Escherichia coli Nissle 1917 strain with a plasmid containing a gene cassette that encoded two proteins PucL and PucM for urate metabolism from Bacillus subtilis, the urate importer YgfU and catalase KatG from E. coli, and the bacterial hemoglobin Vhb from Vitreoscilla sp. The recombinant E. coli strain effectively degraded uric acid under hypoxic conditions. A new method to induce hyperuricemia in mice was developed by intravenously injecting uric acid. The engineered Escherichia coli strain significantly lowered the serum uric acid when introduced into the gut or directly injected into the blood vessel. The results support the use of urate-degrading bacteria in the gut to treat hyperuricemia. Direct injecting bacteria into blood vessels to treat metabolic diseases is proof of concept, and it has been tried to treat solid tumors.Hyperuricemia is the second most prevalent metabolic disease to human health after diabetes. Only a few clinical drugs are available, and most of them have serious side effects. The human body does not have urate oxidase, and uric acid is secreted via the kidney or the intestine. Reduction through kidney secretion is often the cause of hyperuricemia. We hypothesized that the intestine secretion could be enhanced when a recombinant urate-degrading bacterium was introduced into the gut. We engineered an  First, diet control is used to reduce the intake of purines and nucleosides.7 Second, xanthine oxidase inhibitors such as allopurinol, oxypurinol, and febuxostat, are used to inhibit the activity of xanthine oxidase to reduce UA production.8 Third, URAT1 (human urate transporter 1), which is a key UA transporter and is responsible for reabsorbing UA from the renal tubule into cells,10 is inhibited by drugs such as probenecid, benzbromarone, and losartan to reduce UA absorption in the renal tubules.11 Fourth, UA is degraded in the blood by supplementing exogenous UOX.12Hyperuricemia could be treated either by reducing UA production or promoting UA excretion through the kidney, which are two major factors leading to the development of hyperuricemia.14 The addition of UOX can significantly reduce the level of UA in the patient’s body in the short term, but studies have also shown that UOX is disruptive in the body by producing H2O2.15 In addition, long-term uses will cause the body to produce antibodies against UOX.15These clinic therapies have limitations. Restricting diet can significantly reduce the quality of life and the mood of patients, and it must be adhered to for a long time to achieve the therapeutic effect. The use of medicines often has strong side effects, which limits their clinical use.16 Exogenous UOX in various forms, such as being modified by polyethylene glycol, encapsulated with catalase or catalase mimic platinum nanoparticles, has successfully applied to reduce UA in mouse blood.17–19 These designs are aimed to protect UOX and remove toxic H2O2.New strategies have been developed to treat hyperuricemia. Several natural products have been successfully tested as xanthine oxidase inhibitors to inhibit UA production or to reduce UA reabsorption in a mouse model.20 Gut microbiota may be another way to treat hyperuricemia.21 Several Lactobacillus strains may degrade purine and UA in the gut to ameliorate hyperuricemia in mice.23 Since UOX activity requires oxygen,24 its activity may be reduced in the oxygen-limiting gut.26 When an engineered E. coli BL21 (DE3) strain that overproduces secreted UOX is orally given to rats, it enters the intestine and releases UOX in the gut, resulting in reduced levels of serum UA after five weeks of the treatment.27 Yet, the degree of serum reduction is limited. Therefore, an engineered bacterium with enhanced UOX activity under anoxia conditions may be ideal for treating hyperuricemia in the gut.One-third of the produced UA in humans is excreted through the intestinal tract and is further metabolized by the gut microbiota.24 The UOX obtained from several microorganisms does not need any cofactor. It has been used as the clinical diagnostic reagent to detect UA and utilized therapeutically to relieve excessive urate accumulation.28 Especially, the PucL and PucM from Bacillus subtilis are extensively studied. The enzymes catalyze a 3-step UA degradation pathway in B. subtilis29  from E. coli Nissle 1917 (EcN) has been used to treat inflammatory bowel disease and irritable bowel syndrome for over a century.35 Because of its safety record and genetic malleability, it has been engineered to deliver therapeutic payloads to treat several disorders, such as bacterial infection, metabolic disorders, etc.36 Recently, it has been developed as a vector to produce and deliver anticancer agents into tumors by intravenous injection.37 These applications prove that the injection of EcN into the blood is relatively safe.Probiotic We successfully constructed an engineered EcN with PucL, PucM, KatG, Vhb, and YgfU for UA degradation. This strain efficiently degraded UA even with dissolved oxygen (DO) under a 15% level of the normal condition, allowing it to function in the gut. A new method to induce acute hyperuricemia in mice was successfully developed to simulate high UA levels in humans through intravenous injection of UA. The engineered EcN strain was administrated into mice with acute hyperuricemia intragastrically and intravenously, respectively. The serum level of uric acid was sharply reduced by both treatments. Thus, live bacterial therapeutics may have great potentials to treat hyperuricemia.T) catalyzes the step 1 in the UA degradation (T (Table S1). EcN::pMCS2-Ptrc-pucLT with the gene encoding PucLT under the control of the Ptrc promoter in pBBR1MCS-2 offered the highest activity in the cell extract .The UriC domain of PucL  and coexpressed it with PucM in EcN (EcN::pMCS2-Ptrc-pucLMM). With cell extracts, the Vmax of PucLMM increased ~1.8-fold than that of the PucLM. However, the Km of PucLMM increased ~2.0-fold too . Hence, the fastest UA degradation strain EcN::pMCS2-Ptrc-pucLMM-ygfU was used for next step optimization.When YgfU, a UA importer in ncreased . Unexpecncreased . The grokatG gene encoding E. coli catalase was used to convert PucL-generated H2O2 back to O2, and vhb encoding a bacterial hemoglobin protein from Vitreoscilla sp. C1 was applied to allow the engineered strain to degrade UA at low O2 levels. Therefore, EcN::pMCS2-Ptrc-pucLMM-vhb-ygfU-katG was constructed to produce PucLMM, Vhb, YgfU, and KatG in the same bacterium, which reduced the growth rate, but not the final cell density in LB medium . EcN::pMCS2-Ptrc-pucLMM-vhb-ygfU-katG degraded UA at a slightly slower rate than EcN::pMCS2-Ptrc-pucLMM-ygfU did under normal oxygen condition , and the UA concentration in gut is not affected . The induced EcN cells that were administered intragastrically did not ameliorate hyperuricemia , nor did it affect the UA concentration in the gut .39 UA concentration in the 7d-1PO model was 45.2 μM ± 12.1 μM, still significantly lower than that in humans. At 48 μM UA, the engineered EcN degraded UA at a significantly low rate , and potassium oxonate, an inhibitor of UOX, further decreased the UA degradation rate by the engineered EcN cells. Even when potassium oxonate in whole cells was diluted to 10 μM equal to ~ 1% of the amount used for the intraperitoneal administration, it reduced the UA degradation by the EcN cells . These two factors could hamper the engineered EcN strain to treat hyperuricemia mice prepared with the 7d-1PO method.Further inspection revealed that the concentration of serum UA in normal Kunming mice was 14.1 μM ± 6.1 μM , which is 10–24 fold lower than the common range of serum UA concentrations in humans.To increase serum UA in mice to the levels comparable to that in humans, a UA solution was injected into the blood vessel at 70 mg per kg of body weight (mg/kg) of the testing mice. Serum UA was promoted up to ~1 mM, and the concentration gradually decreased over time . No mousMM-vhb-ygfU-katG was used to treat UA-injection hyperuricemia mice. The induced EcN cells were administered by gavage before 70 mg/kg or 5.7 mg/kg of UA was injected intravenously, respectively. In the group injected with 70 mg/kg of UA, the UA level of mice group with oral administration of total 1 × 1011 CFU of the EcN strain with the UA degrading genes was decreased sharply compared with the group with gavage of equal amount of EcN cells with the empty vector  (Control) and commercial mice serum (MM-vhb-ygfU-katG cells (1 x 108 CFU/mL) were added, they rapidly degraded UA in both groups . The engineered EcN strain cannot degrade UA at a meaningful rate at this low level of UA, as the UA degradation rate decreased more than 10 times when the UA concentration was changed from 650 μM to 50 μM . Potassium oxonate, which is frequently used to construct traditional hyperuricemia mice,43 further inhibited UA degradation by the EcN strain. To overcome the two hurdles, a new method to induce hyperuricemia in mice was developed by intravenously injecting UA without potassium oxonate.We developed a new method to induce hyperuricemia in mice by directly intravenous injection of UA, in which the serum UA concentration is closer to that in human serum . The UA uox-deletion mouse increases the serum UA level by about 12 folds, but the level is still lower than that in the human serum.45 Because these UOX-deficient mice are associated with low rate of live births and high mortality, they are not used to test our engineered EcN strain. Intravenous injection of UA sharply increased UA content in the mouse serum by more than 100 times without any death. Since the high UA is kept relatively stable in the mouse serum for the first hour  that produces secreted UOX has been introduced to the gut in hyperuricemia rats, the serum UA is decreased to a limited degree after 5 weeks treatment.27 However, free UOX is easily damaged,17 especially considering that the intestine is a protease-rich environment.50 Oxygen limitation further lowers UOX activity. These factors may contribute to the poor efficacy of using secreted UOX in the gut. In our design, UOX and other enzymes are protected from the unamiable gut environment by EcN cells, allowing effective degradation of UA in the gut and sharply lowering serum UA in hyperuricemia mice . Kanamycin , spectinomycin , and ampicillin  were used at 50 μg/mL, 50 μg/mL, and 100 μg/mL, respectively.The strains and plasmids used in this study are listed in pucL (Gene ID: 936669) and pucM (Gene ID: 937977) from Bacillus subtilis were codon-optimized and synthesized by Beijing Genomics Institute . The 975 bp of pucL encoding the UriC domain was amplified to obtain truncated pucL (TpucL). Asp-44 and Gln-268 of PucL were mutated to Val and Arg, respectively, in pucL and TpucL by using a modified QuikChange site-directed mutagenesis method.72 The mutated pucL and TpucL were named as MpucL and TMpucL. The ygfU (Gene ID: 949017) and the katG (Gene ID: 948431) genes were PCR amplified from E. coli MG1655 genomic DNA. The vgb gene (Protein ID: WP_019959060.1) from Vitreoscilla sp. C1 was codon optimized and synthesized by Beijing Genomics Institute .The 73 The schemes of the constructed plasmids were given in Fig. S6. The detailed plasmid DNA files in the GenBank format were zipped in Dataset S1. Briefly, the TpucL was cloned into pBBR1MCS-2 and pCL1920 under the control of the Trc promoter to generate pMCS::Ptrc-pucLT and pCL::Ptrc-pucLT. The TpucL was cloned into pBBR1MCS-2 under the Plac promoter to obtain pBBR1MCS-2::Plac-pucLT. Plasmids pBBR1MCS-2 and pCL1920 have medium and low copy numbers in E. coli, respectively.75 The Trc promoter has a higher transcription frequency than the Lac promoter.76 The pucL or MpucL and pucM genes were cloned into pBBR1MCS-2 under the control of the Trc promoter to produce pMCS2::Ptrc-pucLM and pMCS2::Ptrc-pucLMM. The ygfU was fused into pMCS2::Ptrc-pucLM and pMCS2::Ptrc-pucLMM to make pMCS2::Ptrc-pucLM-ygfU and pMCS2::Ptrc-pucLMM-ygfU, respectively. Further, the katG and vhb were inserted into plasmid pMCS::Ptrc-pucLMM-ygfU to obtain pMCS::Ptrc-pucLMM-vhb-ygfU-katG. After plasmids were prepared in E. coli XL1-Blue MRF’, they were electroporated into EcN with Eppendorf EporatorTM  by using 2.5-kV pulse.Primers used to amplify DNA fragments are given in Table S2. The TEDA method was used for routine plasmid constructions.600 at 0.05 and incubated at 37°C with shaking (200 rpm). For growth curves, cells were grown for 30 min before 2 mM isopropyl-β-D-thiogalactopyranoside (IPTG)  was added. 1 mL culture was sampled every hour to check the OD600 by using the UV-Vis spectrophotometer . For induction, 2 mM IPTG was added once the OD600 of the cultures reached ~0.6. The cultures were further cultivated at 30°C for 24 hours. The cells were collected, washed, and resuspended into the same HEPES buffer . The cells were concentrated at 5 × 1010 CFU/mL in the potassium phosphate buffer  with 15% glycerol and stored at −80°C. For use, the cells were diluted into the defined OD600 as required. The cell density of OD600 = 1.0 was treated as 1 × 109 CFU/mL.77 We analyzed 12 samples, and results supported this conversion with the average 8.5 × 108 CFU/mL and the standard deviation of 1.2 × 108 CFU·mL−1·OD600.−1The recombinant strains were inoculated in 5 mL LB with indicated antibiotics in a 14-mL culture tube at 37°C with shaking (200 rpm) overnight. The overnight cultures were inoculated in 50 mL fresh LB with the initial OD78 2) When UA and its degradation product (S)-allantoin were determined in complex media, an HPLC method was adopted with minor modifications.79 Briefly, a C18 reverse phase HPLC column  was pre-equilibrated with 100% Solvent A (2.5 mM NH4H2PO4 buffered to pH of 3.5 with phosphoric acid) and 0% Solvent B (5% solvent A and 95% methanol). The column was eluted with the following gradients of Solvent B: 0% from 0 to 3.5 min; 40%–80% from 3.5 to 11.5 min; 80%–100% from 11.5 to 15.3 min; 100%–0% from 15.3 to 18 min; 0% from 18 to 22 min. The flow rate was 1.0 mL/min. UA and allantoin were detected by using a HPLC device  with a diode array detector  at absorbance of 205 nm. 3) When low concentrations of UA (≤180 μM) were determined in blood samples, a UA assay kit  was used according to the manual instruction. 4) When UA concentration in blood sample was higher than 180 μM, a UA meter PD-G001-3-P  was used, which could be operated with simple procedures and low sample volume (<5 μL).Four methods were used to determine the concentration of UA in different cases. 1) UA in enzyme assay mixtures or in resting cell suspensions was determined with a spectrophotometer by directly measuring the absorbance at 293 nm.600 = 1.0 were broken through a Pressure-cell Homogenizer SPCH-18 . The enzyme activity assay of crude extracts was assayed by referring to a published method.38 Briefly, 3.6 mL reaction buffer  containing variable concentrations of UA  at 0, 10, 20, 40, 60, 80, 100, 120, 150 and 200 μM were prepared, and 400 μL crude extract was added to initiate UA degradation. The mixture was incubated at 37°C for 10 min, and the decrease of UA was detected by the spectrophotometric method. The protein concentrations were detected by using a microvolume spectrophotometer . Kinetic parameters of crude extracts were analyzed following the classical Michaelis–Menten equation, and data was fitted by using the ORIGINR 2016 software .Resuspended EcN cells at OD600 was directly transferred into a 50 mL centrifuge tube. UA was added at 1 mM to initiate the reaction, and the tube was incubated at 37°C with shaking (200 rpm) for 60 min. If necessary, a gradient of potassium oxonate  was added into the reaction mixture to check its inhibition effect for UA degradation. Samples were taken at 15 min intervals. After centrifugation at 13000 rpm for 3 min, the concentrations of UA in supernatant were determined by the spectrophotometric method.When UA degradation was assayed in buffer, 20 mL of resuspended induced cells at defined ODThe UA degradation ability of whole cells was also assayed in serum samples or whole blood samples from mice. Commercial mice serum from mice of 4 to 6 weeks of age  was purchased. 2 mL of the whole blood samples were also collected from 6 weeks old and 12 weeks old mice. The prepared whole cells of EcN were harvested and resuspended in the samples of serum or whole blood. Defined concentration of UA was added to initiate the UA degradation assay. The concentrations of UA at defined time intervals were determined by using either the UA assay kit or the UA meter.in vitro. For in vitro test, UA was added at 1 mM to 20 mL of resuspended cells at OD600 = 1.0. The DO in the reaction mixture was continuously recorded for 10 min with a Versastar RDO meter . At defined time intervals, the intracellular ROS was detected by using a ROS assay kit  according to the manufacturer’s instruction. Briefly, 1 mL of cell suspension was mixed with the ROS probe DCFH-DA. The mixture was incubated at 37°C for 20 min. Then recombinant strains were washed thoroughly to reduce background interference. Fluorescence was measured by using a Synergy H1 microplate reader  with excitation at 488 nm and emission at 525 nm.If necessary, the DO and ROS were determined in the UA degradation process 600 = 1.0, and 10 mL Fetal Bovine Serum  was added in a bioreactor as the supplemental nutrient. Following the addition of 2.0 mM IPTG and 50 μg/mL kanamycin, the cells were grown for an additional hour with its DO equal to 15% of the normal condition. Both the eutrophic Brain Heart Infusion Broth and low oxygen were used to simulate intestinal environment. 1.0 mM UA was added in the bioreactor to start UA metabolism under this oxygen-limiting condition. Samples were taken at defined time intervals. After centrifugation, 10 μL of the supernatant was used to measure UA and allantoin by using the HPLC method.Recombinant cells were cultured and harvested in the same way as preparing whole cells. The whole cells were resuspended in 20 mL Brain Heart Infusion Broth . Defined volume of cell mixtures was immediately inoculated with 700 mL autoclaved Brain Heart Infusion Broth in a 1.4-L Multifors parallel bioreactor  at the initial ODFive weeks old (20–25 g) Kunming mice were purchased from Henan Skbex Biotechnology Company. Mice were housed in room (22 ± 2°C) with 65% humidity and 24-hour light-dark cycle. Mice were fed a standard diet. Before the experiments were executed, mice were habituated for a week. Mice were in good health condition at the beginning of the experiments. Mice were randomly divided into groups (n = 6) for subsequent experiments. All animal experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals  and were approved by the Animal Ethics Committee of Shandong University.10 CFU/mL). Both the positive control group and negative control group were administered with the same volume of HEPES buffer by intragastric administration. One hour later, mice in the testing group and the positive control group were intragastrically administered with 1 mL of 20 mM UA solution in saline. The mice in negative control group were administered with same volume of saline. After 30 min, mice were anesthetized and sacrificed. The stomach and small intestine segments were collected, and different organs were frozen in liquid nitrogen and stored at −80°C for further analysis.Eighteen 6-week male Kunming mice were classified into three groups (n = 6). The testing group was administered with 200 μL engineered EcN strain by intragastric administration .Small pieces of stomach, duodenum, jejunum and ileum tissues from these three groups were cut, weighed and homogenized in 1 mL ice-cold PBS  (pH = 7.4) by using a homogenizer , and the lysis mixtures were centrifuged . The supernatants were utilized to analyze the UA and protein concentrations. The UA concentration was determined by using the UA assay kit. The protein level was determined by using OD81 1) Mice induced by the 7d-1PO method were induced by oral administration of adenine (75 mg/kg) for 7 consecutive days, and potassium oxonate at 250 mg/kg was intraperitoneally injected 1 hour after the last adenine administration to inhibit UOX. 2) Mice induced by the 7d-7PO method was similar to the treatment of the 7d-1PO method, but potassium oxonate (250 mg/kg) was intraperitoneally injected 1 hour after each adenine administration. 3) The Ade method orally feeding mice with only adenine (75 mg/kg) for 15 consecutive days. 4) Mice induced by the UA-PO method orally received UA (290 mg/kg) and potassium oxonate (570 mg/kg) for 15 consecutive days. Adenine, UA, and potassium oxonate were suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na). The control group was orally administered with the same volume of 0.5% CMC-Na.The hyperuricemia mouse models were established by four known methods: the 7d-1PO method, the 7d-7PO method, the Ade method, and the UA-PO method, according to publications.10 CFU/mL) 8 hours before adenine administration for 7 consecutive days. In the control group, the mice were given the same volume of the buffer .Mice induced with the 7d-1PO method were selected to receive the treatment with the engineered EcN strain. The mice were divided into three groups, including testing group, model group, and control group. In the model group, the hyperuricemia mice were established according to the 7d-1PO method without treatment with the engineered EcN strain. In the testing group, they were intragastrically administered with 200 μL of the engineered EcN strain  1 hour after the final drug administration, and then blood samples were collected via eyeball enucleation. Blood samples were stored at −20°C until UA analysis; small intestine tissues were obtained after mice were sacrificed, frozen in liquid nitrogen, and stored at −80°C until analyses.Eighteen 6-week male Kunming mice were divided into three groups (n = 6). The two testing groups were tail intravenously injected with a UA solution at 70 mg/kg or 5.7 mg/kg as indicated. The UA injections with the high amount and low amount were used to promote serum UA in a physiologically relevant concentration to humans and mice, respectively. The control group was intravenous injected only with saline. The UA concentration was determined from blood samples taken from mouse tails at defined time intervals. The UA concentrations in these organs were determined by using the UA assay kit . The mice received UA injection had high UA in the sera and termed UA-injection hyperuricemia mice.10 CFU of the EcN cells of two strains for 5 consecutive days, UA solution was intravenously injected 1 hour after the last bacterial administration at day 5. 2) For intravenous treatment, 24 6-week male Kunming mice were divided into four groups: three testing groups and a control group. Defined amount of EcN cells with pMCS2-Ptrc-pucLMM-vhb-ygfU-katG, pMCS2-Ptrc-pucLMM- ygfU, or the empty vector pMCS2 were administered into mice in the three testing groups. The mice in the control group was injected with the same amount of buffer instead. The mice in the intravenous administration of EcN received the bacterial injection first, and then the UA solution was injected into the mice either immediately or 10 hours after the bacterial injection.The engineered EcN strain was used to treat the hyperuricemia mice by either intragastric or intravenous administration. 1) For intragastric treatment, 18 6-week male Kunming mice were divided into three groups: the two testing groups and the control group. Defined amount of the engineered EcN cells and EcN cells with the empty vector were administered into mice in the two testing groups intragastrically. The mice in the control group was injected with the same amount of buffer. All mice in the three groups were intravenously injected with the UA solution at 70 mg/kg. Mice in the intragastric administration group were orally administrated with 2 × 10For both treatments, the UA concentration was determined with UA meter from blood samples taken from mouse tail at defined time intervals. For intravenous treatment, the weight and survival numbers of the mice in each groups were recorded at defined time intervals after EcN was injected to draw survival curves and weight curves.2O2 concentration was determined during UA degradation in vivo. The engineered EcN strains with oxygen-recycling genes or not were used to treat the hyperuricemia mice by either intragastric or intravenous administration, as described above. After the blood samples were taken, the H2O2 levels were detected by using a H2O2 assay kit  according to the manufacturer’s instruction. Meanwhile, the UA concentration was determined with the UA meter.The Hp values of indicated pairs of group. Q values were calculated according to the Benjaminiad Hochberg method to represent the adjusted p value. Q < 0.05 indicated statistical significance. The significant differences between two groups were analyzed using an independent student’s t-test. The p value < .05 indicated statistical significance. Further, the relevant data were fitted by using the ORIGINR 2016 software .Statistical analysis was done using GraphPad Prism 9.0. Data in more than two groups were analyzed using independent one-way analysis of variance (ANOVA) to calculate the Click here for additional data file.


Taz exhibits a gradient expression across the posterior-to-anterior of limb bud mesoderms, similar to Sonic hedgehog (Shh). Importantly, Taz drives PKA to phosphorylate Gli3, resulting in the Gli3 processing into its repressor and attenuation of Hedgehog signaling in the Shh-independent manner. Specifically, Taz deletion in mouse embryonic limb bud mesenchyme not only enhances the Hedgehog signaling but partially restores the phenotypes from Shh deletion in causing severe defects of anteroposterior patterning and digit number and identity. Together, these results uncover Taz-dependent Gli3 processing as a hitherto uncharacterized mechanism controlling Hedgehog signaling, highlighting its cross-regulation by Hippo signaling.The overlapping roles of Hippo and Hedgehog signaling in biological functions and diseases prompt us to investigate their potential interactions. Activation of Hippo signaling enhances the transcriptional output of Hedgehog signaling, and the role of Hippo signaling in regulating Hedgehog signaling relies on the Hippo pathway key effector, Taz. Interestingly, The online version contains supplementary material available at 10.1186/s13619-022-00151-6. Drosophila to humans  signaling has conserved roles in the development of various organs in metazoans ranging from Ctgf) and cystene-rich protein 61 (Cyr61)  antagonizes Hippo signaling in the somatic cells of the ovary to drive germline stem cell differentiation  for further analysis. Smo* or ∆N-Gli2 significantly induced the Gli-luciferase activities and the mRNA levels of Ptc1 and Gli1 without affecting Taz protein levels in C3H10T1/2 cells or in C3H10T1/2Gli2-KO cells, respectively, whereas Taz overexpression or siRNA robustly suppressed or increased the Gli-luciferase activities and the Ptc1 and Gli1 mRNA levels, respectively, in either the presence or absence of Smo* or ∆N-Gli2  treated with or without either N-Shh or Purmorphamine, an agonist of Hh signaling  robustly induced the Gli-luciferase activities and the mRNA levels of hh Figs. . In conthh Figs. . We spechh Figs. , provinghh Figs. . To confi2 Figs. . Interes0% Figs. . Notablyng Figs. . Thus, HIn order to explore further the mechanism underlying Taz suppressing the Hh signaling, we overexpressed or knocked down Taz to examine the potential changes in Smo and Gli proteins in C3H10T1/2 cells. Taz affected neither the protein level nor phosphorylation of Smo Figs. , B. LikePKA plays a conserved role in the regulation of Hh signaling. In the absence of Hh, PKA phosphorylates multiple sites in Gli3 protein Fig. . PKA-medWe then generated the variant of Gli3F that harbored loss-of-function mutations at six PKA consensus serine residues  conserved in mice and humans  by siRNA not only completely abolished Taz-induced Gli3 processing but also reversed the inhibition of Gli-luciferase expression by Taz , and those cells were used as the control for co-immunoprecipitation experiments with Taz antibody or Pka antibody, respectively. Protein complexes precipitated with a Gli3 antibody contained a large amount of both Taz and PKA, which was dampened by the existence of N-Shh  behaved essentially the same as the full-length of Taz (aa 1–1188) in negating Gli-luciferase activities; N-terminus of Taz (aa 1–366) caused no significant change, whereas Taz (aa 1–492) and Taz (aa 367–1188) significantly induced the Gli-luciferase activities Figs. , C. ThusTo determine whether the PKA-sites mutations in Gli3 affect its physical interaction with PKA or Taz, we performed immunoprecipitation experiments. The immunocomplexes of myc-tagged WT contained a large amount of endogenous Taz and PKA, but those of myc-tagged MT contained very little Taz or PKA. Additional experiments confirmed that Gli3 mutations significantly reduced the presence of Gli3 in the Taz or PKA immunocomplexes Figs. F-H. ThusLow cell density inactivates Hippo signaling to stabilize Taz and Yap, while high cell density activates Hippo signaling to induce the proteasomal degradation of Taz and Yap  of mouse limb buds at E10.5. Taz was abundantly expressed in the posterior region and slightly expressed in the anterior region of limb buds, whereas Shh was normally restricted in the posterior domain of the zone of polarizing activity (ZPA)  and Shh−/− (0.016) limb buds  and Taz−/− (8.4) limb buds  patterning of vertebrate limbs  Figs. A, B. Theh−/− 0.01 limb budShhCre allele expresses Cre in place of Shh and therefore is a Shh null allele , to neutralize the BMPs activity that negatively regulates apical ectoderm ridge (AER) function and fibroblast growth factor-8 (FGF8) expression  for whole-mount skeleton preparation with Alcian Blue/Alizarin Red stains. ShhCre+/+ limbs exhibited identical deficits to Shh−/− limbs as described previously  induces the phosphorylation of Lats1/2 and, in turn, leads to the phosphorylation and proteasomal degradation of Taz, which decreases its binding to and driving the PKA to phosphorylate Gli3 at six conserved serine sites, causing the attenuation of Gli3 processing into its C-terminal truncation form (Gli3R) and thereby activating the Hedgehog signaling Fig. . The preDrosophila melanogaster ovarian follicle stem cells and in mouse myofibroblasts of the liver, whereas Drosophila Ci antagonizes Hippo signaling to drive germline stem cell differentiation , SHHCre+/− (ShhCre+/−), and Lats1+/− mouse strains were obtained from Jackson Laboratory  and were described previously . Primary MEF cells were prepared from heterozygous and homozygous mutants of http://crispr.mit.edu/). All specific target sequences were amplified, cloned, and verified by DNA sequencing. After the transient transfection of pX330-sgLats1/Gli2/Pka/Taz plasmids together with a puromycin-resistant plasmid into cells by using Lipofectamine reagent (Invitrogen), puromycin (2 μg/ml) (Invitrogen) treatment for 14 d was employed for selection, and then cells were expanded in the regular culture medium.Expression vectors of sgRNA for mouse Lats1, Gli2, Pka, or Taz were designed as pX330-based plasmids. Targeting sequences were designed using the CRISPR DESIGN tool , Gli1 , Gli2 , Gli3 , c-myc (sc-40), PKAc (sc-903), p-PKA , GSK3β (sc-9166), CK1α , acetylated-α-tubulin , β-actin , Lamin B , glyceraldehyde-3-phosphate dehydrogenase , and rabbit IgG were from Santa Cruz Biotechnology . YAP (#12395), p-GSK3β , Lats1 (#9153), HA (#3724), and Flag (#14793) were from Cell Signaling Technology .Gli3 , Gli3 , Smo (ab72130), p-Ser/Thr (ab117253) and Ki67 (ab15580) were purchased from Abcam . Ptc1 (06–1102) was from Millipore . The IRDye 680 and 800 second antibodies were from LI-COR Bioscience . GST fusion proteins, including GST-Gli3F, GST-Gli3R, and GST-Taz, were generated as previously described . Bioactive Shh recombinant protein (N-Shh) was from PeproTech Inc. , whereas H-89, SB216763, and forskolin were from Sigma .Transient transfection procedures were as previously described  according to the manufacturer’s instructions. Five μg total RNA in a volume of 20 μl was reversely transcribed with SuperScript III reagent (Life Technologies) and the oligo-(deoxythymidine) primer with incubation at 42 °C for 1 hr. After the termination of cDNA synthesis, mRNA levels of target genes were determined by quantitative RT-PCR as described previously  was used to quantify the immunoreactive bands, and the normalized antigen signals were calculated from target protein-derived and β-actin-derived or GAPDH-derived signals. GST pull-down assays were performed as previously described . After the incubation period, the GST fusion proteins were pulled down with glutathione sepharose beads. The beads were washed 4 × with PBS plus 0.2% Tween 20. Subsequently, the washed beads were suspended in SDS-gel loading buffer, and the eluted proteins were resolved by 10% SDS–PAGE. Resolved protein bands were transferred onto PVDF membranes , followed by detection with antibodies.The cytosolic and nuclear fractions of cells were prepared as previously described  was performed according to the manufacturer’s instructions  and described as previously , Lats1 (#9153), Ptc1 (06–1102), Gli3F (ab181130), and biotinylated secondary antibody (Invitrogen) were used, and the diaminobenzidine solution was applied to develop a brown color followed by counterstaining with hematoxylin. Negative controls were performed by using control IgG.C3H10T1/2 cells were fixed for 10 min in ice-cold methanol and permeabilized with 0.1% Triton X-100 in PBS (PBST) for 30 min. After incubation with blocking buffer , cells were incubated with primary antibodies, including c-myc (sc-40) and Gli3 (ab69838). Then, cells were washed with PBST and incubated with appropriate fluorescein isothiocyanate-conjugated secondary antibodies (Invitrogen). Nuclei were counterstained with DAPI. Slides were mounted and analyzed by confocal microscopy using a Carl Zeiss LSM 710 laser scanning microscope . Immunohistochemistry was performed using the Histostain-Plus Kit and diaminobenzidine  as previously described . Additional file 1. Supplementary information includes seven figures and can be found in this article online.


Idiopathic rapid eye movement sleep behavior disorder (iRBD) is an important non-motor complication of Parkinson's disease. At the same time, iRBD is considered to be the prodromal stage of α-synucleinopathy. This high risk of conversion suggests that iRBD becomes a nerve It is a window for early research on degenerative diseases and is the best candidate for neuroprotection trials. A wide range of neuroimaging techniques has improved our understanding of iRBD as a prodromal stage of the disease. In addition, neuroimaging of abnormal iRBD is expected to be a potential biomarker for predicting clinical phenotypic transformation. This article reviews the research progress of neuromolecular imaging in patients with iRBD from the perspective of iRBD transforming synucleinopathies. Idiopathic rapid eye movement (REM) sleep behavior disorder (iRBD) is a parasomnia mainly characterized by the loss of muscular atonia and dream enacting behaviors during REM sleep , 2. The 123Iodine-metaiodobenzylguanidine (123I-MIBG) cardiac imaging.Currently, more and more studies are looking for early neuroimaging evidence of iRBD transformation in order to detect structural and functional abnormalities in the iRBD brain earlier . Among nConsidering all these factors, we review the latest research on molecular imaging between iRBD and α-synucleinopathies. The molecular neuroimaging markers that predict the early transformation of iRBD were the place we pay the closest attention to in this article. A comprehensive understanding and review of neuroimaging techniques to explore phenotypic conversion in iRBD will be able to guide future research in this field and further facilitate the clinical management of this disease.via a direct pathway. Intermediate neurons in turn inhibit glycinergic and gamma-aminobutyric acidergic neurons via spinal anterior horn motor neurons, resulting in skeletal during REM sleep skeletal muscle movements being inhibited during REM sleep  showed behavioral abnormalities during REM sleep similar to those of human RBD . PreviouEM sleep . In addiEM sleep . Under nEM sleep .18F] fluorodeoxyglucose (FDG) positron emission tomography (PET) is an assay targeting glucose metabolism visualization in the brain, with higher metabolic rates presenting high signal changes , which is the priority area for patients with MSA. In contrast, the other five patients showed hypermetabolic changes in the left cingulate gyrus, right frontal lobe and right temporal lobe, which are the preferentially affected regions of PD patients  was determined using ty of PD . Expressty of PD . TherefoSingle photon emission computed tomography is a neuroimaging tool to measure regional cerebral blood flow (rCBF) . Using SThe main pathological alteration in PD is degeneration of the substantial nigra-striatal dopaminergic pathway . StriataIn vivo study of noradrenergic changes in 17 patients with iRBD using 11C-MeNER as a tracer revealed that tracer uptake levels were decreased in the locus coeruleus to the thalamus and red nucleus regions in patients with iRBD compared to normal controls  cardiac imaging is a commonly used imaging technique to diagnose cardiac sympathetic function , and Henan Province Medical Science and Technology Tackling Provincial Ministry Key Projects (SBGJ202102033).The 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.


Premature ovarian failure (POF) is a multifactorial disease that refers to the occurrence of secondary amenorrhea, estrogen decrease, and gonadotropin increase in women under the age of 40. The prevalence of POF is increasing year by year, and the existing instances can be categorized as primary or secondary cases. This disease has adverse effects on both the physiology and psychology of women. Hormone replacement therapy is the recommended treatment for POF, and a multidisciplinary strategy is required to enhance the quality of life of patients. According to recent studies, the primary mechanism of POF is the depletion of ovarian reserve function as a result of increased primordial follicular activation or primordial follicular insufficiency. Therefore, understanding the processes of primordial follicle activation and associated pathways and exploring effective interventions are important for the treatment of POF. Premature ovarian failure (POF), also known as premature menopause, is a condition in which a woman experiences amenorrhea before the age of 40 due to the cessation of ovarian function. POF can lead to infertility in women with physiological or psychological problems , 2. GeneThe ovary is an essential part of the female reproductive system that plays a crucial role in the growth and fertility of the reproductive system by producing mature oocytes and secreting many kinds of hormones . The pri6 primordial follicles at birth, but only a small percentage of them mature into oocytes; the remainder are either kept dormant or disappear. One of the factors that influence the length of a woman's reproductive life is the balance between the primordial follicle's dormant, active, and apoptotic stages , which uses estrogen, progesterone, melatonin and other hormone to alleviate menopausal symptoms caused by POF, is the current preferred treatment , 13. HRTActivation of primordial follicles determines oocyte growth and development, as well as the differentiation and proliferation of surrounding somatic and granulosa cells, both of which are essential for the ovulation process . To provThe primordial follicles are maintained in a dormant state by a certain inhibitory system, which is achieved by a combination of signaling pathways and molecular processes. Studies have shown that this system is necessary for the preservation of the dormant follicle pool . The curAlthough the mechanism of primordial follicle activation is not fully understood, evidence shows that the phosphatidylinositol 3-kinase/protein kinase B pathway (PI3K/Akt) plays a key role in this process. Cell motility, survival, differentiation, growth, and intracellular transport are all regulated by PI3K, which catalyzes the phosphorylation of phosphatidylinositol . ProteinIn the process of follicle development, PTEN has an inhibitory effect on the PI3K/Akt pathway, maintaining primordial follicles in a dormant state for a long time. When the inhibition of PTEN is removed, PI3K is activated in primordial follicles, converting phosphatidylinositol diphosphate (PIP2) to PIP3, thereby stimulating phosphatidylinositol-dependent kinase 1 (PDK1), and activating Akt. This causes the downstream Foxo3 protein to be phosphorylated, resulting in its loss of transcriptional activity and relocation from the nucleus to the cytoplasm for destruction, which activates primordial follicles . As a neAs a downstream molecule of the PI3K/Akt pathway, mammalian target of rapamycin (mTOR) signaling is a crucial intracellular signaling pathway that regulates the cell cycle and has received extensive attention . It is aThe most significant of the various reasons for elevated mTOR activity is the mutation of the PTEN gene. Normally, PTEN inhibits mTOR signaling by blocking the activity of PI3K, an upstream effector of mTOR. When the inhibitory impact of PTEN is weakened, mTOR promotes cell cycle progression, boosts proliferation, and prevents autophagy . Zhang eAs an inhibitor of mTOR, rapamycin can bind to the FKBP12 protein, an intracellular receptor. By directly interfering with the FKBP12 rapamycin-binding domain of mTOR, the FKBP12–rapamycin combination inhibits the activity of mTOR . Among tThe Hippo signaling pathway is composed of a series of conserved kinases and is an important signaling pathway regulator. It can control the size of an organ by regulating cell proliferation and apoptosis, and has the ability to inhibit cell growth . In mammHippo signaling is also impacted by changes in cellular connections, shape, and polarity. Intercellular tension in organs is altered when organs are physically damaged, accompanied by inhibition of the Hippo signaling pathway accumulation of co-activators in the nucleus. This series of modifications affects organ size and controls cell proliferation. Cell proliferation, survival, and growth are promoted when Hippo signaling is inhibited .via the Hippo and Akt pathways, involving signaling pathways such YAP, TAZ, PTEN, mTOR, and Foxo3.The Hippo signaling system has been demonstrated in mouse models to play a function in controlling cell proliferation and death by preserving organ volume at the ideal size by inhibiting cell growth . Genes cThe Notch signaling pathway is a widely distributed and evolutionarily conserved pathway that controls numerous cellular activities, including cell proliferation, migration, differentiation, and death in both healthy and unhealthy conditions . CertainIn addition to many mitotic cycles, the Drosophila oocyte maturation process also includes an endoreplication step in which genomic DNA is duplicated without cell division . The traIn a clinical trial, patients with POF were discovered to have mutations in the Notch2 gene, indicating that these mutations may be connected to the emergence of POF . In a moThe tyrosine kinase receptor KIT and its ligand cytokine KL mediate signaling between granulosa cells and oocytes and are closely associated with female fertility . Follicuin vitro culture assays, and the activation of KIT has been found to inhibit germ cell apoptosis by Sakata et al.  is a multifunctional cytokine that belongs to the transforming growth factor superfamily, which includes three different isoforms of transforming growth factors (TGF-β 1–3) and many other signaling molecules . TGF-β iThe Smads protein is a key downstream medium of TGF-β signaling, which is a group of structurally similar proteins that are the main signal transduction factors of the TGF-β pathway and are essential for regulating cell growth and development. There are three different isoforms of Smads: combinatorial Co-Smads, inhibitory I-Smads, and receptor-regulated R-Smads . The intin vitro mouse ovary culture experiments to further understand the role of TGF-β, in which the control group received no treatment, while experimental groups 1 and 2 received TGF R1 and SD208, respectively. The number and shape of follicles were assessed by sectioning the ovaries after 7 days of culture. It was found that the growth of primordial follicles and oocytes in ovaries treated with TGF R1 was inhibited, the number was decreased, and the proliferation of granulosa cells was significantly weakened. By contrast, in the ovaries treated with SD208, oocytes grew faster, had larger cell diameters, a higher percentage of activated cells, and more granulosa. After TGF R1 expression was significantly reduced by si-RNA, as seen by PCR and western blot, it was observed that the proportion of developing follicles in the ovaries increased, the growth was accelerated, and the volume became larger. Therefore, the researchers came to the conclusion that the TGF-β signaling pathway is crucial for maintaining the dormancy of the primordial follicular pool and that the mechanism of action of SD208 may involve activating the ribosomal protein s6 (RPS6) signaling pathway in mouse ovaries, thereby producing an inhibitory effect to the TGF-β signaling pathway  pathway is considered to be one of the most important signaling pathways in cells. It allows extracellular chemical signals to pass through the cell membrane and be delivered to the DNA promoter in the nucleus to control the expression of the corresponding genes. Several growth factors and cytokines, including interferon, interleukin, and colony-stimulating hormone, are involved in this route, which is related to a number of bodily processes, including immunological adaption, tissue repair, inflammatory response, and apoptosis .The pathway is highly conserved in evolution and consists of JAK, STAT, and the receptor–ligand complex. JAK is a non-acceptor tyrosine kinase that is a subgroup of tyrosine kinases that catalyzes the transfer of phosphate groups from nucleoside triphosphate donors to tyrosine subunits in proteins, regulating cell growth, differentiation, proliferation, death, and migration . JAK hasWhen a ligand binds to the receptor in this pathway, the receptor undergoes a conformational change, so that the two domains in JAK are close to each other and phosphorylation occurs, resulting in conformational change that further activates STAT. The activated STAT signal transfers into the nucleus and regulates the transcription of specific genes . Zhang eThe mitogen-activated protein kinase (MAPK) signaling pathway can transmit signals from the extracellular matrix to the nucleus, which is important for cellular communication. MAPK is a group of serine/threonine protein kinases that can be stimulated by extracellular signals such as cytokines and neurotransmitters to regulate cell growth, proliferation, apoptosis, and other activities . The corThe MAPK pathway is involved in regulating the processes of meiosis, cytoplasmic maturation, nuclear membrane formation, chromatin condensation, and spindle assembly. It can be triggered to promote the resumption and progress of meiosis. Salamone et al. compared prepubertal calf and adult bovine oocytes and found that calf oocytes lacked developmental capacity. They found that calf oocytes contained higher concentrations of MAPK after comparing the levels of the two. This demonstrates that MAPK can regulate oocyte activation by influencing cytoplasmic maturation .in vitro, the majority of cells were unable to complete meiosis, and MAPK remained in a non-phosphorylated form; however, in mature equine oocytes, MAPK was phosphorylated and possibly had kinase activity  techniques have been investigated in recent years to mobilize residual follicles in POF patients. Initially, IVA disrupted Hippo signaling by disrupting the ovaries, and then the fragments were cultured in vitro with an Akt stimulant for 2 days before being transplanted back into the patient. This method has now been developed into a one-step procedure that does not rely on drugs to obtain fertile, mature follicles. When antral follicles develop to the preovulatory stage, mature oocytes are extracted, and embryos are transferred after in vitro fertilization with sperm. IVA has been performed in patients with POF, and the follicles can grow and fertilize successfully  signaling pathway, which is a downstream signal of mTORC1, the TGF- pathway inhibitor SD208 can induce follicular growth and break the dormant state of follicles . Rapamyc pathway , but theessfully . To dateessfully  used autd tissue . FurtherThe process of transformation from primordial follicles into mature follicles is complex. There are a number of intercellular and intracellular signaling pathways that interact in various ways to form a complicated network that can respond to many stimuli, but the precise mechanism remains to be further studied.in vitro are still different for patients with POF. Moreover, current studies are mainly carried out on animal models; thus, due to the differences between human and animal models, the research findings may have limitations.Despite these new advances, regulating primordial follicle activation and controlling oocyte development Some signaling pathways may be related to stem cells. The KL-KIT and TGFβ-Smad and other pathways discussed earlier can regulate the self-renewal of stem cells through different mechanisms and play a role in embryonic development and tissue homeostasis. In recent years, stem cell transplantation has become a hotspot in the treatment of POF. The mechanism by which stem cells improve ovarian function is related to the cytokines or exosomes secreted by them, which regulate immunity and promote follicle development while improving the microenvironment of ovarian tissue, and are related to various signaling pathways.Stem cells are a subtype of cells that maintain an undifferentiated form in both embryonic and adult tissues and can undergo self-renewal and differentiation . By partAlthough the effect of stem cell therapy has been shown in some animals, its clinical application is still severely limited because of the intrusiveness of stem cell collection and possible ethical and immune rejection issues. Further research is therefore needed to explore stem cells that are simple to collect, non-invasive and safe during collection, low in immunogenicity, and do not involve ethical concerns.POF has significant physiological and psychological impacts on women; thus, it is critical to reveal more knowledge about its pathogenic mechanisms and available treatments. It is necessary to have a better understanding of the mechanisms related to follicular pool depletion and to master methods of intervening in ovarian aging. The activation of primordial follicles is a complex but coordinated process that is regulated by multiple factors and pathways. In this review, we focused on describing the roles of PI3K/Akt/Foxo3 signaling pathway, mTOR signaling pathway, etc. in primordial follicle activation, and also discussed other mechanisms that regulate follicle dormancy and activation. Based on our current understanding of follicle activation, the development of drugs that regulate the activation of primordial follicles and target some signaling pathways may provide new possibilities for the treatment of POF. Further studies can also be conducted on other potential regulators that could help provide patients with a more personalized treatment plan. In recent years, many different types of stem cells have been found to have therapeutic effects on POF. For the protection of female fertility, stem cells can be considered treatment options to restore female fertility. In addition, psychological problems caused by impaired fertility also play a certain role in the treatment of POF patients, and the psychological state of patients should also be paid attention to in the treatment of POF . StudiesHowever, there is still much unknown information about the above approaches, and some measures are still limited to the experimental level due to ethical or technical issues. Thus, further research is required. With the advancement of science, technology, and experimental-level strategies, as well as interventions for improving the quality of life of patients with POF through multidisciplinary therapy, we hope to obtain evidence of the safety and efficacy of these methods through large-scale clinical trials.XB conceived the study and wrote the manuscript. SW conceived the study, organized, and edited the text. All authors contributed to the article and approved the submitted version.This work was partly funded by National High Level Hospital Clinical Research Funding (BJ-2021-236).The 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.


Bone morphogenic protein receptor 2 (BMPR2) expression and signaling are impaired in pulmonary arterial hypertension (PAH). How BMPR2 signaling is decreased in PAH is poorly understood. Protein tyrosine phosphatases (PTPs) play important roles in vascular remodeling in PAH. To identify whether PTPs modify BMPR2 signaling, we used a siRNA-mediated high-throughput screening of 22,124 murine genes in mouse myoblastoma reporter cells using ID1 expression as readout for BMPR2 signaling. We further experimentally validated the top hit, PTPN1 (PTP1B), in healthy human pulmonary arterial endothelial cells (PAECs) either silenced by siRNA or exposed to hypoxia and confirmed its relevance to PAH by measuring PTPN1 levels in blood and PAECs collected from PAH patients. We identified PTPN1 as a novel regulator of BMPR2 signaling in PAECs, which is downregulated in the blood of PAH patients, and documented that downregulation of PTPN1 is linked to endothelial dysfunction in PAECs. These findings point to a potential involvement for PTPN1 in PAH and will aid in our understanding of the molecular mechanisms involved in the disease. Pulmonary Arterial Hypertension (PAH) is an obliterative disease of the pulmonary arteries that affects 50–100 individuals in one million people worldwide. The progressive increase in pulmonary vascular resistance ultimately leads to right heart failure, responsible for the high mortality in PAH. On a cellular level, pulmonary arterial endothelial cell (PAEC) dysfunction and apoptosis along with abnormal growth of smooth muscle cells (SMC) leads to medial thickening and neointima formation, causing the occlusive vasculopathy in PAH . While BBased on a high-throughput (HTS) siRNA screen of ~ genes, using a BRE reporter mouse cell line with ID1 expression as readout for increased BMPR2 signaling, in combination with an analysis of publicly available PAH RNA expression data, our group previously identified clinically relevant novel BMPR2 signaling modifier genes, namely, fragile histidine triad (FHIT)  and lympThe PTPN1 encodes for PTP1b was the first-discovered PTP. In a case–control study, PTP1b single nucleotide polymorphisms (SNPs) were shown to contribute to hypertension . ChronicAs endothelial dysfunction plays an important role in PH pathogenesis and as the BMPR2 receptor is highly expressed in endothelial cells, we aimed to investigate the role of PTPN1 in modulating BMPR2 signaling and its effect on PAEC function in PAH. We found that PTPN1 silencing with siRNA decreases BMPR2 signaling, which was associated with impaired proliferation, angiogenesis, and induced apoptosis in PAECs. We also found that PTPN1 RNA expression is downregulated in hypoxic PAECs, lung tissues of sugen5416/hypoxia PH rat models, and in whole blood samples of PAH patients by RNA sequencing. These findings point to a potential involvement for PTPN1 in endothelial dysfunction in PAH and will aid in our understanding of the molecular mechanisms involved in the disease.To identify novel BMPR2 signaling modifiers, a high-throughput siRNA screen of >22,124 genes was performed in an Id1-BRE luciferase containing a C2C12 mouse myoblastoma reporter cell line treated with or without BMP4, as previously described ,9. Follo2, 21% O2, 90% humidity). Cells were sub-cultured at 1:4 ratio and passages between 4 and 6 were used for all PAECs experiments. Human healthy primary pulmonary arterial endothelial cells (PAECs) were purchased from PromoCell GmbH, Heidelberg, Germany (Cat # C12281). Cells were grown in standard endothelial growth medium  with growth factors supplementation and 100 U/mL penicillin–streptomycin solution (Gibco) under standard conditions  were seeded onto 6-well plates and incubated at 37 °C in a humidified 5% CO2 atmosphere. Next day, cells were transfected with 50 nM siRNAs against PTPN1 , BMPR2 (Cat # 4390824), and non-target (NT) controls , with 2 uL of Lipofectamine RNAimax  in a total of 1 mL of OPTIMEM media. Six hours after transfection, the medium was changed to complete growth medium. After 48 h, knockdown efficiency was assessed by qRT-PCR. Knockdown of PTPN1 and BMPR2 was performed in PAECs. Briefly, PAECs , as described previously . Apoptos2 condition in a hypoxia chamber, as described previously [PAECs were grown under 1% Oeviously . HypoxiaPTPN1 mRNA and protein expression in the lung of sugen5416/hypoxia-induced PH experimental rat models was measured by qRT-PCR and western blotting, respectively. Pulmonary vascular remodeling and hemodynamic parameters of the rat samples chosen were previously reported in Dannewitz Prosseda et al. .® Plus Mini Kit . Total RNA from rat lung tissues was isolated using Trizol extraction method, as described previously [TM, Foster City, CA, USA) and gene expression was assessed by TaqMan qRT-PCR with targeted TaqMan assay probes, human PTPN1 (Hs00942477), GAPDH (Hs01786624_g1), 18S (Hs99999901_s1), BMPR2 (Hs00176148_m1), ID1 (Hs03676575_s1), rat PTPN1 (Rn01423685_m1), rat Gapdh (Rn01775763_g1), and TaqMan™ 2× Universal PCR Master Mix (Cat # 4304437).Total RNA from PAECs was extracted and purified with commercially available RNeasyeviously . Total Rn = 359) compared to age- and sex-matched healthy controls (n = 72) [To determine whether PTPN1 expression was downregulated in the blood and PAECs from PAH patients, we analyzed a large RNA-seq data of whole blood of patients with idiopathic, heritable, and drug-induced PAH ((n = 72)  and a pu(n = 72) ). We excWestern blotting was performed, as described previously , with prt-test was performed. p values < 0.05 were considered as significant changes. Number of samples for each experimental condition is presented in each figure and legend.All data were analyzed using GraphPad prism version 9.0 . Data are represented as mean ± standard error mean. For data comparing between two groups, non-parametric student To identify novel modulators of BMPR2 signaling, we previously performed an unbiased siRNA-mediated HTS of 22,124 genes in a BRE-ID1-LUC reporter C2C12 mouse myoblastoma cell line. The screened hits were cross-validated in publicly available gene expression data sets of PBMC and lung tissues of PAH patients. These screening approaches identified three important BMPR2 modifying genes, FHIT , LCK 8,,8,9 and Abnormal proliferation, apoptosis, and tube formation of PAECs is strongly linked with the pathogenesis of PAH ,2. We in2) for 3 weeks and normoxia for 5 weeks (21% O2). The PH model was confirmed by measuring RVSP, RV hypertrophy (Fulton Index: weight ratio of the RV/left ventricle (LV) and septum (LV + S) (Please see the data in [Hypoxia is considered one of the contributing factors to Group 3 PH (PH linked with hypoxia and lung disease) and is used as an insult to produce experimental PH in rodents. Furthermore, a previous study described the regulation of PTP by hypoxia . We ther data in ). We foun = 359) compared to age- and sex-matched healthy controls (n = 72) by RNA-seq. We observed a significantly downregulated PTPN1 expression in PAH patients compared to healthy controls.  and improved pulmonary vascular remodeling in rats . Xu et aLoss of function mutation in BMPR2 occurs in 60–80% of familial cases of PAH patients, but the disease penetrance rate is low ,30, suggWe found a significant downregulation of PTPN1 in the whole blood of a large cohort of PAH patients by RNA-seq analysis, yet the precise cell type or tissue responsible for the observed PTPN1 downregulation is not known. Previous reports describe a link between PTPN1 SNPs and systemic hypertension ,33. HypoWhile we observed a significant downregulation of PTPN1 expression in the whole blood of PAH patients, we did not find these changes in PAECs of PAH patients. This could be due to the fact of relatively lower expression of PTPN1 and variation in PAECs than in the blood cells. Further larger studies would require confirming these findings in PAECs of PAH patients. However, we found a significant correlation between PTPN1 and BMPR2 signaling marker gene expression in the data set C–E, indiThis study has several limitations. First, we demonstrated that inhibition of PTPN1 decreases BMPR2 expression and signaling and induces endothelial dysfunction; however, it would be important to see whether the opposite is true as well: whether overexpression of PTPN1 activates BMPR2 signaling and reverses abnormal endothelial cell behaviors. Second, this study lacks the direct causal link between PTPN1 and PAH in vivo. Further studies need to characterize whether global or cell-specific deletion of PTPN1 causes experimental PH or alternatively predisposes to a more severe PH phenotype in response to injurious agents used to induce experimental PH, such as hypoxia alone, sugen5416/hypoxia, or monocrotaline. Third, we were not able to validate in this pilot study the PTPN1 PAH blood RNA-seq expression data in a second sample cohort. Fourth, mechanisms of how PTPN1 regulates BMPR2 signaling in PAH remains to be explored. The crosstalk between BMPR2 signaling and the other signaling pathways, such as VEGF, ERK/MAPK, PI3K, AKT, p38, JNK, NOTCH, and TAK1, in the context of PTPN1 deficient cells would also need to be explored.In summary, we find PTPN1 expression is downregulated in the whole blood of PAH patients and PAECs exposed to hypoxia and that PTPN1 downregulation is associated with endothelial dysfunction in PAECs. Furthermore, we discovered that PTPN1 is a novel modulator of the BMPR2 signaling pathway. These findings will help investigate the precise role of PTPN1 in PAH.


Sphingolipid molecules have recently attracted attention as signaling molecules in allergic inflammation diseases. Sphingosine-1-phosphate (S1P) is synthesized by two isoforms of sphingosine kinases (SPHK 1 and SPHK2) and is known to be involved in various cellular processes. S1P levels reportedly increase in allergic inflammatory diseases, such as asthma and anaphylaxis. FcεRI signaling is necessary for allergic inflammation as it can activate the SPHKs and increase the S1P level; once S1P is secreted, it can bind to the S1P receptors (S1PRs). The role of S1P signaling in various allergic diseases is discussed. Increased levels of S1P are positively associated with asthma and anaphylaxis. S1P can either induce or suppress allergic skin diseases in a context-dependent manner. The crosstalk between FcεRI and S1P/SPHK/S1PRs is discussed. The roles of the microRNAs that regulate the expression of the components of S1P signaling in allergic inflammatory diseases are also discussed. Various reports suggest the role of S1P in FcεRI-mediated mast cell (MC) activation. Thus, S1P/SPHK/S1PRs signaling can be the target for developing anti-allergy drugs. Sphingolipids are present in the cell membrane and provide structural integrity; they can mediate cell signaling, cell survival and differentiation, immune cell trafficking, and vascular and mucosal integrities ,2. SphinSPHK1 is mainly localized in the cytosol and can be activated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor, hepatocyte growth factor (HGF), and tumor necrosis factor-α (TNF-α) . SPHK2 iCeramide-1-phosphate (C1P) promotes caspase-3-mediated apoptosis and cell cycle arrest while S1P is necessary for cell proliferation and survival ,17. S1P Inhibition of SPHK1 resulted in attenuated allergic airway hyperresponsiveness (AHR) in a model of allergic asthma . SphingoMany of the S1P actions are mediated by five members of the G protein-coupled S1P receptors (S1PR1-S1PR5) with overlapping but distinct couplings to heterotrimeric G proteins ,31. Afte2+ mobilization, and activating protein kinase C (PKC) [S1PR1 can recruit macrophages and MCs to the inflammation sites for tissue repair . S1PR2 r C (PKC) . The MCs C (PKC) . S1PR4 a C (PKC) . S1PR4 c C (PKC) . S1PR5 i C (PKC) ,43. The Alteration of the ceramide composition of the epidermis contributes to the pathogenesis of atopic dermatitis (AD) ,50. AD iDecreased levels of S1P can result in the growth of Pseudomonas aeruginosa and Staphylococcus aureus, which are known to contribute to the pathogenesis of AD . S1PR2 kJTE-013, a selective antagonist of S1PR2, has been shown to suppress 2,4-dinitrochlorobenzene (DNCB)-induced atopic responses in a mouse model. JTE-013 administration can also significantly decrease the levels of pro-inflammatory cytokines  in the ears and lymph nodes as well as the levels of chemokines cc ligand 17 (CCL17) and CCL22 in the ears . DeficieORMDL3) gene as that is responsible for childhood-onset asthma risk [ORMDL3 in asthma.The ER and unfolded protein response (UPR) signals mediate the secretion of bioactive metabolites by the MCs ,61. Genohma risk ,63. ORMDhma risk , ER strehma risk ,66, implORMDL3 expression has been found to be lower in antigen-activated MCs and can suppress MC activation [ORMDL1 and ORMDL2 have been found to be associated with childhood asthma [ORMDL2 and ORMDL3 exhibits increased intracellular levels of S1P in the MCs [ORMDL3 showed a reduced level of S1P [ORMDL3 in allergic inflammation in relation to S1P.tivation . Three Od asthma . The dou the MCs . Transgel of S1P . Targetil of S1P . These rThe S1P levels were observed to be higher in the BAL fluids of patients with asthma . CYM5035In a murine model of chronic asthma, SK1-I, an inhibitor of SPHK1, has been shown to decrease eosinophil numbers as well as levels of cytokines  and chemokines  in bronchoalveolar lavage fluids (BAL) fluids . TreatmeCeramide and S1P can regulate a diverse range of cellular processes concerning immunity and inflammation ,81. S1P FcεRI is necessary for passive cutaneous anaphylaxis (PCA) reaction by activating MAPK signaling . PCA is Allergen (antigen) binding to IgE can activate FcεRI signaling, and IgE-bound FcεRI can activate Src kinases, such as Lyn and Fyn, which in turn can activate PLCγ to convert phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 can increase intracellular calcium concentration by acting through the IP3 receptor on the membrane of the ER. Calcium ions can induce degranulation of MCs, increasing the secretion of histamine and heparin. DAG and calcium ions can activate PKC, which in turn activates P38 mitogen-activated protein kinase MAPK (P38MAPK) as well as increases the expression levels of B cell lymphoma/leukemia 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation protein 1(MALT1). ERK, JNK, and P38 MAPK can activate nuclear factor-κB (NF-kB) to increase the expression levels of T helper 2 (Th2) cytokines. PI3K, which is activated by Lyn and Fyn, can convert PIP2 into phosphatidyl inositol 3,4,5-triphosphate (PIP3), which can activate JNK through RAC and mitogen-activated protein kinase 4 (MAPK4). PI3K can also activate ERK1/2, which in turn can activate phospholipase A2 (PLA2) and increases secretion of lipid mediators, such as leukotrienes and prostaglandins. The prostaglandins may then recruit effector cells, and the leukotrienes can increase vascular permeability. JNK, P38MAPK, and ERK1/2 can increase the production of cytokines in allergen (antigen)-stimulated MCs. Lipid mediators and cytokines also induce MC activation. ↑ denotes increased level/activity; The black and dotted arrows denote the directions of reaction; α, β, and γ denote the subunits of FcεRI; the black stars denote the potential targets of the reactive oxygen species (ROS). MEK, mitogen-activated protein kinase.IgE-treated MCs showed increased levels of S1P and sphingosine-1-phopsphate receptor 3 (S1PR3) . S1P is Activation of P42/P44 MAPK, Akt, and nuclear factor-κB (NF-kB) pathways is essential for S1P-induced COX-2 gene expression . S1PR1/3Anaphylaxis is a severe and life-threatening multisystem syndrome that necessitates immediate medical attention . MCs canHistone deacetylases (HDACs) remove acetyl groups from histones and tighten the binding of DNA to the histones. The regulatory roles of HDACs in gene expression allow HDACs to act as regulators of allergic inflammation. FcεRI signaling is necessary for the induction of HDAC3 and MCP1 (CCL2) , which aSaturated fat-induced NF-κB signaling and elevated expressions of TNFα and MCP1 mRNA in HepG2 cells can be blocked by targeted knockdown of S1PR1 . Since MAntigen stimulation can induce the expression and activity of transglutaminase (TGase II) by activating NF-kB in RBL2H3 cells . TGase ISphingolipid biosynthesis is initiated by SPT. Tubacin, an inhibitor of HDAC6, can inhibit de novo sphingolipid synthesis by suppressing SPT activity . This suPSA can enhance the tumorigenic potential of cancer cells by promoting cellular interactions involving cancer cells, MCs, macrophages, and various stromal cells . This imInhibition of HDAC3 can diminish S1P production in osteoclasts . S1P treS1P is metabolized to ∆2-hexadecenal (∆2-HDE) and ethanolamine phosphate by S1P lyase (S1PL) in mammalian cells . The addThe IgE-bound FcεRI can increase the expression of HDAC3, which can bind to Rac1, and decrease the expression of HDAC2 by inducing ubiquitination of HDAC2. HDAC3 can also bind to the promoter sequences of MCP1 to increase its expression. HDAC3 and MCP1 can mediate PCA, PSA, and triphasic cutaneous reactions (TpCRs). In the absence of antigen stimulation, HDAC2 can bind to the promoter sequences of MCP1 to decrease the expression of MCP1. S1P levels can be increased by SPHKs activated by FcεRI signaling. S1P can inhibit HDAC2 activity by increasing HDAC3 activity, which in turn can increase the expression level of MCP1 to mediate allergic inflammation, such as PCA, PSA, and TpCR.MCs express both SPHK1 and SPHK2 isoforms, which are activated following FcεRI stimulation, and S1P generated by the SPHKs can mediate the IgE-promoted release of mediators and cytokines ,119,120.Src kinases, such as Lyn and Fyn, can interact with and activate SPHK1 in the MCs to promote the recruitment of SPHK1 to FcεRI . DeficieCalcium influx is critical for FcεRI-mediated MC degranulation and cytokine generation . InhibitSPHK1 knockout mice showed reduced plasma S1P levels . FcεRI-mMCs express S1PRs ,129, andThe S1P-S1PR signaling pathway is critical for both MC degranulation and migration . The actMicroRNAs (miRNAs/miRs) are noncoding single-stranded RNAs of 18–24 nucleotides in length. Since microRNAs target multiple genes, they are involved in various life processes such as cellular interactions , cell prmiR-126 can promote IgE-mediated MC degranulation associated with the PI3K/Akt signaling pathway by promoting Ca2+ influx . S1P canmiRNAs can target components of the S1P signaling pathway . For exaIt is known that IgE-bound MCs activate FcεRI signaling, which in turn leads to the activation of ERK, P38 MAPK, JNK, and PI3K. Since FcεRI signaling increases S1P production in MCs, it would be necessary to examine the effects of ERK, P38 MAPK, JNK, and PI3K on S1P production in allergic inflammation both in vitro and in vivo. Since FcεRI signaling also increases the production of Th2 cytokines, it would be interesting to examine the effects of Th2 cytokines on S1P production in allergic inflammation.Based on previous reports, sphingolipid signaling is becoming an important target for developing anti-allergy drugs. It is thus necessary to examine whether S1P can induce the hallmarks of allergic inflammation, such as PCA and PSA, in an IgE-independent manner. Moreover, it may be necessary to identify the S1P-regulated genes, which can provide clues to the understanding of allergic inflammation mediated by crosstalk between FcεRI signaling and S1P signaling.Since S1PR2 is involved in allergic inflammation, the identification of the downstream targets of S1PR2 is necessary for developing anti-allergy drugs. RNA sequencing can identify the downstream targets of S1PR2, which may include components of FcεRI signaling. Proteins that can bind to S1PRs proteins can also serve as targets for developing anti-allergy drugs. It is hence necessary to identify the full-length protein structures of S1PRs, which can enable screening of the inhibitors that regulate the expression and/or activity of S1PRs.Since SPHKs can mediate allergic inflammation, it may be necessary to screen inhibitors of SPHKs based on the full-length protein structures of SPHKs. It is also necessary to identify genes that may be regulated by these inhibitors. These inhibitors can be further developed as anti-allergy drugs and could suppress FcεRI signaling to prevent allergens (antigens) from increasing the production of Th2 cytokines and inducing MC degranulation. It is probable that these inhibitors may also suppress in vivo allergic inflammation, such as anaphylaxis and asthma.Allergic inflammation involves cellular interactions among the MCs, macrophages, and endothelial cells, which are mediated by exosomes. It will be interesting to examine the presence of the components of S1P/S1PR signaling in the exosomes. It is also necessary to examine whether the components of S1P signaling could mediate cellular interactions in allergic inflammation. It is probable that S1P phosphatase and/or S1PL may inhibit allergic inflammation by negative regulation of FcεRI signaling and cellular interactions.MiRNAs are known to regulate expression levels of the components of S1P/S1PR signaling, such as SPHKs and S1PRs ,147. ThuComplete understanding of the crosstalk between FcεRI signaling and S1P signaling in allergic inflammation will thus be necessary for developing anti-allergy drugs in the future. Given the fact that anaphylaxis can enhance the tumorigenic potential of cancer cells, it is expected that the anti-allergy drugs can also be used as anti-cancer drugs


Cognitive function is essential in ensuring the quality of life of the elderly. This study aimed to investigate the effects of Baduanjin imagery and Baduanjin movement  on cognitive function in the elderly using functional near-infrared spectroscopy (fNIRS).72 participants with a mean age of 66.92 years (SD = 4.77) were recruited for this study. The participants were randomly assigned to three groups: the Baduanjin imagery, the Baduanjin exercise, and the Control. Stroop task was used to record the accuracy and reaction times, and a near-infrared spectral brain imaging system was used to monitor the brain's oxy-hemoglobin concentration responses.F = 114.076, p < 0.001) and the interaction effect between test phase and group  were all significant. The simple effect analysis further demonstrated that the reaction times of the Baduanjin imagery group and Baduanjin exercise group in the post-test was faster than that in the pre-test (ps < 0.001); (2) Analysis of fNIRS data showed the significant interaction effect  between the test phase and group in the left dorsolateral prefrontal cortex. Further analysis showed that, during the post-test incongruent tasks, the oxy-Hb variations were significantly higher in participants of the Baduanjin imagery group (p = 0.005) and Baduanjin exercise group (p = 0.002) than in the control group; For the right inferior frontal gyrus, the interaction between the test phase and group was significant . Further analysis showed that, during the post-test incongruent tasks, the oxy-Hb variations were significantly higher in participants of the Baduanjin imagery group than in the control group (p = 0.001).(1) For the reaction times of Stroop incongruent tasks, the main effect of the test phase (Baduanjin imagery and exercise positively affect cognitive performance; Baduanjin imagery and exercise activated the left dorsolateral prefrontal cortex; Baduanjin imagery activated the right inferior frontal gyrus, while Baduanjin exercise could not. According to China's National Bureau of Statistics, by 2020, there are already 260 million people aged 60 years and older in China, accounting for 18.7% of the total population . With thNumerous studies show that physical exercise positively affects cognitive and motor function in older adults –12. BaduIn addition to traditional exercise interventions to improve physical and mental health, another widely researched and applied approach is motor imagery training, which has the characteristics of high safety and operability –20. As aThe prefrontal lobe (PFC) is an important brain region for executive functions, with the dorsolateral prefrontal (DLPFC) and the inferior frontal gyrus (IFG) being the main activation areas , 25. TheFunctional near-infrared spectroscopy (fNIRS) is widely adopted in cognitive neuroscience to measure the cortical oxy-hemoglobin concentration in real-time, reflecting the individual brain activation status . It is aThe current study monitored changes in the PFC blood oxygenation in the elderly before, during, and after the intervention using fNIRS. We aimed to explore whether Baduanjin imagery has the same effect as actual movement and examine the mechanism of cerebral blood oxygenation in improving cognitive function by Baduanjin imagery to provide a practical basis for enhancing cognitive function in the elderly.We set up the Baduanjin imagery group, the Baduanjin exercise group, and the Control group and proposed the following hypotheses: (1) Baduanjin imagery improves cognitive function in the elderly, as reflected in the Stroop task reaction times; (2) Baduanjin exercise improves cognitive function in the elderly, as reflected in the Stroop task reaction times; (3) Baduanjin imagery practice enhances oxy-hemoglobin concentration in the left DLPFC and the right IFG during the inhibition task; (4) Baduanjin exercise enhances oxy-hemoglobin concentration in the left DLPFC and right IFG during the inhibitory control task.f = 0.25 and power 1-β = 0.95, we calculated the minimum sample size to be 66 people and planned to recruit 70 people considering sample attrition. Furthermore, we recruited 76 participants from the local community in Jinan, China, of whom one quit without completing the experiment, one slept during the intervention, and two had a low ACC rate (9.26 and 13.89%). Finally, data from 72 participants  were included in the data analysis. The participants were randomly assigned to the Baduanjin imagery group, the Baduanjin exercise group, or the Control group. The eligibility criteria were age 60 and above, having Baduanjin exercise experience ≥3 years, and MMSE score ≥24. The exclusion criteria were the presence of neurological disease, vision problems, or previous similar experimental experiences. This study was previously approved by the Institutional Ethics Committee. All participants signed informed consent before the experiment.According to G-power 3.1 , to estiThe MMSE is prepared by Folstein  and reviThe PARS-3 revised by Liang and Liu  includesThe ability to kinesthetic and visual imagery was assessed using the MIQ-R revised by Zhang and Mao . The queWe used the Stroop test as the cognitive test , 49, whiThe experimental protocols included a pre-test, an intervention, and a post-test . In the In the intervention, three groups of participants performed the Baduanjin imagery training, the Baduanjin exercise, and the reading of neutral material, respectively. We gave participants in the Baduanjin imagery group a brief introduction to the content of the imagery training. We asked them to wear an eyeshade to reduce light stimulation and enhance visual imagery. Relaxation imagery was given to participants in the Baduanjin imagery group before the Baduanjin imagery to reduce stress and anxiety, thus increasing the effectiveness of the imagery intervention . The audBefore the formal intervention, “Preparation Instructions” was used to tell participants to be ready to enter an intervention. The instructions for the Baduanjin imagery group were: “For the next 15 min, please sit comfortably with an eyeshade, close your eyes and fully participate, do not talk or fall asleep, and do not shake or move your body. Follow the audio for relaxation imagery and Baduanjin imagery. The process is uninterrupted, signal me if you're ready, and we'll start.” The instructions for the Baduanjin exercise group were: “For the next 15 min, please follow the audio for warm-up activities and Baduanjin exercise without interruption throughout. Signal me if you're ready, and we'll start.” For the control group, the instructions were: “For the next 15 min, please have a rest for a while and then read the material I have distributed to you. Signal me if you're ready, and we'll start.” Furthermore, we gave the participants in the Baduanjin imagery group “Baduanjin imagery guidelines” to guide them in forming a visual-motor imagery of the Baduanjin, while the exercise and control groups did not have the guide. According to the “Baduanjin imagery guidelines”, participants should sit in a chair and listen carefully to the Baduanjin movement commands with their eyes closed, follow the commands to imagine as clearly as possible the Baduanjin movements as they had practiced, and “watch” the Baduanjin movement process in their minds like watching a movie.In addition, the effectiveness of the imagery intervention was assessed by a motor imagery manipulation check , 52 afteThe fNIRS data were collected by the NirSmart II system . The fNIRS system uses the 2 × 7 Prefrontal Cortex Layout  and Baduanjin exercise group  were significantly faster in the post-test than in the pre-test, However, there was no significant difference between the pre-test and the post-test (p = 0.071) for the Control group. In addition, there were no between-group differences in both the pre-test and post-test (ps > 0.05). These results indicate that the Baduanjin imagery and Baduanjin exercise significantly shortened participants' reaction time to complete an inhibitory control task and increased practitioners' cognitive response speed.For the RTs, the main effect of the test phase was significant under the congruent task, ps > 0.05). In the incongruent task, the main effects of the test phase and group were not significant (ps > 0.05). The interaction between the test phase and group was significant, F  = 2.554, p = 0.013, η2 = 0.132. The results of the simple effect test showed that there were significant differences in the post-test task among the three groups, F  = 6.254, p = 0.003, η2 = 0.153. The oxy-Hb variations in the left DLPFC in participants of the Baduanjin imagery group (p = 0.005) and Baduanjin exercise group (p = 0.002) were significantly higher than that in the Control group. In the pre-test task, there was no significant difference between participants of the three groups (p = 0.965). The oxy-Hb concentrations of participants in three groups at all five phases were not significant (ps > 0.05). These results indicate that the Baduanjin imagery and the Baduanjin exercise significantly increased the oxy-Hb concentration in the participants' left DLPFC in the post-test cognitive state . In the incongruent task, the main effect of the test phase was not significant (ps > 0.05); the main effect of the group was significant, F  = 3.356, p = 0.041, η2 = 0.089. The multiple comparison results showed that the oxy-Hb variation of the Baduanjin imagery group was significantly higher than that in the Control group (p = 0.016). The interaction between the test phase and group was significant, F  = 2.060, p = 0.044, η2 = 0.110. The simple effect test showed significant differences in the post-test task among the three groups, F  = 5.933, p = 0.004, η2 = 0.147. The oxy-Hb variation in the right IFG in participants of the Baduanjin imagery group was significantly higher than that in the Control group (p = 0.001), while not in the Baduanjin exercise group (p = 0.069). In the pre-test task, there was no significant difference between participants of the imagery and control groups (p = 0.665). The oxy-Hb concentrations of participants in three groups at all five phases were not significant (ps > 0.05). These results indicate that the Baduanjin imagery significantly increased the oxy-Hb concentration in the participants' right IFG in the post-test cognitive state  more likely to contribute to increased cerebral oxygenation in the elderly  can be found in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the School of Public Health of Shandong University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.LY and YH provided the ideas. LY participated in writing the introduction, methods, results, discussion, the experiments' conducting, and the data processing. JW participated in the revisions of the article. GS completed the editing and review of the article. All authors contributed to the article and approved the submitted version.This study was funded by the Social Science Fund of Shandong Province (No. 22CTYJ07), the Young Scholars Program of Shandong University, the Teaching Research Project of Shandong University (No. XYJG2020025), and the Graduate Research Fund of Shandong University College of Physical Education (No. 2021YJKTYB29).The 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.