Impaired decidualization was recognized as one of the crucial pathomechanisms accounting for unexplained recurrent spontaneous abortion (URSA). Currently, the exact molecular mechanism and targeted clinical decision are still in exploration. Bushen Huoxue decoction (BSHXD) has previously been proved effective in treating URSA, but its mechanism remains to be elucidated. This study aimed to explore the regulation mechanism of BSHXD in decidualization from its intervention in autophagy so as to rationalize its potential as a novel therapeutic regime for URSA. Decidua tissues were collected from patients with URSA and healthy pregnant women who underwent legal terminations for non-medical reasons at the first trimester. Besides, cell line T-hESCs was utilized to establish induced decidualization model, and were randomly divided into ESC group, DSC group, 3-MA group, AMPK siRNA group, scrambled siRNA group and AMPK siRNA + BSHXD group. Transmission electron microscopy, Monodansylcadaverine (MDC) assay, qRT-PCR, immunohistochemistry, immunofluorescence and Western blotting were used to evaluate the level of decidualization, autophagy and activation of AMPK signaling pathway in decidua tissues and cell experiments. Experiments on decidua tissues showed that decidualization was impaired in URSA with inhibited autophagy. Besides, pAMPK T172 and pULK1 S556 were decreased, and pmTOR S2448 and pULK1 S757 were increased. Cell experiments showed that the level of autophagy increased during induced decidualization, but when autophagy was inhibited, decidualization was impaired. In addition, AMPK/mTOR/ULK1 affected decidualization by mediating autophagy, and BSHXD improved decidualization through this mechanism. In conclusion, this study clarified that the inhibition of autophagy mediated by AMPK/mTOR/ULK1 was associated with impaired decidualization, and the intervention of BSHXD on this pathological process may be a vital mechanism for its treatment of URSA. This study laid the foundation for further research and application of BSHXD. Recurrent spontaneous abortion (RSA) is defined as three or more consecutive abortions prior to the 20th week of gestation. Data show that RSA affects approximately 1–5% of reproductive women worldwide . Now sevAMP activated protein kinase (AMPK) is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis, and one of the mechanisms is to activate autophagy by inactivating mTOR complex-1or by directly phosphorylating ULK1 . StudiesAstragalus mongholicus Bunge and Salvia miltiorrhiza Bunge. Previous studies in vitro or in vivo have discovered that BSHXD is capable of preventing miscarriage by playing a comprehensive role in angiogenesis, decuvialization, maternal and fetal immune regulation , pmTOR S2448 Rabbit pAb , mTOR Rabbit pAb , ULK1 Rabbit pAb , Anti-AMPK alpha 1 (phospho T183) + AMPK alpha 2 (phospho T172) , pULK1 S757 Rabbit pAb , GAPDH , Goat Anti-Mouse IgG Antibody , Goat Anti-Rabbit IgG Antibody , Alexa FluorTM 594 Goat Anti-Rabbit IgG (H + L) Antibody , Alexa FluorTM 488 Goat Anti-Mouse IgG (H + L) Antibody , TB Green Premix Ex Taq II , PrimeScript RT reagent Kit with gDNA Eraser .DMEM/F12 , Fetal bovine serum (FBS) , Penicillin , Streptomycin , 8-Br-cAMP , MPA , 3-MA (3-methyladenine) , TritonX-100 , Vimentin Monoclonal Antibody , pULK1 S556 antibody , p62/SQSTM Polyclonal Antibody , LC3 Polyclonal Antibody , AMPKBSHXD was composed of six kinds of botanical drugs, and the information and ingredient doses were shown in The UHPLC-QE-MS analysis of BSHXD was performed on a 1290 UHPLC system with a Waters UPLC BEH C18 column (1.7 μm 2.1*100 mm). The column temperature was kept at 55°C and the sample injection volume was 5 μL. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Linear gradient elution was applied  at a flow rate of 0.5 ml/min. MS detection were performed by using an Q Exactive Focus mass spectrometer  coupled with an Xcalibur software to maintain the MS and MS/MS data based on the IDA acquisition mode. The operating parameters were as follows: Sheath gas flow rate, 45 Arb; Aux gas flow rate, 15 Arb; Capillary temperature, 400°C; Full ms resolution, 70000; MS/MS resolution, 17500; Collision energy, 15/30/45 in NCE mode; Spray Voltage, 4.0 kV (positive) or −3.6 kV (negative). Identification of chemical compounds from peaks containing MS-MS data was performed by using the secondary mass spectrometry database “BIOTREE PWT database” provided by Shanghai BIOTREE biotech Co., Ltd. and the corresponding cleavage law matching method.The URSA group consisted of 16 women who had experienced twice or more miscarriage at early pregnancy, and the certain cause of miscarriage had been ruled out, such as endocrine, anatomical or genetic abnormalities, infection and other factors. The control group included 14 healthy women who voluntarily terminated pregnancy at an early stage due to non-medical factors. Besides, they had at least one healthy child without abortion history. The fetal heart rate of this pregnancy was normal identified by ultrasound examination, and the fetal development was consistent with the gestational age. Besides, all the subjects should meet the following requirements: 25–40 years old, 6–9 weeks of gestation, BMI between 18-29, normal menstrual history, no serious comorbidities and no bad habits such as smoking or drinking. There were no statistical differences in age, gestation time, and BMI between the two groups, as shown in In our practice, curettage was routinely performed in both URSA and the control group, and the decidua tissue samples were obtained immediately after the operation. The tissue was washed with cold PBS and divided into three parts. All tissues used for quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analysis were cryopreserved at −80°C. Transmission electron microscopy (TEM) samples were immersed in 2.5% glutaraldehyde. Immunohistochemical and immunofluorescence samples were fixed with 4% paraformaldehyde.in vitro. The induction process lasted for 6 days with medium changing every 2 days. The decidualization was evaluated by morphological phenotype and the increased level of decidual markers PRL mRNA and IGFBP1 mRNA .5 cells/well. After about 40% of the adherent cells were fused, three small interfering RNA (siRNA) oligos specific for human AMPK  or scrambled siRNA , were transfected into cells by using transfection reagents riboFECT™ CP Reagent  according to the manufacturer’s instructions. The final concentration of each siRNA was 50 nM. And the T-hESCs were transfected on day 0 and day 4 after MPA and 8-Br-cAMP treatment , DSC group (treatment of 1uM MPA +0.5 mM 8-Br-camp for 6 days), 3-MA group , AMPK siRNA group , scrambled siRNA group  and AMPK siRNA + BSHXD group .3 [Fe(CN)3] for 1 h. The samples were immersed in a dioxy solution of 2% acetic acid at 4°C overnight, and then experienced dehydration in gradient ethanol solution and soaked in different proportions of pure acetone and embedding agent. The samples were polymerized at 60°C, and made into ultra-thin sections. Uranyl acetate and lead citrate (3%) were utilized to stain the sections. The formation of autophagosomes was observed under a electron microscope .The decidua tissue or the cells digested by trypsinase were rapidly fixed in precooled 2.5% glutaraldehyde solution for 24 h, and then washed with PBS and transferred to 1% osmium and 1.5%KAfter the cells in each group were treated for corresponding time, discard the supernatant cleaned with 1×Wash Buffer. Each cell was added with 90 μL 1×Wash Buffer and 10 μL MDC staining solution, and incubated in darkness at room temperature for 30 min. After incubation, the cells were washed with 1×Wash Buffer for 2 times and observed under a fluorescence microscope immediately.β-actin were determined by qRT-PCR using TB Green Premix Ex Taq II. The primer sequences used for real-time PCR were as follows:Total RNA from decidual tissue or cells were isolated by RNAiso Plus  according to the manufacturer’s recommendations. 1 μg RNA was reverse transcribed using PrimeScript™ RT reagent Kit with gDNA Eraser . Relative mRNA levels of PRL, IGFBP1 and PRL-Human-F:ACCTCTCCTCAGAAATGTTCAGCGA; PRL-Human-R:TCTGTTGGGCTTGCTCCTTGTCTT; IGFBP1-Human-F:AAAGCCCAGAGAGCACGGAGATAA;IGFBP1-Human-R: ATGGCGTCCCAAAGGATGGAATGA.The decidual tissues were fixed with 4% paraformaldehyde, and then dehydrated, and embedded in paraffin. Paraffin sections (5 μm) were prepared, dewaxed, and rehydrated in ethanol. After antigen repair, the slides were blocked with 3% hydrogen peroxide for 10min to inhibit endogenous peroxidase activity, and then nonspecific binding sites were blocked with 5% bovine serum albumin for 30 min. After that, sections were incubated overnight with rabbit anti-pAMPK T172 antibody and anti-pmTOR S2448 antibody at 4°C overnight, and anti-pULK1 S556, anti-pULK1 S757 then incubated with goat anti-rabbit immunoglobulins for 1 h at room temperature. Afterwards, sections were subsequently re-stained with hematoxylin. Finally, the tissue sections are dehydrated and sealed, and images were captured with an optical microscope.α2 T172, 1:250; LC3, 1:200; Vimentin, 1:200), and incubated with secondary antibody for 2 h (Alexa FluorTM 488 Goat Anti-Mouse IgG (H + L) Antibody, 1:250; Alexa FluorTM 594 Goat Anti-Rabbit IgG (H + L) Antibody, 1:250). At last, DAPI was utilized to counterstain the decidual tissues and then the tissues were observed under the fluorescence microscope.The decidual tissues were fixed with 4% paraformaldehyde in PBS for 10 min, and then infiltrated with 200 μL 0.3% Triton X-100 for 10 min. After that, the cells were then blocked with 5% bovine serum albumin (BSA) for 30 min, stained with primary antibody overnight , ULK1, pULK1 (S757), pULK1 (S556), pmTOR (S2448), mTOR in cells were detected by Western blotting. First, lysates of decidua tissue or cells were prepared in high efficiency cell tissue rapid lysis buffer . Then, protein concentration was determined by the BCA Protein Detection Kit . After that, sample proteins were isolated by sodium dodecyl sulphate-polyacrylamide gel elec-trophoresis and then transferred to polyvinylidene difluoride (PVDF) membrane at 100V, 65 min. And then the PVDF blots were incubated with primary antibodies at 4°C overnight: p62 (1:1000), LC3 (1:1000), AMPKα2 (1:1000), pAMPKα2 T172 (1:1000), ULK1 (1:1000), pULK1 S757 (1:1000), pULK1 S556 (1:1000), pmTOR S2448 (1:1000), mTOR (1:1000). Subsequently, the membranes were washed and further incubated with secondary antibodies. Finally, the PVDF membrane was immersed in ECL luminescent solution and scanned by the developer after 2 min of shading.The expressions of p62, LC3 in decidual tissues and p62, LC3, AMPKt test or one-way ANOVA with Dunnett’s multiple comparisons. A value of p < 0.05 was regarded as statistically significant.All results were presented as the mean ± SEM and analyzed by GraphPad Prism 8 . Statistical analyses were performed by two-tailed unpaired In order to identify the main components of BSHXD, UHPLC-QE-MS analysis was applied. The total positive  and negap < 0.05 and p < 0.01, respectively)  , indicatp < 0.05). Considering that DSCs are the main components of decidua tissue, immunofluorescence co-location analysis of LC3 and vimentin (a marker of DSCs) was performed. And the results showed that LC3 and vimentin were colocalization, indicating that LC3 was expressed in DSCs. In addition, the intensity of LC3 fluorescence in URSA group was significantly lower than that in the control group (p < 0.05)  , suggestp < 0.01 and p < 0.05, respectively), while the positive area of pmTOR S2448 and pULK1 S757 in URSA was significantly increased (p < 0.05) (p < 0.01)  . Conside < 0.01) , suggestIn order to screen out the optimal concentration and intervention time of BSHXD on T-HESC, CCK-8 assay was utilized to calculate the cell viability of each group treated by different concentrations of BSHXD under different time. The results showed that the cell activity was highest when 0.25 mg/ml BSHXD was administered for 48 h, and this intervention was used in the follow-up experiment, as shown in In order to select AMPK siRNA oligos with the best knockdown efficacy, three siRNA were respectively transfected into T-hESCs, and the results were shown in the p < 0.01) (p < 0.01). Besides, LC3II/LC3I ratio was increased (p < 0.05), and p62 was significantly decreased (p < 0.05)  (p < 0.05) (p < 0.05)  , and aut < 0.01)  and its  < 0.01)  was sign < 0.05) , indicatnder TEM  and the  < 0.01) , and the < 0.05) . Meanwhi < 0.05) , indicatp < 0.01), and the level of pmTOR S2448/pmTOR and pULK1 S757/ULK1 was increased when compared with the scrambled siRNA group (p < 0.01) (p < 0.01). Besides, the expression of p62 was increased and the ratio of LC3II/LC3I decreased in the AMPK siRNA group when compared with the scrambled siRNA group  (p < 0.01)  . Moreove < 0.01)  and the  < 0.01)  was signctively) . Further < 0.01) . Besides < 0.01) . These rp < 0.05) (p < 0.05). Besides, the ratio of LC3II/LC3I increased and the expression of p62 was decreased in AMPK siRNA + BSHXD group when compared with the AMPK siRNA group (p < 0.05) (p < 0.01) (p > 0.05), while pmTOR/mTOR and pULK1757/ULK1 in the AMPK siRNA + BSHXD group were significantly higher than those in the scrambled siRNA group (p < 0.05)  . Moreove < 0.05)  and the  < 0.05)  were sig < 0.05) . Moreove < 0.01) . These r < 0.05) , suggestin vitro. And AMPK knockdown can inhibit autophagy by affecting the phosphorylation of mTOR and ULK1, thus affecting decidualization, while BSHXD can restore autophagy and improve decidualization by regulating AMPK/mTOR/ULK1. In summary, our study identified a novel mechanism of BSHXD in URSA treatment from the aspect of autophagy and provided a laboratory basis for its clinical promotion.In this study, we observed impaired decidualization, decreased autophagy level and down-regulation of AMPK/mTOR/ULK1 pathway in the decidua tissue of URSA. Subsequently, we confirmed that the inhibition of autophagy can lead to impaired decidualization Decidualization provides growth factors and cytokines for embryo development and protects the blastocyst from immune rejection, which is a key prerequisite for embryo implantation and development . Previouin vitro. We displayed that the level of autophagy was increased in DSC group when compared with the ESC group, while the decidualization markers PRL and IGFBP1 in 3-MA group were significantly decreased after the successful inhibition of autophagy when compared with the DSC group. Therefore, it can be inferred that moderate level of autophagy are necessary for decidualization, which has also been confirmed by other researchers. Mestre et al. found that autophagy flux increased during decidualization (The essence of decidualization is the transformation of fibroblast-like ESCs into secretory DSCs, leading to the decline of cellular ATP and intracellular remodeling, both of which are key signals for initiating autophagy . In addilization , while dlization . Su et alization . Therefoin vitro. The results showed that pAMPK/AMPK and pULK1 S556/ULK1 were significantly decreased in AMPK siRNA group when compared with the scrambled siRNA group, while pmTOR S2448/mTOR and pULK1 S757/ULK1 were significantly decreased in AMPK siRNA group. And, as a result, the levels of autophagy and decidualization were correspondingly decreased. Combined with the above finding on the relationship between autophagy and decidualization, we concluded that AMPK/mTOR/ULK1 can affect decidualization by regulating autophagy.AMPK is a highly conserved serine/threonine isotrimer kinase in eukaryotic cells, and is widely involved in the metabolic process of various substances as well as in maintaining cell and systemic energy metabolism. It can activate autophagy and help with energy recycling by decomposing aging organelles and misfolded proteins so as to cope with energy stress and meet the high energy requirements of decidualization. mTOR and ULK1 are two important downstream factors of AMPK in the initiation of autophagy. mTOR is a vital regulator of cell growth, apoptosis, energy metabolism and autophagy as well as the growth of various tumors . ULK1 isin vitro, we treated cells with BSHXD after transfection with AMPK siRNA and compared it with AMPK siRNA group and scrambled siRNA group. The results showed that BSHXD significantly increased the levels of pAMPK/AMPK and pULK1 S556/ULK1, degraded the level of pmTOR S2448 and pULK1 S757/ULK1, enhanced the level of autophagy, and improved decidualization impaired by AMPK knockdown. Therefore, we speculated that BSHXD could improve autophagy by regulating AMPK/mTOR/ULK1, thus contributing to decidualization in vitro. In addition, when we compared phosphorylation state of AMPK/mTOR/ULK1 between AMPK siRNA + BSHXD group and scrambled siRNA group, we found that ULK1 S556 was more sensitive to AMPK phosphorylation mediated by BSHXD than mTOR. That was to say, the regulation effect of BSHXD in AMPK/pULK1 S556 was superior to AMPK/pmTOR S2448/pULK1 S757. The possible explanation for this phenomenon is that the phosphorylation of mTOR is affected by multiple factors, and BSHXD is multi-targeted. And some upstream factors of mTOR may interfere with its phosphorylation by AMPK under the influence of BSHXD. However, this speculation needs further confirmation.Next, we focused on the regulation mechanism of BSHXD on decidualization from the aspect of autophagy mediated by AMPK/mTOR/ULK1. During decidualization in vivo.Taken together, we concluded that BSHXD improved decidualization by activating autophagy via AMPK/mTOR/ULK1 signaling pathway, thus rationalizing its potential as a novel therapeutic regime for impaired decidualization and pregancy loss . In futu


Cyanidioschyzon merolae. The ND deletion changed the localization of CmMYB1 from the cytoplasm to the nucleus, enhanced the binding efficiency of CmMYB1 to promoters of nitrate assimilation genes, and increased the transcripts of nitrate assimilation genes under +N condition. A pull-down assay using an ND-overexpressing strain combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis helped us to screen and identify an unknown-function protein, the CmNDB1. Yeast two-hybrid analysis demonstrated that CmNDB1 interacts with ND. Similar to ND deletion, CmNDB1 deletion also led to the nucleus localization of CmMYB1, enhanced the promoter-binding ratio of CmMYB1 to the promoter regions of nitrate assimilation genes, and increased transcript levels of nitrate assimilation genes under +N condition. Thus, these presented results indicated that ND and CmNDB1 negatively regulate CmMYB1 functions under the +N condition in C. merolae.Nitrogen assimilation is an essential process that controls plant growth and development. Plant cells adjust the transcription of nitrogen assimilation genes through transcription factors (TFs) to acclimatize to changing nitrogen levels in nature. However, the regulatory mechanisms of these TFs under nitrogen-repleted (+N) conditions in plant lineages remain largely unknown. Here, we identified a negative domain (ND) of CmMYB1, the nitrogen-depleted (−N)-activated TF, in a unicellular red alga  Nitrogen is a component of most basic macromolecules, including proteins and nucleic acids. Thus, it is an essential element for all living organisms. Plants uptake nitrogen sources, mainly ammonium and nitrate through ammonium transporter (AMT) and nitrate transporter (NRT), respectively, from their growing environments. These nitrogen sources are then assimilated into amino acids through nitrogen assimilation enzymes, and these amino acids would be further utilized .Arabidopsis, recent studies have uncovered that the Nodule INception (NIN)-Like Protein 6 and 7 , could positively regulate the transcripts of AtNRT1.1, as well as the gene-encoding nitrate reductase (NR), AtNIA1 and AtNIA2, under −N conditions through forming a complex with TCP20 in the nucleus (OsNRT2.3 and gene-encoding glutamine synthetase (GS), OsGS1;1, under nitrogen-deficient condition but not +N condition in roots  positively regulate in controlling the expressions of nitrogen assimilation genes under nitrogen-deficient or −N conditions. In  nucleus . Althoug nucleus , the inain roots . The mecCyanidioschyzon merolae is a unicellular red alga, which was isolated from a hot spring in Italy , which negatively controls the function of CmMYB1 under the +N condition in C. merolae T1, BZU1, BZ1, CmMYB1 partially truncated transformants , MΔ95a and BZ6 strains were grown at 40°C under continuous white light (50 μmol m–2 s–1) in a liquid MA2 medium  was added into the cell pellet followed by 0.15-g glass beads . Cells were disrupted by vortexing , and supernatant  was collected by centrifuging at 15,000 rpm for 20 min at 4°C and stored at −80°C until use. Protein samples were boiled at 95°C for 5 min and later cooled on ice for 2 min. The proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride  membrane . The membrane was blocked with 5% w/v skim milk in 0.1% TBS-Tween at 4°C overnight. In case of detecting FLAG-fused CmMYB1, the membrane was briefly washed three times with 0.1% TBS-Tween before being incubated with an anti-FLAG antibody  for 1 h at room temperature. Next, the membrane was washed as mentioned above and incubated with antimouse IgG (H + L) and an HRP conjugate antibody  for 1 h at room temperature. The washing step was repeated before visualization using the ImmunoStar Zeta .immunoblot analysis.A total soluble protein fraction was collected as described in section “Protein extraction and immunoblot analysis.” PVDF membrane was firstly treated with methanol for 60 s and washed with RO water two times. Then, a blotting buffer  was used for PVDF membrane incubation for 5 min; a 16.6-μg/sample total soluble protein fraction was used. The membrane was dried for 10 min and incubated in a blocking solution (5% w/v skim milk in 0.1% TBS-Tween) at 4°C overnight. The remaining steps were the same as mentioned in ® qPCR RT Master Mix with gDNA Remover  using 500 ng RNA of each sample as input. Synthesized cDNA was then subjected to Quantitative Real-Time PCR (qRT-PCR) analysis. qRT-PCR analysis was performed as described previously  were collected and fixed with a cold fixation buffer (n buffer  and incuC. merolae logarithmic growth cells (25 ml at OD750 = 0.5) were collected and fixed as described previously (eviously . The fixeviously . Togetheeviously  was addeAPCC promoter (The pSUGA_CmMYB1_1-380 was first created with primer set MYB1OE380_F (listed in promoter .––1 flow rate with a linear gradient generated by aqueous solvent A (0.1% formic acid in water) and organic solvent B (0.1% formic acid in acetonitrile): 5% B to 35% B in 40 min, to 45% B in 46 min, to 95% B in 48 min, 95% B from 48 to 49 min, and, finally, to 5% B in 50 min. MS1 scans from m/z = 321--1,500 were performed in the Orbitrap mass spectrometer with the resolution set to 120,000 with lock masses at m/z = 445.12003 and 391.28429, which was followed by the acquisition of higher energy collisional dissociation (HCD)-MS2 using the ion trap. The settings for the MS2 scans were as follows: an intensity threshold = 1,000, charge states = + 2 to + 6, isolation width = 1.2 m/z, AGC target = 5,000, maximum ion injection time = 50 ms, normalized collision energy = 35%, and dynamic exclusion enabled with 50-s exclusion duration. The MS/MS cycle time was set to 4 s. The MS/MS data were acquired over 55 min after the LC gradient was started. Peptide identification was done by searching the C. merolae protein database1 and a common contaminating protein list using a MASCOT search engine . A maximum of four trypsin miscleavages were allowed. The peptide mass tolerance and MS/MS tolerance were set at 5 ppm and 0.5 Da, respectively. Propionamidated cysteine (+71.0371) was set as a fixed modification. Protein N-terminal acetylation (+42.0106 Da) and oxidation of methionine (+15.9949 Da) were considered as variable modifications. The Mascot ion score threshold was set to 0.05. Obtained MS/MS data are presented in Proteins from Negative Domain-Overexpressing Strain (ND_OE) and TFc were extracted as described in section “Protein extraction and immunoblot analysis.” From each sample, the 750-μg protein was subjected to the pull-down assay with 50-μl/sample anti-FLAG antibody magnetic beads . The beads were washed six times with a lysis buffer  and were boiled for 5 min in an SDS sample buffer. The eluted proteins were separated by SDS-PAGE using a 5–20% polyacrylamide gradient gel  and were visualized by Coomassie brilliant blue staining. Each lane in the gel was entirely cut out, was transferred to a 14-ml round-bottom tube, and was shaken sequentially in 30, 50, and 100% acetonitrile solutions for destaining and dehydration. The gel strips were incubated in 25-mM ammonium bicarbonate containing 10-mM dithiothreitol (DTT) at 56°C for 1 h and then in 25-mM ammonium bicarbonate containing 55-mM acrylamide at room temperature for 1 h for reduction and alkylation of the sulfhydryl groups. The samples were washed in water and were then dehydrated in the acetonitrile solutions before tryptic digestion. The dried gel strips were reconstituted by a solution containing 90-ng Trypsin Gold , 50-mM ammonium bicarbonate, and 10% acetonitrile, and were incubated overnight at 37°C. The resulting tryptic peptides were eluted three times in 75% acetonitrile and 1% formic acid. After concentration in a speedvac, the samples were finally dissolved in 45 μl of 0.5% formic acid. From each sample, 15 μl was subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Orbitrap Fusion mass spectrometer equipped with an Easy-nLC 1,000 high-performance liquid chromatography (HPLC) system . The peptides were separated on a C18 tip column  at a 300-ml minC. merolae genomic DNA as templates. The PCR-amplified genes were then cloned into SmaI-digested pGADT7, respectively, using the Gibson Assembly Master Mix  to create each AD vector. To create the BD plasmid, the CmMYB1 ORF was firstly amplified with the primer set MYB1y2h_F, MYB1y2h_R listed in C. merolae genomic DNA as a template. The PCR-amplified CmMYB1 ORF was then cloned into SmaI-digested pGBT9 using the same method mentioned above to create the pGBT9_CmMYB1 vector. The pGBT9_CmMYB1_1–380 plasmid was then created using the same method mentioned above with primer set MYB1y2h_381_F, MYB1y2h_381_R listed in D-galactopyranoside [  and Aureobasidin A (AbA)  being added] and SD−LWHA  lacking with X-α-Gal and AbA being added) plates and SD−LW plates and incubated at 30°C for 1 week. Photos of SD−LW plates were taken on the second incubation day. Photos of SD−LWH and SD−LWHA plates were taken on the 7th incubation day.The open-reading frames (ORFs) of the top 15 candidates’ encoded genes picked up from LC-MS/MS analysis were amplified with the primer sets listed in C. merolae genomic DNA as templates, respectively. Then, Fragment_1 and Fragment_2 were used to create pMKT_CAT plasmid using the Gibson Assembly Master Mix . Restriction enzyme HpaI was used to cut the pMKT_CAT plasmid, and two linear DNA fragments, “pMKT” DNA fragment and “CAT” DNA fragment, were obtained. Finally, “pMKT” DNA, “CAT” DNA, CmMYB1_ORF DNA, and CmMYB1_ter DNA were used to create pMKT_CAT_CmMYB1 plasmid using the Gibson Assembly Master Mix . The primer set MCS_J282C_orf_F, MCS_J282C_ter_R was used to amplify CmMYB1_CAT DNA using KOD Plus NEO . The CmMYB1_CAT DNA was then transformed into a WT strain as described previously . Finally, the DNA fragment used for transformation was amplified using the primer set URA5.3pro_F, URA5.3ter_R with pBluescript _URA5.3KO plasmid as a template. The amplified DNA fragment was then transformed into the WM1 strain as described previously , CmNDB1orf_R, and CmNDB1ter_F, CmNDB1ter_R were used to amplify Fragment_1 and Fragment_2, using KOD Plus NEO  and ed pMKTm  plasmid ed pMKTm , and traCmMYB1 background strain combined with a plasmid series expressing partially truncated CmMYB1s would be a good strategy. Since CmMYB1 would only be expressed exogenously; different phenotypes among the strains must be caused by the different CmMYB1 sequences. Firstly, we designed and obtained a CmMYB1 knockout strain named BZ1  and after the −N condition in each constructed strain by Northern blot analysis , was higher in pPD1–310 but lower or no change in pPD1–380, pPD1–460, and pPD1–99, 201–523 than in pPD1–523 under the +N condition  and after the −N condition. In addition to the strains used in CmNRT under the −N condition but not the distinct region , because, in ondition . To inveondition . We perfondition . Among tondition .C. merolae database2), annotated as a hypothetical protein with a comment as a “cyanobacterial protein,” could grow on SD−LWH media and SD−LWHA, stronger selection media than SD−LWH  analysis. Results showed that transformants coexpressing ND and CMR469C  analysis using a strain in which Myc-fused CmNDB1 and FLAG-fused CmMYB1 were expressed. We could not observe the interaction between CmNDB1 and CmMYB1 in C. merolae cells under many experimental conditions (see section “Discussion”).Based on the results observed from the Y2H analysis, we investigated whether CmNDB1 interacts with CmMYB1 in the CmNDB1 knockout strain named BZ6   . These rCmNRT, CmNR, and CmNIR, was significantly increased in BZ6 against in MΔ95a under the +N condition  mechanism , 2010. Tl strain . In the m medium , 4F,G, wondition , 4G. Thiondition . The othromosome , suggestCmNDB1 deletion resulted in CmMYB1 localization change  can be found in the article/BZ and SI designed the research. BZ, HS, and SI performed the research and wrote the manuscript. BZ, HS, KI, KT, and SI analyzed the data. All the authors contributed to the article and approved the submitted version.SI was employed by Nippon Telegraph and Telephone Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


P. pastoris. Out of ten initially selected sequences, three enzymes (one from Aspergillus niger and two from Candolleomyces aberdarensis) were actively expressed. Cultivation of respective expression clones in a bioreactor led to production titers of up to 300 mg L−1. Enzymes were purified to near homogeneity and characterized regarding their specific activities and pH-optima for typical UPO substrates. This work demonstrated that directed evolution is not necessarily required to produce UPOs in P. pastoris at respective titers. The heterologous producibility of these three UPOs will expand the toolbox of available enzymes and help to advance their synthetic application.Unspecific peroxygenases (UPOs) catalyze the selective transfer of single oxygen atoms from peroxides to a broad range of substrates such as un-activated hydrocarbons. Since specific oxyfunctionalizations are among the most-desired reactions in synthetic chemistry, UPOs are of high industrial interest. To broaden the number of available enzymes, computational and experimental methods were combined in this study. After a comparative alignment and homology modelling, the enzymes were expressed directly in  Although their exact physiological function is unknown, they have been widely studied regarding their application potential, since specific oxyfunctionalizations are among the most-desired reactions in synthetic chemistry [Unspecific peroxygenases UPOs; EC 1.11.2.1), members of the heme-thiolate proteins, catalyze the selective transfer of single oxygen atoms from peroxides  aegerita, Marasmius rotula, or Chaetomium globosum, recombinantly produced UPOs from Coprinopsis cinerea, Collariella (Chaetomium) virescens, Daldinia caldariorum, Hypoxylon sp. EC38, or evolved variants from Candolleomyces (Psathyrella) aberdarensis and Cyclocybe aegerita [Leptoxyphium  fumago forms an exception, since it phylogenetically belongs to the UPOs (but not according to its EC classification) and can be produced as a wild-type enzyme in larger amounts (>500 mg L−1).Nowadays, thousands of UPO sequences can be found in genome-sequenced fungi, but only a handful of wild-type enzymes and a few recombinantly expressed UPOs have been characterized and used in lab-scale studies. Examples are wild-type UPOs from aegerita ,7,8,9. C−1). In particular, the common Escherichia coli expression system has not properly worked for many years and still does not when it comes to UPOs. Only recently has it become possible to produce small amounts of active UPOs using a modified E. coli expression system, although this did not work in the case of all UPO constructs tested and always occurred without glycosylation [Saccharomyces cerevisiae and Pichia pastoris  appear to be more promising for recombinant expression and specifically for enzyme evolution. Examples for the successful expression and evolution are the UPOs from Cyclocybe aegerita and Candolleomyces aberdarensis [P. pastoris, these corresponding genes had to be evolved, particularly in the signal peptide and sometimes also in the core domain [One major bottleneck so far remains the lack of recombinant expression of a representative selection of UPO genes with satisfactory expression levels  aegerita variant PaDa-I (AaeUPO-PaDa-I) and Coprinopsis cinerea (rCciUPO) belong to the “long” UPO subset [In this study, we took another approach by selecting candidates after comparative alignments and homology modelling, and then proceeding with their direct expression in PO genes  were ideThe overall selection followed an UPO database and alignments including more than 2000 protein sequences used in Hofrichter et al. . EspeciaPichia pastoris X33 and zeocin were purchased from ThermoFisher . Competent Escherichia coli NEB 5-alpha cells were purchased from New England Biolabs . Expression plasmid pPpB1 was provided by the group of Anton Glieder .Chemicals were purchased from either Sigma-Aldrich , Carl-Roth , or VWR  in >97% purity. M, SI −1 at 72 °C, and a final extension at 72 °C for 120 s. PCR products were cleaned up using the DNA clean and concentrator-5 kit . Amplified genes and plasmid backbone were mixed in 3:1 molar ratio, digested by DpnI (NEB), ligated without further purification using isothermal assembly [E. coli NEB 5-alpha according to the manufacturer’s protocol for heat shock transformation. E. coli NEB 5-alpha colonies were selected on low-salt LB  agar (15 g L−1) containing 25 mg L−1 zeocin. Plasmids were purified from cultures after overnight cultivation in low-salt LB + zeocin at 37 °C and 180 rpm using the GeneJET Plasmid MiniPrep Kit . Sequences of the cloned UPO-genes were verified by Sanger sequencing . Plasmids were linearized by digestion with PmeI and cleaned up (vide supra). Competent P. pastoris X33 cells were prepared as follows: 500 mL of YPD.media  was incubated inoculated from an overnight culture and grown overnight to an optical density determined at 600 nm (OD600) of 1–1.5. Cells were pelleted at 1500 g, washed twice with 250 mL ice-cold water, washed once with 20 mL ice-cold 1 M sorbitol, and finally resuspended in 1 mL 1 M sorbitol. Linearized plasmid (4–8 µg) and 80 µL competent cells were mixed in a pre-cooled 2 mm electroporation cuvette and incubated for 5 min. Electroporation was carried out using a Gene Pulser Xcell  with settings for Saccharomyces cerevisiae. Immediately after electroporation, 1 M sorbitol was added and cells were transferred to 1.5 mL reaction vials and incubated at 30 °C for about 60 min, after which an equal volume of YPD was added and incubation was continued for 30–60 min. Aliquots were spread onto YPD agar containing 1 M sorbitol and 100 mg L−1 zeocin and incubated at 30 °C for several days until colonies were visible.Genes of putative UPO genes were synthesized by BioCat  in pUC19. Coding sequences (CDSs) are given in the assembly , and traP. pastoris X33 colonies (24 colonies for each UPO-expression plasmids) from zeocin selection plates were picked into 96 deep-well plates (DWPs) and cultivated at 30 °C, 800 rpm, >80% humidity in a Microtron (InforsHT) according to the screening strategy described by [®  system. Following the initial methanol-addition, additional methanol was fed every 17–24 h. Samples for activity determination (50 µL) were taken before feeding of methanol. Cultivation was terminated after about 100 h. Enzyme activity in the culture supernatant was determined using the 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and 5-Nitro-1,3-benzodioxole (NBD) assays (vide infra) [ribed by  with thee infra) .P. pastoris X33 clones that exhibited UPO activity in the screening were cultivated in a 1-L (working volume) 4× DASGIP parallel bioreactor system (Eppendorf). Cultivations were carried out as described by Cino et al. 1999 [600 of 1–2 at an initial cultivation volume of 500 mL (50% working volume). Glycerol and methanol feed rates during the respective fed-batch phases were based on the dissolved oxygen (DO) concentration which had a set-point of 30% and was controlled by stirring rate (≤1200 rpm), aeration rate (≤50 L min−1), and oxygen content (21–100%); pH was held above 5 by the addition of 15% ammonia. Temperature was set at 30 °C. DO-based feeding rates for glycerol were 0–20% DO, 2–4 mL h−1, 20–30% DO, 4–8 mL h−1, 30–50% DO, 8–10 mL h−1, >50% DO, and 10 mL h−1. For methanol, the profile was: 0–25% DO, 0–3 mL h−1, 25–30% DO, 3–4 mL h−1, 30–40% DO, 4–6 mL h−1, >40% DO, and 6 mL h−1.In order to produce the proteins, those al. 1999  with theg. Further clarification was carried out by crossflow-filtration using a 0.2 µm membrane cassette . High-molecular weight compounds were concentrated using a 10 kDa cut-off crossflow-ultrafiltration membrane module  followed by a further concentration using ultrafiltration centrifugal concentrators . Fast protein liquid chromatography (FPLC) was carried out using an Azura FPLC system . Concentrated samples were clarified using 0.2 µm PES syringe filters  and mixed with an equal volume of 10 mM potassium phosphate buffer (KPi) pH 7 with 2 M ammonium sulfate prior to hydrophobic interaction chromatography (HIC). HIC was carried out using a 250 mL (3.6 cm inner diameter) column packed with Toyopearl butyl 650 M . Samples were applied directly to the column at a flow-rate of 4 mL min−1. Proteins were eluted using a binary gradient with buffers consisting of 10 mM KPi pH 7 with 1 M ammonium sulfate (buffer A) and without ammonium sulfate (buffer B) at a flow rate of 8 mL min−1. The elution profile was 1 column volume (CV) buffer A followed by a linear gradient of 100% buffer A to 100% buffer B over 1.4 CV. Fractions with a sufficiently high Reinheitszahl (AbsorptionSoret/Absorption280nm) (RZ) were collected, concentrated, and dialyzed against size exclusion chromatography (SEC) buffer consisting of 10 mM KPi pH 7 with 150 mM NaCl. SEC was carried out using a HiPrep 16/60 Sephacryl S-300 HR column . Samples were applied to the column using a sample loop (1.1 mL) at a flow-rate of 0.5 mL min−1, which was kept constant for 0.7 CV and increased to 1 mL min−1 afterwards. Samples with a sufficiently high RZ were collected, concentrated, and dialyzed against 100 mM KPi pH 7 for characterization experiments.Culture broth was removed from bioreactors and initially clarified by centrifugation at 15,000× P. pastoris X33 clones, assays were carried out in 96-well flat bottom microtiter plates (MTPs) using an Infinite 200 Pro . Assay volume in MTPs was 0.2 mL with the same composition as given for assays carried out in semi-micro cuvettes.Enzyme activity assays were carried out using a UV-1700  in semi-micro poly(methyl methacrylate) (PMMA) cuvettes (pathlength 1 cm) at room temperature. For screening of 2HPO4/citric acid buffer pH 4.4, 100 µL 3 mM ABTS, 50 µL 40 mM H2O2, and 100 µL sample. For the determination of pH-optima, 750 µL McIlvaine buffer (200 mM Na2HPO4/100 mM citric acid) was used. Absorption was determined at 420 nm; product concentration was calculated with an extinction coefficient of 36,000 M−1 cm−1 [Routine ABTS  assay consisted of 750 µL 100 mM NaM−1 cm−1 .2O2, and 10 µL sample. Absorption was determined at 310 nm; product concentration  was calculated with an extinction coefficient of 9300 M−1 cm−1 [The pH optima of unspecific peroxygenases (UPOs) for veratryl alcohol (VA) were determined with the following assay mixture: 840 µL McIlvaine buffer, 100 µL 50 mM VA, 50 µL 40 mM HM−1 cm−1 .i pH 7, 100 µL 5 mM NBD  in acetonitrile, 200 µL H2O, 50 µL 20 mM H2O2, and 100 µL sample. pH optima of the UPOs for NBD were determined with the following assay composition: 750 µL McIlvaine buffer, 100 µL 5 mM NBD in acetonitrile, 50 µL 20 mM H2O2, and 100 µL sample. Absorption was determined at 425 nm; product concentration (4-nitrocatechol) was calculated with an extinction coefficient of 9700 M−1 cm−1 [The routine NBD assay (following demethylenation) consisted of 550 µL 100 mM KPM−1 cm−1 .Protein concentrations were determined using the Bio-Rad Protein Assay Kit II  according to the manufacturer’s protocol using a bovine serum albumin dilution series for calibration. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using Mini-PROTEAN TGX precast gels and Laemmli sample buffer  with β-mercaptoethanol as a reducing agent, according to the manufacturer’s recommendations. Gels were stained with colloidal coomassie stain . For glyCyclocybe aegerita and Marasmius rotula . As a first premise, we decided to select genes phylogenetically close to known and “working” reference UPOs, like those of the enzymes from s rotula . Secondls rotula . PhylogePichia pastoris expression vector pPpB1 [aox1-promoter, which has been successfully utilized to express AaeUPO-PaDa-I [P. pastoris transformants were successfully generated for all 10 expression constructs. After transformation, 24 clones per construct were picked and cultivated in deep-well plates (DWPs) for 94 h. Supernatant from clones was tested for UPO activity using the ABTS and NBD assays. Clones transformed with expression plasmids for two UPOs from Candolleomyces aberdarensis (CabUPO1 and CabUPO2) and UPO from Aspergillus niger (rAniUPO) exhibited activity with both assays, while no activity could be determined for either assay for all other constructs , as exemplarily shown in CabUPO 2. Activity profiles of all cultivations are given in The best-performing clones identified in the screening, i.e., those that exhibited the highest activities towards ABTS and NBD, were cultivated in a 1-L bioreactor in order to produce sufficient amounts of enzyme for subsequent purification and characterization. Fermentation of oduction , which cCyclocybe aegerita variant PaDaI (rAaeUPO-PaDa-I) [Cultivations had to be terminated when the culture volume exceeded the reactors’ working volume due to continuous feeding of substrate. Judging from the protein production pattern exhibited, it might well be possible to further increase total protein titers with longer cultivation times. The volumetric ABTS activity that could be produced was in the same order of magnitude for all three proteins and about an order of magnitude lower than that of routine fermentations of recombinant UPO from -PaDa-I) . In ordeCabUPO1, rCabUPO 2, and rAniUPO was first clarified by centrifugation, then by 0.2 µm cross-flow filtration, and was afterwards concentrated via cross-flow ultrafiltration using a 10 kDa cut-off membrane before chromatographic purification by FPLC. The concentrated culture broth was first subjected to hydrophobic interaction chromatography (HIC). rCabUPO 2 eluted in a single peak  protein band. Size exclusion chromatography (SEC) was carried out after concentration and dialysis of the pooled fractions from the HIC step to further purify the enzymes. While this was not strictly necessary for rCabUPO 2, it was deemed appropriate to treat all enzymes similarly before biochemical characterization. While SEC for rCabUPO 2 .CabUPO 1 and rCabUPO 2 bands were in range of predicted weight, while rAniUPO appeared as a non-distinct band at around 55 kDa and 40 kDa, which is significantly over the predicted weight of 27.6 kDa for the processed protein , which is often used as an indicator for the purity of heme-containing enzymes, was well above two for rCabUPO 1, in accordance with the high degree of purity determined by SDS-PAGE. While rCabUPO 2 was similarly pure by SDS-PAGE, its RZ was notably lower, which could have been the result of partial loss or lack of heme-incorporation [Z of rAniUPO might be caused (at least to some extent) by the high glycosylation degree. Characteristic features of the purified proteins are summarized in Spectra of the purified enzymes exhibited the heme-thiolate typical Soret region around 420 nm, as well as the charge transfer bands at about 530 nm and 570 nm . The Reiporation . While eCabUPO 1, rCabUPO 2, and rAniUPO were compared regarding their specific activities and pH-optima for typical UPO substrates. Storage of the enzymes at 4 °C after purification for several days led to partial inactivation, most notably in the form of irreversible protein denaturation (which became visible by precipitation). For this reason, pH-optima and activities of all samples were normalized and ‘absolute’ specific activities are not given. pH-Optima were determined with the substrates ABTS, NBD, and VA . The 3D models of the evolved rCabUPOs have recently been published [AniUPO had a C-score (confidence score) of 1.98  and an estimated TM-score  of 0.99 ± 0.04.None of the here-studied UPO enzymes currently have an experimentally resolved tertiary structure . Their respective 3D structures were instead simulated by using the computational C-I-TASSER pipeline  for conublished . The topAniUPO model and the resolved MroUPO in a structural alignment yielded a TM-score of 0.93, indicating a high global structural similarity between both proteins . In MroUPO’s channel-bottleneck was squarish, while rAniUPO’s bottleneck was oblong. Taking MroUPO’s left and right bottleneck flanks were mostly defined by sidechain residues F160 and I84 and the top flank by A59. The bottom flank was defined mainly by the peptide backbone between E157 and S156. rAniUPO’s channel-bottleneck was squished oblong by a much bulkier L66 that protruded into the channel from the top (A59 in MroUPO), while the bottleneck was at the same time offset to the left due to F161 (I153 in MroUPO). This diminished the influence of L93 in rAniUPO’s bottleneck topology (I84 in MroUPO). The bottom flank of rAniUPO’s bottleneck was mostly defined by the peptide backbone of E165 and G164, similar to MroUPO. Additionally, the left flank of rAniUPO’s bottleneck was opened up relatively to that of MroUPO (substituting F160 in MroUPO by A168 in rAniUPO). This in turn allowed rAniUPO’s L62 to participate in the topology of the bottleneck flank (whereas I55 hardly did in MroUPO).Comparing the simulated rAniUPO’s and as comparison MroUPO’s substrate access channel , will be required in the future. Nonetheless, the results presented here increase the toolbox of UPOs available for genetic engineering and heterologous expression—a general requirement for the industrial application of this enzyme type. The described expression of new UPOs, the (evolutionary) optimization of the enzymes [Out of a selection of ten putative UPO genes, three enzymes exhibited activities towards UPO-typical screening substrates. These enzymes were successfully produced in ent with ). Nevert enzymes ,29,30,31 enzymes ,35,36,37


Forkhead box transcription factors have been shown to be involved in various developmental and differentiation processes. In particular, members of the FoxP family have been previously characterized in depth for their participation in the regulation of lung and neuronal cell differentiation and T-cell development and function; however, their role in adipocyte functionality has not yet been investigated. Here, we report for the first time that Forkhead box P4 (FoxP4) is expressed at high levels in subcutaneous fat depots and mature thermogenic adipocytes. Through molecular and gene expression analyses, we revealed that FoxP4 is induced in response to thermogenic stimuli, both in vivo and in isolated cells, and is regulated directly by the heat shock factor protein 1 through a heat shock response element identified in the proximal promoter region of FoxP4. Further detailed analysis involving chromatin immunoprecipitation and luciferase assays demonstrated that FoxP4 directly controls the levels of uncoupling protein 1, a key regulator of thermogenesis that uncouples fatty acid oxidation from ATP production. In addition, through our gain-of-function and loss-of-function studies, we showed that FoxP4 regulates the expression of a number of classic brown and beige fat genes and affects oxygen consumption in isolated adipocytes. Overall, our data demonstrate for the first time the novel role of FoxP4 in the regulation of thermogenic adipocyte functionality. Excessive adipose tissue expansion leads to obesity and is associated with increased risk to develop a number of metabolic disorders, including type 2 diabetes, hypertension, and cardiovascular disease . It has Forkhead box factors belong to a large family of transcription factors comprising more than 50 members characterized by a homologous DNA-binding domain. They have been involved in development and differentiation and, specifically, members of the Foxo and Foxa family have been shown to participate in metabolism. In particular, transcription factors such as Foxo1 were found to modulate uncoupling protein 1 (UCP1) expression  in adipoThe members of the FoxP subfamily, which includes four members—FoxP numbered 1 through 4, contain a conserved leucine zipper domain and a zinc finger motif, in addition to the characteristic winged helix/forkhead DNA-binding module  that givg. The pelleted cells representing the SVF were resuspended in culture medium. To induce brown-like fat differentiation, confluent 10T1/2 cells and SVF cells were treated with induction medium containing DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 20 nM insulin , 1 nM T3 , 125 μM indomethacin , 1 μM dexamethasone , 1 μM rosiglitazone , and 0.5 μM isobutylmethylxanthine . The culture medium was replaced with maintenance medium containing DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1 nM T3, and 20 nM insulin after 2 days for 10T1/2 cells or after 4 days for mouse SVF cells. We subsequently replaced the maintenance medium every 2 days, until cells were fully differentiated. To test the role of cAMP modulators in the regulation of FoxP4 levels, we treated brown fat differentiated cells with either vehicle or with 5, 10, or 20 μM of forskolin , 5, 10, or 20 μM of isoproterenol  or 125, 250, or 500 μM of IBMX, for 4 h before cells were harvested for molecular analysis or with doses and times otherwise noted in the legends. To obtain mature adipocytes, we dissected ScWAT fat pads of 9-week-old C57Bl/J6 male mice and washed in PBS containing 1% antibiotic and digested for 45 min in a solution containing 1 mg/ml of collagenase type IV ; the resulting cells were filtered through a 100-μm cell strainer  to eliminate clumps and centrifuged for 10 min at 200 g. Middle layer containing mature adipocytes and the pelleted cells representing the SVF were resuspended in lysis buffer for RNA extraction .10T1/2 and human embryonic kidney-293 (HEK-293) cells were obtained from American Type Culture Collection. Stromal vascular fraction (SVF) cells were isolated from scWAT adipose tissues of male mice and maintained in growth medium using DMEM , supplemented with 10% (v/v) FBS  and with 1% penicillin/streptomycin . Isolation of mouse SVF cells was performed as previously described . Briefly9 Pfu of each adenovirus diluted in saline were injected subcutaneously bilaterally into the scWAT fat pads of 9-week-old male mice, twice a week, for 2 weeks. Mice were sacrificed at the fourth day after the final viral delivery. The Institutional Animal Care and Use Committee at New York University Langone Medical Center approved the mouse experimental protocol for this study.Male and female C57Bl/J6 mice  were housed at 24°C in standard cages and fed standard chow diet, exposed to a 12-h light/12-h dark cycle and had free access to food and water. For cold exposure experiments, 9-week-old male and female mice were either kept at room temperature or at 4°C, with full access to fresh water, for up to 6 h. Mice were euthanized at the end of the cold exposure, and fat adipose tissues were collected for mRNA and protein analysis. For studies of the effects of β-adrenergic stimulation, 100 μl of either CL316,243 dissolved in saline, at the concentration of 1 mg/kg of body weight , or saline alone, were injected in 9-week-old mice intraperitoneally. Fat depots were collected 3 h after the injections. To study the effect of HSF1 gain of function in vivo on the levels of FoxP4, we injected adenoviruses expressing either control (CMV-GFP) or mouse HSF1 (HSF1-CMV-GFP) (generated by Vector Biolabs) as previously described (Renilla luciferase control reporter vector (#E2231) were purchased from Promega. The UCP1 (2.1 kb) luciferase plasmid was a kind gift of Dr Evan Rosen  and Dr Sona Kang (U.C. Berkley).The expression plasmid for FoxP4 was obtained from Origene (MR 221681) and the HSF1 plasmid from Addgene (#71724); the pGL3 luciferase reporter vector (#E1751) and the pRL For gene expression studies, we transfected differentiated 10T1/2 cells using DharmaFECT transfection reagent , according to the manufacturer's instructions. Specifically, for gain-of-function experiments, brown-like differentiated 10T1/2 cells were transfected with 5 μg of either vector, HSF1, or FoxP4 expression plasmids and harvested 2 days later. For knockdown experiments, brown-like differentiated 10T1/2 cells were transfected with 35 nM of siRNA luciferase , siRNA HSF1, or siFoxP4 and harvested 3 days later. To test the effect of FoxP4 on brown fat differentiation, undifferentiated 10T1/2 cells were transfected with 35 nM of siLuc or siFoxP4. About 24 hours after transfection, cells were induced to differentiate and harvested 5 days later.Extraction of total RNA from cultured cells and fat tissues was achieved using RNeasy Midi Kit  and TRIzol , respectively. About 1 μg of total RNA was reverse transcribed using the iScript™ cDNA Synthesis Kit . For gene expression analysis, we performed real-time quantitative PCR in triplicate using 25 ng of complementary DNA and 300 nM of primers with the iQ™ SYBR® Green Supermix , according to manufacturer's protocols. The relative mRNA quantification was calculated via the ΔΔCt method with normalization of each sample to the average change in cycle threshold value of the control 36B4 gene. We used the following primers for quantitative PCR analysis: FoxP4—Forward: CGACATGATGGTGGAATCTG, Reverse: TGTTTGCTGTCATTGTTCCC; HSF1—Forward: AGGCAGGAGCATAGATGAGA, Reverse: AGGATGGAGTCAATGAAGGC; Ucp1—Forward: GGCCCTTGTAAACAACAAAATAC, Reverse: GGCAACAAGAGCTGACAGTAAAT; 36B4—Forward: GCTTCATTGTGGGAGCAGAC, Reverse: ATGGTGTTCTTGCCCATCAG; PGC1α—Forward: ACCATGACTACTGTCAGTCACTC, Reverse: GTCAC AGGAGGCATCTTTGAAG; PRDM16—Forward: CCACCAGCGAGGACTTCAC, Reverse: GGAGGACTCTCGTAGCTCGAA; Cidea—Forward: TGACATTCATGGGATTGCAGAC, Reverse: CGAGCTGGATGTATGAGGGG; CytC—Forward: AAATCTCCACGGTCTGTTCGG, Reverse: GGGTATCCTCTCCCCAGGTG; Mcad—Forward: ATGACGGAGCAGCCAATGAT, Reverse: TCGTCACCCTTCTTCTCTGCTT; Cpt1α—Forward: TTGCCCTACAGCTCTGGCATTTCC, Reverse: GCACCCAGATGATTGGGATACTGT; Cox4β—Forward: CTGCCCGGAG TCTGGTAATG, Reverse: CAGTCAACGTAGGGGGTCATC; Elovl3—Forward: TTCTCACGCGGGTTAAAAATGG, Reverse: TCTCGAAGTCATAGGGTTGCAT; ZNF638—Forward: ATTGAGAGCTGTCGGCAGTTA, Reverse: GGAATGAGAACGT CTTCTTGGAG; PPARα—Forward: TCGAGGAAGGCACTACACCT, Reverse: TCTTCCCAAAGCTCCTTCAA; aP2—Forward: ACACCGAGATTTCCTTCAAACTG, Reverse: CCATCTAGGGTTATGATGCTCTTC; C/EBPα—Forward: GAACAGCAACGAGTACCGGGTA, Reverse: GCCATGGCCTTGACCAAGGAG; and PPARγ—Forward: AGTCTGCTGATCTGCGAGCC, Reverse: CTTTCCTGTCAAGATCGCCC.Whole cell extracts were obtained from cultured cells and tissues with RIPA buffer , supplemented with a cocktail of protease inhibitors . About 20 μg of protein lysates were run on a 10% SDS-polyacrylamide gels and transferred onto 0.45 μm PVDF membranes . Membranes were blocked using a solution containing 5% nonfat dry milk (w/v) resuspended in TBST 0.1% buffer  for 1 h at room temperature and were subsequently incubated with primary antibodies at 4°C overnight in a solution containing 1% BSA resuspended in TBST 0.1% buffer . We used the following antibodies at the dilutions indicated: anti-FoxP4  at 1:1,000; anti-HSF1  at 1:1,000; anti-UCP1  at 1:1,000, and antivinculin  at 1:2,000. The membranes were washed three times in TBST 0.1% (v/v) after incubation with the primary antibody and subsequently incubated at room temperature for 1 h with a 1:20,000 dilution of antirabbit (#1706515) or antimouse (#1706516) IgG horseradish peroxidase-conjugated (BioRad) in TBST 0.1% containing 2% nonfat dry milk (w/v). Immunoblots were developed after four additional washes in TBST 0.1% (v/v), using an enhanced chemiluminescence kit  on Hyblot CL autoradiography films , using an X-ray film developer .oC overnight using an anti-HSF1 antibody or an anti-FoxP4 antibody or with an anti-IgG control antibody . After the cross-linked protein–DNA complexes were incubated with protein A agarose beads , they were eluted for 15 min at room temperature using Elution Reagent C . DNA fragments were purified and amplified by RT-PCR using the primers listed later, designed specifically to recognize the area spanning the putative heat shock element (HSE) identified in the proximal region of the FoxP4 promoter, in position -240 to -233, and the putative forkhead response element identified in the proximal UCP1 promoter in position -479 to -472: PromFoxP4—Forward: CTTCAACAGCCTTGGACCAG, Reverse: CTTCAACAGCCTTGGACCAG; PromUCP1—Forward: CTTCAACAGCCTTGGACCAG, Reverse: CTTCAACAGCCTTGGACCAG; and Actb—Forward: TGAGGTACTAGCCACGAGAGAG, Reverse: ACACCCGCCACCAGGTAAGCA.Chromatin immunoprecipitation (ChIP) analysis of 10T1/2 cells differentiated into brown-like adipocytes was performed according to the manufacturer's protocol , as previously described . BrieflyFor luciferase assays, we plated 20,000 HEK-293 cells per well of a 12-well tissue culture plate, and 24 h later, cells were cotransfected with 0.5 μg of the UCP1 luciferase reporter plasmid and with vector expression plasmid or FoxP4 (0.5 μg) and 50 ng of Renilla reporter plasmid, using Lipofectamine 2000 . About 48 h after transfection, cells were harvested for luciferase assays. The luciferase reporter activity was measured using the Dual-Luciferase Reporter Assay System Kit . Luciferase activity was detected using the SpectraMax 96 well Plate Reader , and the values were normalized to Renilla activity.Cellular metabolic rates were measured using an XFe24 Analyzer (Seahorse Agilent). For FoxP4 overexpression studies, differentiated 10T1/2 cells were transfected with either vector or FoxP4 expression plasmid, whereas for knockdown studies, either control siRNA (siLuc) or siFoxP4 were transfected in these cells. Respiration was measured under basal conditions, following the addition of ATP synthase inhibitor oligomycin, the mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone, or the complex III inhibitor antimycin A/rotenone, as per vendor's protocol .t-test or by one-way analysis of variance. P values <0.05 were considered statistically significant. All statistical analyses were performed using Prism 7 software .All the results were expressed as mean ± SE, unless otherwise noted. Different groups were compared via Student's FoxP4 has been previously shown to be expressed, and to exert its function, in neurons, intestine, and immune cells , 21, 22,Given that transcription factors and cofactors such as PGC1α, ZFP516, PRDM16, PPARα, ZNF638 , 30, andWe have previously shown that HSF1 is induced by cold exposure and agents that increase cAMP levels . FurtherGiven our demonstration that FoxP4 levels are regulated in adipose tissues, we assessed the potential relevance of FoxP4 in inducing the expression of the critical marker of brown fat function UCP1. FoxP4 gain-of-function and loss-of-function experiments in differentiated 10T1/2 cells demonstrated that modulation of FoxP4 affects UCP1 mRNA and protein levels, as shown in Since FoxP4 regulates the classic thermogenic marker UCP1, we determined the importance of FoxP4 in adipocyte function by assessing the impact of FoxP4 gain-of-function and loss-of-function studies in differentiated 10T1/2 cells on a panel of thermogenic genes. Our gene expression analysis in cells overexpressing FoxP4 revealed that the levels of genes such as PGC1α, Dio2, HSF1, ZNF638, CytC, Cox4β, Prdm16, Evolv3, and Mcad are significantly induced A. ConverForkhead box transcription factors have been shown to regulate development and differentiation processes in a number of cell types; however, less is known about the role that these proteins play in adipocyte differentiation and thermogenic functionality. While to date, members of the Foxo, Foxa, and Foxc subfamilies, such as Foxo1 , 14, 32,Our studies demonstrating that FoxP4 is present in adipose tissues is novel given that FoxP4 has been mainly reported to be expressed in neuronal, pulmonary, gut, and T cells , 35, 36.Our data analyzing the inducibility of FoxP4 in response to classic thermogenic cues and promoter occupancy studies in combination with gain-of-function and loss-of-function analyses mechanistically demonstrate that FoxP4 expression is regulated through a functional HSE motif present in its proximal promoter region, both in vitro and in vivo, and that this regulation involves the stress-induced transcription factor HSF1. These data not only specify a novel mechanism of FoxP4 regulation previously not characterized but also expand our current knowledge on the mechanisms through which HSF1 regulates brown fat programs, beyond the role that our laboratory has described for HSF1 as a regulator of PGC1α expression . In addiOur results demonstrate for the first time that FoxP4 is involved in thermogenic functionality via the direct control of the UCP1 promoter through a forkhead-binding motif not previously shown to be involved in the regulation of UCP1 expression. Future studies will determine whether forkhead box factors may complement each other by acting promiscuously at the forkhead-binding motif we have identified at the UCP1 promoter or whether they may compete for their binding in virtue of their differential affinity to this site.Our data obtained through RNA analysis and Seahorse studies demonstrate that modulation of FoxP4 levels not only affects thermogenic gene expression but also functionally alters oxygen consumption in adipocytes, thereby implicating FoxP4 for the first time in thermogenic fat activity. In conclusion, our studies provide the first demonstration of FoxP4 involvement in adipocyte tissue functionality, neither previously known nor appreciated.The data underlying this article are available in the article. Additional data underlying this article will be shared on reasonable request to the corresponding author.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


We found that YQJPF significantly ameliorates liver injury in vivo and in vitro that is associated with the regulation of hepatocyte necroptosis. Specifically, YQJPF decreased expression of receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3) and pseudokinase mixed lineage kinase domain-like (MLKL) to inhibit the migration of RIPK1 and RIPK3 into necrosome. YQJPF also reduces the expression of inflammatory cytokines IL-6, IL-8, IL-1β, and TNF-α, which were regulated by RIPK3 mediates cell death. RIPK1 depletion was found to enhance the protective effect of YQJPF. Furthermore, we showed that YQJPF significantly downregulates the mitochondrial reactive oxygen species (ROS) production and mitochondrial depolarization, with ROS scavenger, 4-hydroxy-TEMPO treatment recovering impaired RIPK1-mediated necroptosis and reducing the expression of IL-6, IL-8, IL-1β, and TNF-α. In summary, our study revealed the molecular mechanism of protective effect of YQJPF on hepatocyte necroptosis, targeting RIPK1/RIPK3-complex-dependent necroptosis via ROS signaling. Overall, our results provided a novel perspective to indicate the positive role of YQJPF in ACLF.Acute-on-chronic liver failure (ACLF) is described as a characteristic of acute jaundice and coagulation dysfunction. Effective treatments for ACLF are unavailable and hence are urgently required. We aimed to define the effect of Yi-Qi-Jian-Pi Formula (YQJPF) on liver injury and further examine the molecular mechanisms. In this study, we established CCl Acute-on-chronic liver failure (ACLF) is a syndrome characterized by acute decompensation of chronic liver disease, which has a high short-term mortality rate, and it seriously threatens the lives of patients with chronic liver disease . HoweverCorethrodendron multijugum (Maxim.) B. H. Choi and H. Ohashi (Huangqi in Chinese) and Pseudostellariae Radix, Pseudostellaria heterophylla (Miq.) Pax (Taizishen in Chinese) are Monarch drugs; Angelicae Sinensis Radix, Angelica sinensis (Oliv.) Diels (Danggui in Chinese), Wolfiporia extensa (Peck) Ginns (Fulin in Chinese), Atractylodes lancea (Thunb.) DC (Cangzhu in Chinese) and Atractylodes macrocephala Koidz (Baizhu in Chinese) are Ministerial drugs; and Citrus Reticulata, Citrus × aurantium L. (Chenpi in Chinese), Scutellariae Radix Scutellaria baicalensis Georgi (Huangqin in Chinese), and licorice, Glycyrrhiza uralensis Fisch. ex DC., G. glabra L., and G. inflata Batalin (Gancao in Chinese) are adjuvants. A retrospective cohort study on 60 patients with HBV-ACLF reported that YQJPF could prevent the development of liver failure and improve the Model for End-Stage Liver disease score /LPS injection that recapitulate some features of clinical ACLF. In these models, we assessed the features of ACLF in laboratory settings. Further, ACLF models were treated with YQJPF to assess its effect on liver injury. Additionally, we explored the role of inflammation infiltration and hepatocyte necroptosis and the potential mechanisms for the treatment of liver injury in ACLF.In the present study, we developed β-actin (20536-1-AP), RIPK1 (17519-1-AP), and RIPK3 (17563-1-AP) were purchased from Proteintech Group, Inc . Caspase-3 (#9662), cleaved-caspase-3 (#9664), caspase-8 (#9746), cleaved-caspase-8 (#9748), TNF-α (#11948), IL-18 (#54943), IL-1β (#12242), and IL-6 (#12153) were procured from Cell Signaling Technology . Mixed lineage kinase domain-like protein  were purchased from Abcam Technology .Methylprednisolone was obtained from the Nanjing Hospital affiliated to Nanjing University of Chinese Medicine  and was dissolved in PBS to a concentration of 30 mg/ml and was stored at 4°C. Necrotatin-1 (Nec-1) and 4-hydroxy-TEMPO (Tempol) were bought from Sigma-Aldrich . Dulbecco’s modified essential medium (DMEM), fetal bovine serum (FBS), Opti-MEM medium, phosphate-buffered saline (PBS), and trypsin-EDTA were purchased from GIBCO . The antibodies against YQJPF was provided by Nanjing University of Chinese Medicine, Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine. YQJPF was prepared from nine commonly used Chinese herbal medicines . All theThen, all herbs were decocted twice and the decoction liquids were concentrated to a density of 2.86 g/ml and was stored at 4°C. For quality control, HPLC analysis was used as previously described . YQJPF w4 and olive oil [1:1 (w/v)] was used to induce liver cirrhosis in rats (1.5 ml/kg body weight). LPS (100 μg/kg) and D-Gal (400 mg/kg) were used to induce acute liver injury. The mice were randomly divided into seven groups (n = 8 for each group). The experimental groups were as follows  for 2 weeks followed by i. p. with LPS and D-Gal once.Group 5: YQJPF low-dose treatment group; i. p. with CCl4 for 12 weeks and i. g. with YQJPF (28.6 g/kg) for 2 weeks followed by i. p. with LPS and D-Gal once.Group 6: YQJPF high-dose treatment group; i. p. with CCl4 for 12 weeks and i. p. with methylprednisolone (15 mg/kg) for 2 weeks followed by i. p. with LPS and D-Gal once.Group 7: methylprednisolone treatment group; i. p. with CClA total of 56 male Sprague-Dawley rats, with the body weight of 180–220 g, were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd . All animals were housed with laboratory chow and tap water at 22 ± 2°C under 55 ± 5% humidity-controlled environment and a 12-h light–dark cycle. The experimental design was approved by the Institutional and Local Committee on the Care and Use of Animals of Nanjing University of Chinese Medicine . All animals were provided care in compliance with the National Institutes of Health (United States) guidelines. A mixture of CCl follows :Group 1:2 and 95% air humidified atmosphere at 37°C.Human LO2 cell line was obtained from the Cell Bank of Chinese Academy of Sciences . Cells were cultured in DMEM with 10% FBS and 1% antibiotics and were incubated in 5% COLiver hematoxylin and eosin (H&E) and Masson and Sirius Red staining were performed as per the previously described methods . RepreseBlood samples were collected and incubated at room temperature for 1 h, and extracted serum was collected after centrifugation. Levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in serum were measured using enzyme-linked immunosorbent assay kits  according to the manufacturer’s protocols.Liver tissues were treated according to the treatment regimen mentioned earlier After treatment, hepatic mtDNA, ATP, malondialdehyde (MDA), and superoxide dismutase (SOD) were quantified using corresponding kits  according to the manufacturer’s instructions.Immunohistochemical staining of liver tissue was performed using antibodies against caspase 8, MLKL, RIPK1, and RIPK3, as previously described . Represe3 cells per well. After incubating for 24 h, the cells were treated with LPS (10 μg/ml) and D-Gal (10 mg/ml) for 4 h for the hepatocyte injury model. Further, the cells were treated with serial concentration gradients of YQJPF and atractylone for another 24 h, with triplicates for each concentration. Then 5 mg/ml MTT was added in cells, and cells were incubated at 37°C for 4 h, and the absorbance was measured at a wavelength of 490 nm. Independent experiments were performed in triplicates.To assess the effects of different treatments on cell proliferation, Cell viability analysis was performed using MTT. In brief, The LO2 cells were seeded into 96-well plates at a density of 3 × 10Cell death was determined using the trypan blue staining assay . In brief, after YQJPF treatment, the cells were digested using trypsin without EDTA followed by centrifugation. The cells were further washed twice with PBS and were resuspended in PBS. Cell suspension and trypan blue solution were mixed gently in 1:1 ratio and allowed to stand for 3 min. A few of the stained cells were counted with a hemocytometer, and photographs were taken in a blinded fashion at random fields. Stained blue cells represented dead cells.Total RNA was extracted from liver tissues and LO2 cells by using Trizol reagent according to the manufacturer’s protocol  and further reverse-transcribed into cDNA by using PrimeScript RT reagent kit . Real-time PCR was performed using the SYBR Green I fluorescent dye , according to the manufacturer’s protocol. Glyceraldehyde phosphate dehydrogenase (GAPDH) served as an invariant control, and mRNA levels were expressed as fold changes after normalizing to GAPDH. The experiment was performed in triplicates. Primers  are listed in Cells and liver samples were lyzed using a mammalian lysis buffer  and were denatured in SDS loading buffer. Then, western blot analysis was performed according to the manufacturer’s instructions . Thereafter, Cells and liver samples were incubated overnight with antibodies against caspase 3, cleaved-caspase-3, caspase 8, cleaved-caspase-8, MLKL, RIPK1, RIPK3, IL-6, IL-18, IL-1β, and TNFα at 4°C. Protein detection, band visualization, and quantification were performed as per the manufacturer’s instructions. Anti-β-actin antibody  was used as a loading control.The immunoprecipitation assay was performed using co-immunoprecipitation (Co-IP) kit (Thermo) as per manufacturer’s instructions. Briefly, the cell lysates were incubated with anti-RIPK1 and anti-RIPK3 antibodies. Protein G-agarose beads were added and incubated overnight to precipitate the protein complexes. Western blot analysis was performed to detect the protein complexes expression.LO2 cells were seeded on 24-well plates and cultured in DMEM with 10% FBS. Afterward, they were treated with corresponding reagents for 24 h. Then, the cells were fixed with 4% PFA for 30 min at 37°C, permeabilized with PBS-T (0.1% Triton x-100 dissolved in PBS), and blocked with PBS-B (4% BSA dissolved in PBS). The cells were stained with the corresponding antibody (1:200 dilution) overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit IgG (1:100 dilution) for 2 h. Finally, 4′,6-diamidino-2-phenylindole (DAPI) staining was performed by incubation in dark for 5 min, and the fluorescence was observed using a fluorescence microscope  to visualize the nuclei.Levels of IL-6, IL-8, IL-1β, and TNF-α in serum were measured using ELISA kits  according to the manufacturer’s instructions.The level of intracellular ROS was determined using an oxidation-sensitive fluorescent probe, 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) . Briefly, LO2 cells were seeded in a 24-well plate and pre-treated with ROS scavenger Tempol (10 μM) for 1 h before exposing to YQJPF or selective RIPK1 inhibitor Nec-1. DCFH-DA probe was subsequently incubated with LO2 for 30 min. Subsequently, ROS levels were detected according to the manufacturer’s instructions.We determined mitochondrial ROS production by using a fluorescent probe, MitoSOX, which is a red mitochondrial superoxide indicator for live cell imaging . Briefly, LO2 cells were treated with YQJPF or selective RIPK1 inhibitor Nec-1. Further, LO2 cells were incubated with MitoSox reagent (5 μM) for 10 min at 37°C and washed with PBS. The red fluorescence density was detected using a fluorescence microscope  with the rhodamine channel.The mitochondrial membrane potential (MMP) was determined using 5.5′,6.6′-tetrachloro-1.1′,3.3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1)  staining as per the manufacturer’s protocol. LO2 cells were washed with PBS and incubated with JC-1 working solution at 37°C in dark for 20 min. After removing JC-1 solution, the cells were washed with PBS, and images were taken using a fluorescence microscope  with both red and green channels. The MMP is represented by the average red/green fluorescence intensity ratio.LO2 cells were seeded in six-well plates . After the corresponding treatment, transmission electron microscopy (TEM) images were obtained using a JEM 1010 transmission electron microscope . Briefly, LO2 cells were fixed in Karnovsky fixative solution  during 1 h and then washed three times in a cacodylate buffer for 15 min. Then pellets were embedded into 2% agar and postfixed in 1% osmium tetraoxide in a cacodylate buffer during 1 h. Samples were then dehydrated in 50, 70, 95, and 100% of acetone for 2, 10, 30, and 60 min, respectively, and embedded into Durcupan ACM . And the ultrathin sections (70 nm) were placed on nickel grids and examined at 100 keV. For each group, electromicrographs were recorded at a microscope magnification of ×10,000.2 at 37°C. After that, cells were trypsinized, washed in PBS and stained according to kit according to the manufacturer’s instructions. Stained cells were analyzed using BD Accuri C6 flow cytometer  and data was processed with FlowJo_v10.6.2 software. Each sample was assessed using a collection of 10,000 events. The mean values and standard deviations were calculated from three independent experiments.FITC Annexin V apoptosis detection kit  was used for detection of cell apoptosis. Briefly, LO2 cells were seeded in six-well plates in complete medium and allowed to attach for 24 h. Then, cells were treated as indicated. Cells were further incubated for 24 h, in 5% COp value of <0.05 was considered statistically significant.Two-tailed Student’s tests and one-way ANOVA analysis were performed using GraphPad Prism software v. 7.0 . The values and data are presented as the mean ± SEM from three independent experiments. A 4 for 12 weeks. Further, acute liver injury was induced by using a combination of LPS and D-Gal. And YQJPF was given for 2 weeks to investigate the protective effect on ACLF rats. In addition, methylprednisolone (Sou-Medrol) was used as a positive control drug, which has been confirmed as a therapeutic agent for ACLF  and D-Gal (10 mg/ml) to induce liver injury in LO2 cells. MTT assay showed that LPS and D-Gal inhibit cell viability, whereas YQJPF and atractylone could promote cell viability in a dose-dependent manner. YQJPF above 10 μg/ml and atractylone at 5 μM showed a significant effect . Light mNecrosis and apoptosis are the most common pathways of death during liver injury . Hence, The RIPK1/RIPK3 signaling pathway has been reported to be involved in the induction of necroptosis . Thus, wNumerous studies have shown that ROS production is necessary for necroptosis in several cell lines such as macrophages , fibrosain vivo studies on the effect of YQJPF on hepatocyte necroptosis were the same as those of in vitro results. Protein levels of key caspases did not change under YQJPF treatment. Therefore, reduction in cell death due to YQJPF was independent of apoptosis. Further, we proceeded to investigate whether YQJPF could disrupt hepatocytic death by regulating necroptosis. Western blot analysis of RIPK1, RIPK3, and MLKL, which governed necroptosis, indicated that necroptosis is increased in ACLF in vivo  and D-Gal (10 mg/ml). YQJPF and atractylone (the main component of YQJPF) could restore the damage to cell viability induced by LPS and D-Gal and decrease ALT, AST, and LDH levels in cell supernatant. These data clearly indicated that YQJPF confers protection against ACLF.The pathobiology of ACLF is characterized by hepatic and systemic inflammation; progressive, unrelenting hepatocyte injury; and hepatocyte death. Hepatocyte death typically follows two patterns: necrosis and apoptosis . In our Liver failure is characterized by massive loss of parenchymal cells, and cell death can occur in this pathophysiological context . NecroptOxidative stress, a major contributor of liver injury, can be induced by excessive ROS. Studies have shown that ROS may be considerably involved in the pathogenesis of liver diseases . ROS havvia Keap1/Nrf2 pathway  pattern is a physiological regulation network, which contains a regulation center, overall effective target, material basis and functional unit. Its mechanism needs to be indicated by means of TCM pattern prevention and treatment characteristics, multiple organ integration effectiveness, and functional unit network so as to guide clinical practice as well as for inspiring basic research. It is urgently to exploring the efficacy mechanism and regulation with the molecular biology and systems biology. In our research, we conducted a deep study of the pathogenesis, TCM pattern feature and efficacy evaluation of TCM in ACLF. And results showed that YQJPF attenuates liver injury in ACLF. Recently, Chen et al.’ reported that flavonoids have been to possess a wide variety of biological activities such as antioxidant activity, anti-inflammatory activity, hepatoprotective effect, antibacterial activity, antiviral activity, anticancer activity, and antidiabetic activity . Chen et pathway . In our in vivo and in vitro. Mechanistically, YQJPF inhibits hepatocyte necroptosis by blocking the RIPK1/RIPK3 complex formation followed by relieving mitochondrial dysfunction, which ultimately prevents hepatocytic cell death. Most importantly, we confirmed that YQJPF can be used as a medicine for ACLF treatment and provided a scientific basis for the research on the application of YQJPF in ACLF.In summary, YQJPF attenuates liver injury by inhibiting hepatic inflammation and necroptosis of hepatocytes