leptin and inflammation jointly regulate kisspeptin expression on the HPT axis.

Autophagy-deficient mice are more susceptible to engrafted leukemogenesis.

Autophagy is primarily considered as an important survival mechanism for both normal cells and cancer cells in response to metabolic stress or chemotherapy; but the role of autophagy in leukemogenesis is not fully understood. The aim of this study is to explore the role of intrinsic autophagy in the leukemogenesis of B-cell acute lymphoblastic leukemia (B-ALL). In this study, conditional knockout mice Atg7f/f;Ubc-Cre, in which an autophagy-essential gene Atg7 is universally deleted, were used as recipients, B-ALL cell line 697 was used as donor cells to generate leukemia mouse model. Compared to wild-type mice, Atg7 knockout mice were more susceptible to engrafted leukemogenesis, shown by increase in white blood cells, lymphocytes, and platelets, decrease in HSPC number and its colony-forming unit (CFU). The liver and spleen displayed hepatosplenomegaly and inflammatory cell infiltration. Furthermore, second competitive transplantation revealed dysfunction of the HSPC in Atg7-knockout leukemia mice represented by destructive self-renew ability (CFU) and reconstitution ability including decreased B220, Ter 119 cells, and increased Gr-1 cell percentage. In summary, Mice with universal deletion of Atg7 are more inclined to the occurrence of engrafted human leukemia, which is largely attributed to the deterioration of the function of HSPC in autophagy deficient mice.

[Analysis of dietary iron intakes of pregnant and lactating women based on food composition databases from China and US].

OBJECTIVE: To evaluate the difference between estimations on dietary iron intake based on the China and US food composition databases. METHODS: Total 368 records of 24-h dietary recall on mid-term pregnant women and lactating rural women were analyzed for their iron intakes with the China Food Composition Table 2002 and the USDA National Nutrient Database for Standard Reference release 25, respectively. The values of dietary iron intake derived from two composition databases were compared statistically. RESULTS: The dietary iron intakes of total 368 dietary records estimated with China and US databases were (24.37 +/- 9.66) mg and (16.20 +/- 9.13) mg respectively (with paired t test, t = 20.081, P < 0.01, correlation coefficient r = 0.657, P < 0.01), with average ratio of 1.69 +/- 0.55 between China and US values. In terms of food classification, the most significant differences were with dairy products, fishes, fruits, meats and cereals, with ratios of (10.66 +/- 2.24), (5.10 +/- 5.51), (3.01 +/- 2.26), (3.01 +/- 7.85) and (2.33 +/- 0.77), respectively. Only iron intake values from tuberous crops and soy products had inverse ratio as (0.83 +/- 0.50) and (0.75 +/- 0.53). CONCLUSION: The averaged dietary iron intake value for total records was very close to the reported national levels when estimated with the China Food Composition Table 2002, but much decreased when estimated with US food composition database with values as only approximately 66% of reported levels.