Dual Key-Activated Nir-I/II Fluorescence Probe for Monitoring Photodynamic and Photothermal Synergistic Therapy Efficacy.

As cancer markers, hydrogen peroxide (H2 O2 ) and viscosity play an essential role in the development of tumors. Meanwhile, based on the performance of near-infrared (NIR) fluorescence imaging and the high efficiency of photodynamic therapy (PDT) and photothermal therapy (PTT) synergistic therapy, it is urgent to develop a dual-key (H2 O2 and viscosity) activated fluorescence probe for cancer phototherapy. Herein, a NIR-I/II fluorescence probe named BX-B is reported. In the presence of both H2 O2 and viscosity, the fluorescence signal of NIR-I (810 nm) and NIR-II (945 nm) can be released. In the presence of H2 O2 , the PDT and PTT effects are observed. BX-B is used to monitor its therapeutic effects in cancer cells and tumor-bearing mice due to the increased viscosity caused by PDT and PTT. In addition, the tumors of mice treated with BX-B are almost completely ablated after the laser irradiation based on its PDT and PTT synergistic therapy. This work provides a reliable platform for effective cancer treatment and immediate evaluation of therapeutic effects.

Caffeoyl-coenzyme A O-methyltransferase mediates regulation of carbon flux fluctuations during phenylpropenes and lignin biosynthesis in the vegetative organ roots of Asarum sieboldii Miq.

Asarum sieboldii Miq. possesses remarkable medicinal value due to its essential oil enriched with phenylpropenes (e.g., methyleugenol and safrole). Although the biosynthesis of phenylpropenes shares a common pathway with lignin, the regulation mechanisms in carbon flux allocation between them are unclear. This study is the first to genetically verify the carbon flux regulation mechanism in A. sieboldii roots. We regulated the expression of Caffeoyl-coenzyme A O-methyltransferase (CCoAOMT), an essential enzyme in the common pathway, to investigate carbon flux allocation in vegetative organs. Here, the lignin and phenylpropene content fluctuation was analyzed by wet chemistry and GC-MS methods. A bona fide CCoAOMT gene from A. sieboldii was firstly cloned and verified. Preliminary heterologous expression validation in transgenic Arabidopsis thaliana showed that RNAi-induced CCoAOMT down-regulation significantly decreased lignin content by 24% and increased the S/G ratio by 30%; however, AsCCoAOMT over-expression in A. thaliana resulted in a 40% increase in lignin content and a 20% decrease in the S/G ratio when compared to the wild type. Similar trends were noted in homologous transformation in A. sieboldii, although the variations were not conspicuous. Nevertheless, the transgenic A. sieboldii plants displayed substantial differences in the level of phenylpropene compounds methyleugenol and safrole leading to a 168% increase in the methyleugenol/safrole ratio in the over-expression line and a 73% reduction in RNAi-suppression line. These findings suggest that the biosynthesis of phenylpropene constituents methyleugenol and safrole seems to be prioritized over lignin. Furthermore, this study indicated that suppression of AsCCoAOMT resulted in marked root susceptibility to pathogenic fungal disease, implying a significant additional role of CCoAOMT in protecting plant vegetative parts from diseases. Overall, the present study provides important references and suggests that future research should be aimed at elucidating the detailed mechanisms of the carbon flux allocation between phenylpropenes and lignin biosynthesis, as well as the disease resistance competency.

Herbal small RNAs in patients with COVID-19 linked to reduced DEG expression.

In China, more than 80% of patients with coronavirus disease 2019 (COVID-19) received traditional Chinese medicine (TCM) as a treatment and their clinical efficacy have been reported. However, the underlying molecular mechanism remains unclear. Previous studies have identified herbal small RNAs (sRNAs) as novel functional components. In this study, a cohort of 22 patients with COVID-19 treated with Toujie Quwen (TQ) granules was analyzed. We observed thousands of herbal small RNAs that entered the blood cells of patients after the consumption of TQ granules. In response to this treatment, the reduced differentially expressed genes (DEGs) were highly correlated with the predicted target genes of the most prevalent herbal sRNAs detected in the blood. Moreover, the predicted target genes of the top 30 sRNAs from each of the 245 TCMs in the Bencao sRNA Atlas overlapped with 337 upregulated DEGs in patients with mild COVID-19, and 33 TCMs, with more than 50% overlapping genes were identified as effective TCMs. These predicted target genes of top 30 sRNAs from Juhong, Gualoupi and Foshou were confirmed experimentally. Our results not only elucidated a novel molecular mechanism of TCM potential clinical efficacy for COVID-19 patients, but also provided 33 effective COVID-19 TCMs for prescription optimization.

Identification and characterization of a heme exporter from the MRP family in Drosophila melanogaster.

BACKGROUND: The heme group constitutes a major functional form of iron, which plays vital roles in various biological processes including oxygen transport and mitochondrial respiration. Heme is an essential nutrient, but its pro-oxidant nature may have toxic cellular effects if present at high levels, and its synthesis is therefore tightly regulated. Deficiency and excess of heme both lead to pathological processes; however, our current understanding of metazoan heme transport is largely limited to work in mammals and the worm Caenorhabditis elegans, while functional analyses of heme transport in the genetically amenable Drosophila melanogaster and other arthropods have not been explored. RESULTS: We implemented a functional screening in Schneider 2 (S2) cells to identify putative heme transporters of D. melanogaster. A few multidrug resistance-associated protein (MRP) members were found to be induced by hemin and/or involved in heme export. Between the two plasma membrane-resident heme exporters CG4562 and CG7627, the former is responsible for heme transit across the intestinal epithelium. CG4562 knockdown resulted in heme accumulation in the intestine and lethality that could be alleviated by heme synthesis inhibition, human MRP5 (hMRP5) expression, heme oxygenase (HO) expression, or zinc supplement. CG4562 is mainly expressed in the gastric caeca and the anterior part of the midgut, suggesting this is the major site of heme absorption. It thus appears that CG4562 is the functional counterpart of mammalian MRP5. Mutation analyses in the transmembrane and nucleotide binding domains of CG4562 characterized some potential binding sites and conservative ATP binding pockets for the heme transport process. Furthermore, some homologs in Aedes aegypti, including that of CG4562, have also been characterized as heme exporters. CONCLUSIONS: Together, our findings suggest a conserved heme homeostasis mechanism within insects, and between insects and mammals. We propose the fly model may be a good complement to the existing platforms of heme studies.

Drosophila ZnT1 is essential in the intestine for dietary zinc absorption.

Zinc is an essential trace element and participates in a variety of biological processes. ZnT (SLC30) family members are generally responsible for zinc efflux across the membrane regulating zinc homeostasis. In mammals, the only predominantly plasma membrane resident ZnT has been reported to be ZnT1, and ZnT1-/ZnT1- mice die at the embryonic stage. In Drosophila, knock down of ZnT1 homologue (dZnT1//ZnT63C/CG17723) results in growth arrest under zinc-limiting conditions. To investigate the essentiality of dZnT1 for zinc homeostasis, as well as its role in dietary zinc uptake especially under normal physiological conditions, we generated dZnT1 mutants by the CRISPER/Cas9 method. Homozygous mutant dZnT1 is lethal, with substantial zinc accumulation in the iron cell region, posterior midgut as well as gastric caeca. Expression of human ZnT1 (hZnT1), in the whole body or in the entire midgut, fully rescued the dZnT1 mutant lethality, whereas tissue-specific expression of hZnT1 in the iron cell region and posterior midgut partially rescued the developmental defect of the dZnT1 mutant. Supplementation of zinc together with clioquinol or hinokitiol conferred a limited but observable rescue upon dZnT1 loss. Our work demonstrated the absolute requirement of dZnT1 in Drosophila survival and indicated that the most essential role of dZnT1 is in the gut.

Effects of light irradiation on essential oil biosynthesis in the medicinal plant Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim) Kitag.

Asarum heterotropoides Fr. var. mandshuricum (Maxim) Kitag (Chinese wild ginger) is an important medicinal herb. Essential oil extracted from its roots is the key ingredient and is mainly composed of phenylpropanoid compounds. As a skiophyte plant, light is a crucial factor for A. heterotropoides var. mandshuricum growth and metabolism. To investigate the effects of light irradiation on the essential oil biosynthesis in A. heterotropoides var. mandshuricum, the plants were cultivated in four light irradiation treatments (100, 50, 24 and 12% full sunlight). The photosynthetic capacity, essential oil content and composition, activities of several enzymes and levels of some secondary metabolites involved in the shikimic acid and cinnamic acid pathways were analyzed. The leaf mass per area, average diurnal net photosynthetic rate, and the essential oil content increased significantly with increasing light intensity. Phenylalanine, cinnamic acid, and p-coumaric acid in the cinnamic acid pathway were at their highest levels in plants cultivated in 100% full sunlight. The highest content of shikimic acid in the shikimic acid pathway was obtained in plants grown in 50% sunlight transmittance. The activity of the enzymes 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase, phenylalanine ammonia lyase, cinnamate-4-hydroxylase and 4-coumarate:CoA ligase increased proportionally with light intensity. Overall, we conclude that high light irradiation promotes high net photosynthetic rate, high activity of enzymes and high amounts of phenylpropanoid precursor metabolites leading to significant biosynthesis of essential oil in A. heterotropoides var. mandshuricum.

Erratum to: Herbal decoctosome is a novel form of medicine.

Following the published article, we noticed an error duplication in Figure 5G "control" and "PGY-6" that was introduced during the revised process, with an attempt to replace it with higher-resolution images. Here we provide the original data in the first submitted manuscript (Figure 5G).

Iron Overload Damages the Endothelial Mitochondria via the ROS/ADMA/DDAHII/eNOS/NO Pathway.

It has been recognized that iron overload may harm the body's health. Vascular endothelial cells (VECs) are one of the main targets of iron overload injury, and the mechanism involved was thought to be related to the excessive generation of reactive oxygen species (ROS). However, the subcellular and temporal characteristics of ROS generation, potential downstream mechanisms, and target organelles in VECs injured by iron overload have not been expounded yet. In this study, we elucidated the abovementioned issues through both in vivo and in vitro experiments. Mice were fed pellet diets that were supplemented with iron for 4 consecutive months. Results showed that the thoracic aortic strips' endothelium-dependent dilation was significantly impaired and associated with inflammatory changes, noticeable under brown TUNEL-positive staining in microscopy analysis. In addition, the serum content of asymmetric dimethylarginine (ADMA) increased, whereas nitric oxide (NO) levels decreased. Furthermore, the dimethylarginine dimethylaminohydrolase II (DDAHII) expression and activity, as well as the phosphorylation of endothelial nitric oxide synthase (eNOS) in aortic tissue, were inhibited. Human umbilical vein endothelial cells were treated with 50 µM iron dextran for 48 hours, after which the cell viability, NO content, DDAHII expression and activity, and phosphorylation of eNOS decreased and lactate dehydrogenase and caspase-3 activity, ADMA content, and apoptotic cells significantly increased. After the addition of L-arginine (L-Arg) or pAD/DDAHII, the abovementioned changes were reversed. By dynamically detecting the changes of ROS generation in the cytoplasm and mitochondria and interfering with different aspects of signaling pathways, we have confirmed for the first time that excessive ROS originates from the cytoplasm and activates the ROS-induced ROS release (RIRR) mechanism, leading to mitochondrial dysfunction. Together, our data suggested that excessive free iron ions produced excess ROS in the cytoplasm. Thus, excess ROS create one vicious circle by activating the ADMA/eNOS/DDAHII/NO pathway and another vicious circle by activation of the RIRR mechanism, which, when combined, induce a ROS burst, resulting in mitochondrial dysfunction and damaged VECs.

[Photosynthetic characteristics and active ingredients differences of Asarum heterotropoides var. mandshuricum under different light irradiance].

Chlorophyll content,leaf mass to per area,net photosynthetic rate and bioactive ingredients of Asarum heterotropoides var. mandshuricum,a skiophyte grown in four levels of solar irradiance were measured and analyzed in order to investigate the response of photosynthetic capability to light irradiance and other environmental factors. It suggested that the leaf mass to per area of plant was greatest value of four kinds of light irradiance and decreasing intensity of solar irradiance resulted in the decrease of leaf mass to per area at every phenological stage. At expanding leaf stage,the rate of Chla and Chlb was 3. 11 when A. heterotropoides var. mandshuricum grew in full light irradiance which is similar to the rate of heliophytes,however,the rate of Chla and Chlb was below to 3. 0 when they grew in shading environment. The content of Chla,Chlb and Chl( a+b) was the greatest value of four kinds of light irradiance and decreasing intensity of solar irradiance resulted in its decreasing remarkably( P<0. 05). The rate of Chla and Chlb decreased but the content of Chla,Chlb and Chl( a+b) increased gradually with continued shading. The maximum value of photosynthetically active radiation appeared at 10: 00-12: 00 am in a day. The maximum value of net photosynthetic rate appeared at 8: 30-9: 00 am and the minimum value appeared at 14: 00-14: 30 pm at each phenological stage if plants grew in full sunlight. However,when plants grew in shading,the maximum value of net photosynthetic rate appeared at about 10: 30 am and the minimum value appeared at 12: 20-12: 50 pm at each phenological stage. At expanding leaf stage and flowering stage,the average of net photosynthetic rate of leaves in full sunlight was remarkably higher than those in shading and it decreased greatly with decreasing of irradiance gradually( P < 0. 05). However,at fruiting stage,the average of net photosynthetic rate of leaves in full sunlight was lower than those in 50% and 28% full sunlight but higher than those in 12% full sunlight. All photosynthetic diurnal variation parameters of plants measured in four kinds of different irradiance at three stages were used in correlation analysis. The results suggested that no significant correlation was observed between net photosynthetic rate and photosynthetically active radiation,and significant negative correlation was observed between net photosynthetic rate and environmental temperature as well as vapor pressure deficit expect for 12% full sunlight. Positive correlation was observed between net photosynthestic rate and relative humidity expect for 12% full sunlight. Significant positive correlation was observed between net photosynthetic rate and stomatal conductance in the four light treatments. Only,in 12% full sunlight,the net photosynthetic rate was significantly related to photosynthetically active radiation rather than related to environmental temperature,vapor pressure deficit and relative humidity. In each light treatment,a significant positive correlation was observed between environmental temperature and vapor pressure deficit,relative humidity as well as stomatal conductance. Volatile oil content was 1. 46%,2. 16%,1. 56%,1. 30% respectively. ethanol extracts was 23. 44%,22. 45%,22. 18%,21. 12% respectively. Asarinin content was 0. 281%,0. 291%,0. 279% and 0. 252% respectively. The characteristic components of Asarum volatile oil of plant in different light treatments did not change significantly among different groups.

Herbal decoctosome is a novel form of medicine.

Traditionally, herbal medicine is consumed by drinking decoctions produced by boiling herbs with water. The functional components of the decoction are heat stable. Small RNAs (sRNAs) were reported as a new class of functional components in decoctions. However, the mechanisms by which sRNAs survive heat treatment of the decoction and enter cells are unclear. Previous studies showed that plant-derived exosome-like nanoparticles (ELNs), which we call botanosomes, could deliver therapeutic reagents in vivo. Here, we report that heat-stable decoctosomes (ELNs) from decoctions have more therapeutic effects than the decoctions in vitro and demonstrate therapeutic efficacy in vivo. Furthermore, sRNAs, such as HJT-sRNA-m7 and PGY-sRNA-6, in the decoctosome exhibit potent anti-fibrosis and anti-inflammatory effects, respectively. Decoctosome is comprised of lipids, chemical compounds, proteins, and sRNAs. A medical decoctosome mimic is called bencaosome. A single lipid sphinganine (d22:0) identified in the decoctosome was mixed and heated with the synthesized sRNAs to form the simplest bencaosome. This simple bencaosome structure was identified by critical micelle concentration (cmc) assay that sRNAs coassembled with sphinganine (d22:0) to form the lipid layers of vesicles. The heating process facilitates co-assembly of sRNAs and sphinganine (d22:0) until a steady state is reached. The artificially produced sphinganine-HJT-sRNA-m7 and sphinganine- PGY-sRNA-6 bencaosomes could ameliorate bleomycin-induced lung fibrosis and poly(I:C)-induced lung inflammation, respectively, following oral administration in mice. Our study not only demonstrates that the herbal decoctosome may represent a combinatory remedy in precision medicine but also provides an effective oral delivery route for nucleic acid therapy.

Plant-derived phosphocholine facilitates cellular uptake of anti-pulmonary fibrotic HJT-sRNA-m7.

Pulmonary fibrosis, a progressive chronic disease with a high mortality rate, has limited treatment options. Currently, lung transplantation remains the only effective treatment. Here we report that a small RNA, HJT-sRNA-m7, from a Chinese herbal medicine Hong Jing Tian (HJT, RHODIOHAE CRENULATAE RADIX ET RHIZOMA, Rhodiola crenulata) can effectively reduce the expressions of fibrotic hallmark genes and proteins both in alveolar in vitro and in mouse lung tissues in vivo. We also discovered over one hundred oil-soluble chemicals from HJT decoctions, most of which are found in lipid extracts from other Chinese herbals decoctions, including Pu Gong Ying (PGY, TARAXACI HERBA, Taraxacum mongolicum), Chuan Xin Lian (CXL, changed to "ANDROGRAPHIS HERBA, Andrographis paniculata"), and Jin Yin Hua (JYH, lonicera japonica or Honeysuckle). We identified the active component in these decoctions as two forms of phosphocholines, PC (18:0/18:2) and PC (16:0/18:2). These PCs potentially could form liposomes with small RNAs to enter human alveolar and gastric cells. Our experimental results suggest an unprecendent lipid complex route through which botanic small RNA can enter human bodies. Our results provide an innovative treatment strategy for oral delivery of siRNAs as therapeutic medication.

Saponins (Ginsenosides) from the Leaves of Panax quinquefolius Ameliorated Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) overdose is one of the most common inducements of drug-induced liver injury (DILI) in the world. The main purpose of this paper was to investigate the liver protection activity of saponins (ginsenosides) from the leaves of Panax quinquefolius (PQS) against APAP-induced hepatotoxicity, and the involved mechanisms were demonstrated for the first time. Mice were pretreated with PQS (150 and 300 mg/kg) by oral gavage for 7 days before being treated with 250 mg/kg APAP. Severe liver injury was exerted at 24 h post-APAP, and hepatotoxicity was assessed. Our results showed that pretreatment with PQS significantly decreased the serum alanine aminotransferase (ALT), aspartate transaminase (AST), tumor necrosis factor (TNF-α), and interleukin-1ß (IL-1ß) levels in a dose-dependent manner as compared to the APAP administration. Meanwhile, compared with that in the APAP group, PQS decreased hepatic malondialdehyde (MDA) contents and 4-hydroxynonenal (4-HNE) expression and restored reduced glutathione (GSH) content and superoxide dismutase (SOD) activity in livers of mice. PQS inhibited the overexpression of pro-inflammatory factors cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the liver tissues. Furthermore, Western blotting analysis revealed that PQS pretreatment inhibited the activation of apoptotic signaling pathways via increase of Bcl-2 and decrease of Bax and caspase-3 protein expression levels. Liver histopathological observation provided further evidence that PQS pretreatment significantly inhibited APAP-induced hepatocyte necrosis, inflammatory cell infiltration, and congestion. Biological indicators of nitrative stress such as 3-nitrotyrosine (3-NT) were inhibited after PQS pretreatment, compared to the APAP group. The present study clearly demonstrates that PQS exerts a protective effect against APAP-induced hepatic injury because of its antioxidant, anti-apoptotic, and anti-inflammatory activities. The findings from the present investigation show that PQS might be a promising candidate treatment agent against drug-induced ALI.

[A study of relationship between dialectical classification according to traditional Chinese medicine of acute coronary syndrome with serum osteoprotegerin and its ligand].

OBJECTIVE: To investigate the relationship between osteoprotegerin (OPG) system and acute coronary syndrome (ACS) and its classification according to traditional Chinese medicine (TCM). METHODS: A prospective study was conducted. The patients with ACS (n=210) and the patients with stable angina pectoris (SAP, n=200) were enrolled. The serum OPG and its ligand (sRANKL) were determined by enzyme-linked immunosorbent assay (ELISA), the OPG/sRANKL ratio was calculated, and the number of coronary vessels was involved, finally their relationship with the typing according to TCM was evaluated. One hundred and fifty non-coronary heart disease patients were enrolled as control. RESULTS: The serum OPG, OPG/sRANKL in ACS and SAP groups were significantly higher than those in control group, and the sRANKL was significantly lower than that in control group (all P<0.01). The OPG, OPG/sRANKL in ACS groups were significantly higher than those in SAP group (both P<0.01). Serum OPG, OPG/sRANKL in ACS patients with different number of coronary vessel disease were significantly higher than those in control group, and the sRANKL was significantly lower than that in control group (all P<0.01). OPG and OPG/sRANKL were gradually increased with increase in number of diseased coronary vessels, but the sRANKL descended (P<0.05 or P<0.01). Serum OPG and OPG/sRANKL were descended according to dialectical classification of TCM: Yang Qi weakening syndrome>Qi and blood stagnation syndrome>Qi deficiency with blood stasis syndrome>stagnation of phlegm blocks the heart-vessels syndrome>Yin deficiencies of the heart and the kidney syndrome>deficiency of both Qi and Yin syndrome, among them they were significantly higher in Yang Qi failure syndrome and Qi and blood stagnation syndromes than those of both Qi and Yin syndrome [OPG(ng/L): 621.38±32.86, 617.63±39.60 vs. 593.86±36.19, OPG/sRANKL(g/mol): 1 018.98±106.03, 1 011.27±121.61 vs. 942.16±115.82, P<0.05 or P<0.01]. Among different types of TCM in ACS group the serum sRANKL was significantly lower than that in control group (all P<0.01), but the difference among different types was not significant. CONCLUSIONS: Serum OPG, sRANKL, OPG/sRANK levels were related with incidence and severity of coronary lesions in ACS patients. Serum OPG and OPG/sRANKL ratio may have correlation with Yang Qi weakening syndrome and Qi deficiency with blood stasis syndrome.

[Study on compatibility extraction technology of Rhei Radix et Rhizoma, Scutellariae Radix and Phellodendri Chinensis Cortex].

OBJECTIVE: To determine the optimum compatibility extraction technology of Rhei Radix et Rhizoma, Scutellariae Radix and Phellodendri Chinensis Cortex. METHOD: The drugs were combined respectively firstly, then use HPLC to determinate extraction rates of evaluating indicators including emodin, chrysophano, baicalin and berberine hydrochloride. Orthogonal experiment was used to detect the effect on concentration and volume of ethanol, times of extraction. RESULT: The extraction rates of evaluating indicators were relatively high in combined extraction of Rhei Radix et Rhizoma with Scutellariae Radix and single extraction of Phellodendri Chinensis Cortex. The optimized combined extraction were 12 times more than the volume of 60% ethanol solution, by boiling for 3 times, each for 1.0 hour, and that of single extraction were 10 times more than the volume of 60% ethanol solution, by boiling for 3 times, each for 1.0 hour. CONCLUSION: The optimized compatibility extraction technology is reasonable, stable and practical.

[Effect of light intensity on growth and quality of Asarum heterotropoides var. mandshuricum].

To study the infection rate of leaf spot disease, the drying rate of root and volatile oil content of Asarum heterotropoides var. mandshuricum at the unwrapping stage, blooming stage, the initial fruit stage, fructescence and wither stage during the growth period under the different sunlight intensity of 100% (I), 50% (II), 28% (III), 12% (IV). The volatile oil content was measured according to the method of Chinese Pharmacopoeia and the oil composition was determined by GC-MS. The unwrapping stage, blooming stage and the early fruit stage postponed about 2 days with decrease of the sunlight intensity. The infection rate of leaf was 88.46%, 70.00%, 0.23%, 0.07% under light intensity of I, II, III and IV, respectively, the drying rate was 25.14%, 28.27%, 30.23%, 31.57% under light intensity of I, II, III and IV, respectively, and the volatile oil content was 18.1, 17.6, 16.3, 15.3 mL x kg(-1) under light intensity of I, II, III and IV, respectively. The composition of the oil determined by GC-MS was different between the groups, but the content did not changed significantly with the decrease of the light intensity.