Allium schoenoprasum L.) treated with 1-methylcyclopropene (1-MCP) during storage were investigated in this study. The 1-MCP treatment  effectively inhibited tissue respiration and H2O2 production, enhanced the ascorbic acid (ASA) and glutathione (GSH) content, and promoted the activity of antioxidant enzymes  during the 5-day storage period at 20 °C. The result further showed that the 1-MCP treatment inhibited chlorophyll degradation, alleviated cell membrane damage, and delayed the chive senescence, with the yellowing rate being reduced by 67.8% and 34.5% in the 1-MCP treated chives on days 4 and 5 of storage at 20 °C, respectively. The free amino acid content of the chive was not affected by the 1-MCP treatment at 20 °C. However, the senescence rate of the chive was not reduced by the 1-MCP treatment when stored at 3 °C. The liquid chromatography data further showed that the 1-MCP treatment induced a 15.3% and 13.9% increase in the isoalliin and total S-alk(en)ylcysteine sulfoxides (ACSOs) content of the chive on day 2 at 20 °C, respectively. Furthermore, there was a strong positive correlation between ACSOs content and CAT/APX activity, indicating that ACSOs probably played a key role in enhancing the antioxidant capacities of the chive during storage at 20 °C. Thus the study efficiently demonstrates that 1-methylcyclopropene preserves the quality of chive (Allium schoenoprasum L.) by enhancing its antioxidant capacities and organosulfur profile during storage.The quality, antioxidant capacities, and organosulfur profile of chives ( Allium genus includes several hundred species and is widely utilized as a drug in folk medicine for its potent antibacterial, hypoglycemic, hypolipidemic, hypocholesterolemic, cardiovascular, antithrombotic, and antitumor activities [Allium schoenoprasum L (chive) is rich in carbohydrates, proteins, amino acids, vitamins, and minerals and is cultivated as vegetables or seasoning herbs all around the world [Allium plants, chives have a unique flavor, and the precursor for flavor and therapeutic compounds are S-alk(en)ylcysteine sulfoxides (ACSOs) [The tivities ,2,3,4. Ahe world . Like ot (ACSOs) .2−, H2O2, and·OH−) [Allium genus plant.Similar to many green leafy vegetables, postharvest chives have a high respiration and senescence rate which often leads to quality deterioration, including leaf yellowing, wilting, and nutrient loss at ambient temperature storage. The most obvious characteristics of senescence are degradation of chlorophyll, breakdown of chloroplasts, increased oxidation, and hydrolysis of macromolecules, such as proteins . During and·OH−)  and its and·OH−) . When ROand·OH−) . ResearcAllium genus has been rarely reported in the literature. Thus, a study on the effect of 1-MCP treatment on the storage quality of chive will be practically useful in the postharvest management of the Allium genus plant.The current consensus is that the postharvest treatment-dependent delay in leaf senescence in the vegetables is mostly attributed to decreased ROS production . To dateAllium plant during storage. The objective of this study is to determine the impact of 1-MCP treatment on leaf quality, antioxidant capacity, and organosulfur compounds of chive during storage at temperatures (20 °C and 3 °C). The relationship between ACSOs and the antioxidant capacity of the chive leaf was also studied. The results of this work will provide insight into the mechanisms of senescence physiology in Allium vegetables as well as the role of ACSOs in Allium vegetable senescence.The physiological and biochemical properties of similar plant species such as garlic and onion have been extensively studied ,18,19,20Allium schoenoprasum L. cv. Xinghua), grown in Zhujia village, Xinghua County , Jiangsu Province, China, was used for this study. Harvested chives (day 0) were transported to the laboratory within 3 h at 20 °C. The senescent leaves were removed while the leaves with uniform sizes were selected and dried with a fan. The sample was then separated into four groups, CK 20 °C (non-treated), CK 3 °C (non-treated), 1-MCP 20 °C and 1-MCP 3 °C (treated with 1-MCP).The chive (w/v) KOH aqueous solution was kept inside the plastic container to prevent the accumulation of CO2 by chives. After fumigation (day 0.5), the CK 20 °C group and 1-MCP 20 °C group were stored at 20 °C (RT) with 85–90% humidity for 5 d, while the CK 3 °C group and 1-MCP 3 °C group were stored at 3 °C (LT) with 85–90% humidity for 20 d  for quality parameter analysis, physiology and ROS level. After the above determinations, samples of size 0.5–1 cm were collected, immediately frozen in liquid nitrogen, and stored at −80 °C for further analysis. As the white shaft part was very short, chives were sampled starting from the white and green transition to 2 cm of the top of the leaves, which represent the whole plant.S-methyl-L-cysteine sulfoxide  was purchased from Nanjing Chemlin Chemical Industrial Co., Ltd.; S-allyl-L-cysteine sulfoxide  was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; S-propyl- L-cysteine sulfoxide  was purchased from Jiangsu Aikon Biopharmaceutical R&D Co., Ltd.; (E)-S-(1-propenyl)- L-cysteine sulfoxide (PeCSO) was identified by high-resolution mass spectrometry (MS). Twelve amino acids, asparagine (Asn), glutamine (Gln), serine (Ser), arginine (Arg), threonine (Thr), alanine (Ala), proline (Pro), methionine (Met), valine , tryptophan (Trp), leucine (Leu), phenylalanine (Phe), HPLC ≥ 98%) were all purchased from Shanghai Yuanye Bio-Technology Co., Ltd.v/v) and then centrifuged at 12,000 g for 15 min at 4 °C. The chlorophyll content was quantified by measuring the absorbances at 665 and 649 nm in the supernatant and the content was expressed as g kg−1. Except for the content of free amino acids, PeCSO and ACSOs, all the other results were expressed on a fresh weight basis.Chlorophyll content was measured as reported by  with sliAt each time point, 1 kg chives from each sampling treatment were used to measure the yellowing rate. The leaf yellowing scales were divided into five grades: grade 0: no yellowing area; grade 1: approximately 1–10% yellowing area; grade 2: approximately 10–25% yellowing area; grade 3: approximately 25–50% yellowing area; and grade 4: more than 50% yellowing area. Leaf yellowing rate = ∑ (yellowing grade × number of leaves at this grade)/.2 gas analyzer . Approximately 400 g chives from each biological replicate were enclosed in 7.4 L glass jars at 20 °C or 3 °C for 1 h to measure respiration rate.Respiration rate was directly measured by a CO2O2) content and malondialdehyde (MDA) content were measured using the method of [2O2 content and MDA content were expressed as mmol kg−1.Hydrogen peroxide (Hethod of . H2O2 co−1. GSH (reduced glutathione) and GSSG (oxidized glutathione) concentrations were measured by an enzymatic cycling assay method described by [−1.ASA concentration was determined by the method as described by  and was ribed by  and wereSuperoxide dismutase (SOD) and ascorbic peroxidase (APX) activity measurements were adapted from . One uni2O2) and 0.2 mL enzyme extract, with a total volume of 3.0 mL. H2O2 decomposition was measured by the decrease in absorbance at 240 nm. One unit was defined as the amount of enzyme that caused one absorbance change per minute under the assay conditions.Catalase (CAT) activity was determined according to the method reported by  with a s2O2 at 470 nm. The absorbance at 470 nm was recorded every 30 s using a spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme required to increase the absorbance by 0.001 min−1 under the assay conditions.Peroxidase (POD) activity was assayed as previously described by . POD actThe γ-glutamyl transpeptidase (GTP) activity was determined according to the method reported by  with a s2, 25 μmol/L pyridoxal phosphate, pH 7.0), then centrifuged at 12,000 g for 15 min at 4 °C. The reaction mixture was 0.1 mL above enzyme extracts and 10 mmol/L S-methyl cysteine sulfoxide. The reaction mixture was incubated at 37 °C for 10 min. The enzymatic reaction was terminated by the addition of 1 mL of 2 mol/L HCl containing 5 mmol/L dinitrophenyl hydrazine. Subsequently, the mixture was incubated at 37 °C for 5 min, afterwards, 5 mL of 2.5 mol/L NaOH was added to the solution, incubated for 10 min and the absorbance was measured at 420 nm against a blank (enzyme extract substituted by distilled water). One unit of allinase activity is defined as the amount of enzyme that produces 1 μmol pyruvate per min under standard conditions.Allinase activity was determined according to the method reported by  with a sThe organosulfur compounds and amino acids were extracted and determined according to the method reported by  with a sDansyl derivatives were prepared by mixing 100 μL of the sample extract with 250 μL of the dansyl chloride (Dns-Cl) reagent (10 mmol/L Dns-Cl in acetonitrile) and 0.65 mL of 20 mmol/L borate buffer (pH 9.2). The mixture was briefly shaken, allowed to stand at room temperature for 30 min, filtered through a 0.45 μm nylon filter, and analyzed by HPLC.A high-performance liquid chromatography (HPLC)  equipped with a ZORBAX SB-Aq  column and PDA detector  was used to determine the content of organosulfur compounds and amino acids. The chromatographic condition was as follows: 50 mmol/L sodium acetate buffer  and methanol (solvent B) were used as the mobile phase, with a flow rate of 0.9 mL min−1 and the gradient A/B 70/30 (0 min), 70/30 (2 min), 60/40 (in 12 min), 50/50 (in 32 min), 40/60 (in 42 min), 5/95 (in 42.01 min), 5/95 (in 51 min), 70/30 (in 51.01 min), and 70/30 (in 60 min), detection wavelength of 250 nm. The injection volume was 20 μL. The content of free amino acids, PeCSO and ACSOs, were expressed on a dry weight basis.For identification of each peak, the eluents corresponding to each peak of HPLC were collected separately, desalted by C-18 solid-phase extraction column and analyzed by LC-MS system . A 2 μL solution was injected into the UPLC column (2.1 × 100 mm ACQUITY UPLC BEH C18 column containing 1.7 μm particles) with a flow rate of 0.3 mL/min. Buffer A consisted of 0.1% formic acid in the water, and buffer B consisted of 0.1% formic acid in acetonitrile. The gradient was 5% buffer B for 0.5 min, 5–95% buffer B for 11 min, 95% buffer B for 2 min.Mass spectrometry was performed using an electrospray source in positive ion mode with MS acquisition mode, with a selected mass range of 50–1200 m/z. The lock mass option was enabled using leucine-enkephalin (m/z 556.2771) for recalibration. The ionization parameters were the following: the capillary voltage was 2.5 kV, sample cone was 40 V, source temperature was 120 °C (and desolvation gas temperature was 400 °C). Data acquisition and processing were performed using Masslynx 4.1.p < 0.05). Origin 2021  was employed to create figures.All values are reported as the mean ± standard errors of three biological replicates. Statistical analysis was performed with the SPSS 18.0 software . The significant differences between mean values were analyzed using the LSD’s test , ASA content C in chivAntioxidant enzymes in fruit and vegetables include SOD, CAT, APX, and POD. SOD is one of the most important antioxidant enzymes, and its activity declined in the first 12 h, increased afterward, dropped again during storage at RT. Nevertheless, SOD activity A was 15.There was no correlation between SOD/CAT/APX activity and POD activity. The POD activity increased dramatically in the first 12 h after harvest in all groups D. When sThe results strongly demonstrate that 1-MCP treatment can efficiently enhance non-enzymatic and enzymatic antioxidant capacities in chives during RT storage, but do not affect the non-enzymatic and enzymatic antioxidant capacities of chives during LT storage.Based on the results of the preliminary experiment (1-MCP treatment had no effect on chives during LT storage), the content of ACSOs and free amino acids of 1-MCP treated chives stored at 3 °C were not determined. ACSOs and free amino acid content of the extract from chive samples are as shown in the HPLC chromatogram in Allinase, which is located in the cell vacuoles, hydrolyzes ACSOs, producing a variety of antibacterial sulfur-containing compounds . The allIsoalliin (PeCSO) was identified using LC-MS coupled with high-resolution mass spectrometry, as shown in Through preliminary experiments, we found that chives could only be stored for 5 days at 20 °C and only 20 days at 3 °C. Thus we chose 5 days of study for 20 °C and 20 days of study for 3 °C. Vegetables do not require ethylene action for normal senescence to take place ,32. In t2O2 content in 1-MCP treated chives during RT storage. However, during LT storage, 1-MCP treated chives showed no difference in respiration rate and H2O2 content compared with untreated chives. This result strongly indicates that 1-MCP treatment does not affect the physical activity and enzyme activity of chive when stored at LT. Similar results have been reported in a combined 1-MCP and heat treatment on peach [Respiration is an indication of the tissue metabolic rate and the major factor that contributes to the deterioration and loss of quality of plant produce during postharvest storage . Chive lon peach .2O2 in 1-MCP treated vegetables correlated with the lower MDA level during RT storage. This finding suggests that 1-MCP treatment effectively maintained the membrane integrity of the chive by suppressing oxidative stress during RT storage, while the 1-MCP treatment did not affect the MDA and ROS levels of the chives during LT storage in this study. It has been reported that combined 1-MCP and heat-treatment even induced oxidative stress during LT storage in peach [The MDA concentration and ROS level are often used as an index to measure membrane integrity and oxidative stress in vegetables . In thisin peach .2O2), and maintained the cell structural integrity. The accumulation of ROS in plants during senescing can activate the enzymatic and non-enzymatic antioxidant system to scavenge ROS down to a safe range [2O2 content and MDA content was lower in 1-MCP treated chives during RT storage. The 1-MCP treatment had little effect on the antioxidant system during LT storage, except for CAT activity. It has been reported in the literature that POD activity increases as the leaves are senescing, the role of which is dual [The 1-MCP preserved a high content of antioxidants (ASA and GSH) and high activities of antioxidant enzymes  in chives stored at 20 °C which might have contributed to scavenging ROS , is the result of chlorophyll degradation. The 1-MCP treatment delayed chlorophyll degradation in chives during RT storage. Similar findings have been reported ,39. The Pleurotus eryngii showed no effects on total free amino acids [When plant cells undergo senescence, they suffer from oxidative stress and produce a large amount of ROS, causing membrane lipid peroxidation, degradation of protein, and production of free amino acids ,41. The no acids .In summary, the results indicate that 1-MCP enhances antioxidant capacity, reduces oxidative stress, and delays senescence in postharvest chives during storage at RT, while 1-MCP treatment had no effect on the antioxidant capacity, oxidative stress, and senescence levels on chives during storage at LT.S-alk(en)ylcysteine sulfoxides (ACSOs) are bioactive compounds that are beneficial for both the plant itself and human health due to their antibiotic properties. It has been widely reported that alliin is an effective hydroxyl (OH) scavenger ,45,46, iOverall, ACSOs showed a strong positive correlation with the CAT and APX, indicating that ACSOs may act as antioxidants in chives. The 1-MCP increased the content of ACSOs during storage at RT. However, compared with RT storage, chives stored at LT storage had higher ACSOs content.Allium schoenoprasum L.) by enhancing its antioxidant capacities and organosulfur profile during storage.The possible senescence mechanism of chive leaves and mechanism of yellowing inhibition of chives during storage by 1-MCP treatment is shown in


Among viruses that promote γδ T cell mobilisation in humans, herpes viruses (HHVs) occupy a particular place since they infect the majority of the human population and persist indefinitely in the organism in a latent state. Thus, other infections should, in most instances, be considered co-infections, and the reactivation of HHV is a serious confounding factor in attributing γδ T cell alterations to a particular pathogen in human diseases. We review here the literature data on γδ T cell mobilisation in HHV infections and co-infections, and discuss the possible contribution of HHVs to γδ alterations observed in various infectious settings. As multiple infections seemingly mobilise overlapping γδ subsets, we also address the concept of possible cross-protection. Viruses of the human herpes viridae (HHV) group have been co-existing with humans for millions of years, and existed in primates before the split of hominids and chimpanzees . Eight sThis review focusses on a particular subset of non-conventional T cells which use γ and δ clonotypic TCR chains in association with CD3 components to form their TCR. γδ T cell subsets are mobilised in acute infections by several HHVs, with sometimes-major expansions. They are at the interface between innate and adaptive immunity, and share with NKT or MAIT cells a limited TCR diversity due to their usage of a small set of TCR-variable genes. This restricted diversity reflects the recognition of relatively frequent ligands for which antigen-specific clones are present at a relatively high frequency. This allows them to perform their function after limited clonal expansion. This, added to the faculty of some of them to acquire an effector potential prior to antigenic challenge, allows them to respond quickly to infections. However, some subsets show adaptive-like behaviour, with massive clonal expansions and functional plasticity ,10,11,12Although γδT cell subsets share common properties in different mammal species, they have diverged significantly. Differences between human and murine systems are reported for thymic differentiation processes as well as major γδ T cell subsets residing in blood and tissues . SimilarAlterations of γδ subsets have been reported in the context of HHV infections and HHV-unrelated viral infections, including HIV, HCV, HEV, HBV, influenza, myxoviruses, flaviviruses and the vaccinia virus ,17,18. H+ precursors in multiple inflammatory contexts, in particular in situations where a strong allogeneic stimulation occurs, such as solid organ transplantation (SOT) or hematopoietic cell transplantation (HCT) [HHV latency is characterised by the persistence of the viral genome in an episomal form in the nucleus of infected cells. This is associated with no or very low viral replication, and with the expression of a restricted set of viral latency-associated genes. Viral reactivation from latently infected cells can be induced by factors which promote cell differentiation or activation as well as extrinsic environmental stress, such as hypoxia or inflammation . Factorson (HCT) ,21. Impaon (HCT) ,23. In ton (HCT) ,25. Repoon (HCT) ,27. Afteon (HCT) . Multiplon (HCT) ,29. The on (HCT) ,31. In the absence of obvious immunosuppression, reactivations of latent viruses are reported during sepsis with a frequency similar to that observed in HCT patients ,33,34, dposVδ2pos T cells (Vγ9Vδ2) are usually present, but are outnumbered by other γδ cells expressing mainly Vδ1 and, less frequently, Vδ2 (Vγ9neg) and Vδ3, which pair with multiple Vγ genes with some tissue-dependent skewing [γδ T cells are the first T cells to appear in primary lymphoid organs during foetal development. In humans, six variable γ genes (TRGV) and seven variable δ genes (TRDV) are functional ,38. In t skewing . With rare exceptions, the molecules identified as γδ TCR ligands are non-conventional MHC-related  or MHC-unrelated self-proteins . Several of them are endogenous proteins which can be upregulated or expressed at the cell membrane under inflammatory or metabolic stress, or following malignant transformation. These ligands are recognised independently of associated peptides or lipids, and do not require the antigen processing machinery used for antigen presentation to αβTCRs. Few foreign antigens from pathogens have been reported to stimulate γδ TCRs . These expansions display features of adaptive responses, and mainly involve private sequences ,59. Vγ9VCo-stimulatory, inhibitory and lymphokine receptors regulate γδ T cell functions , and subsequently in other contexts of immunosuppression [neg cells in blood, stable expression of a TEMRA late effector phenotype  and increased expression of activation markers  and of co-inhibitory receptors PD-1, TIM-3 and LAG-3 [Expansions of Vδ2pression ,85,86,87pression  and incrpression . A highepression ,89,90. And LAG-3 .neg cells from HSCT patients by spectratyping CDR3δ [negVδ2neg following the reactivation of CMV after HSCT [neg γδ cells from patients experiencing the reactivation of CMV, but not those from patients experiencing the reactivation of EBV, recognised specifically CMV-infected fibroblasts [neg cells from patients having undergone HSCT/CBT for B cell chronic leukaemia (CLL) reacted in vitro against autologous B-CLL cells, and this reactivity required CD8αα in addition to Vδ1 TCR. Strikingly, however, transduction of the TCR from two clones highly reactive to CMV into αβ T cells conferred reactivity towards the leukemic cells, but not against CMV-infected fibroblasts. In another study, expanded Vδ1 did not react against autologous B-CLL cells [neg clones from patients could have been selected by alternative determinants, with a possible influence of the infected tissue. The consequences of CMV infection on γδ T cells have also been examined after hematopoietic stem cell transplantation (HSCT) or cord blood transplantation (CBT). The reconstitution of the γδ T cell pool after HSCT or CBT occurs faster than the reconstitution of the αβ T cell compartment, and even faster in patients with reactivating CMV, whereas the reconstitution of Vδ2 cells is not changed . Initialng CDR3δ ,92, but ng CDR3δ . Howeverter HSCT ,94,95. Cter HSCT ,94. Puriroblasts ,93,96. Troblasts . In the neg cells in blood when compared to CMVneg individuals [neg subset [neg cells by CMV comes from their expansion in neonates after in utero infection [An imprint of CMV is also observable in healthy carriers, who usually display higher numbers of Vδ2ividuals ,92. Asymg subset ,82,91,97nfection : althougnfection . As thesnfection .An MHC-like protein, endothelial protein C receptor (EPCR), has been identified as a ligand for a Vγ4Vδ5 highly expanded clone representing 25% of blood T cells in a CMV-infected patient. The expression of this antigen does not seem to be modified by CMV infection . NeverthChronic inflammation as well as aging are associated with increased oxidative stress, which promotes the reactivation of CMV; conversely, the reactivation of CMV promotes oxidative stress ,102. RecThus, the non-Vγ9Vδ2 γδ T cell compartment displays an adaptive behaviour and CMV appears to be one major driver of clonal amplifications, possibly by inducing the expression of multiple stress ligands, which are also expressed on tumours.+, LMP1+ and LMP2+) is found in germinal centres of EBV carriers, Hodgkin’s lymphoma and some in-vitro-generated EBV-infected lymphoblastoid cell lines (LCLs). Latency III  is associated with EBV infection of naive B cells, lymphomas occurring in EBV-associated lymphoproliferative disease (EBV-PTLD) and most EBV-LCLs [EBV seroprevalence is over 90% in the worldwide population ,107,108.EBV-LCLs . The virEBV-LCLs ,116,117.EBV-LCLs .+ Burkitt’s lymphoma cell lines Daudi and Akata, whereas no response is observed against latency II or latency III EBV-LCLs [A specific reactivity of γδ T cells against some EBV-infected targets is substantiated by in vitro studies. γδ T cells expressing Vγ9-JP/Vδ2 are responsive to latency I EBVEBV-LCLs ,120. MorEBV-LCLs , or by bEBV-LCLs ,119,120.Healthy donors segregate into two populations according to the magnitude of their in vitro response to PhAgs and Akata ,119,120.pos cells are amplified in blood, display an activated phenotype  and persist for several months at the convalescent phase [+ healthy children who presumably encountered EBV asymptomatically, suggesting that high Vγ9Vδ2 responses in IM are linked to a reduced symptomatology [At the acute phase of IM, patients have elevated numbers of CD8, CD4, NK, iNKT and γδ cells ,123,124.nt phase ,126. IM matology .neg patients on an EBV-DNAemia basis and 22 patients with the post-HSCT reactivation of EBV [+ cells selectively in EBVpos patients. Thus, Vδ2 cells were activated following the reactivation of EBV, and may have participated in the control of EBV infection/reactivation. In contrast, another study linked an increased oligoclonality within the Vδ1 subset to the reactivation of EBV after allo-HSCT. However, the role of CMV was not excluded [Analyses of γδ T cells after HSCT also support the involvement of Vγ9Vδ2 cells in EBV responses. In a large cohort including 110 EBVn of EBV , all patexcluded .IM is generally resolutive. In rare cases, reported mainly in East Asia, it evolves on a chronic mode called chronic active EBV infection (CAEBV), with recurrent IM-like episodes. In this paediatric disorder, T and NK cells get infected by EBV in a context of high viral load, possibly after contacting infected B cells, and infiltrate multiple organs. These infected T/NK cells then apparently disappear to become undetectable after one year. Starting as a systemic inflammation, the disease can become malignant, leading to hemophagocytic lymphohistiocytosis (HLH) or lymphoma transformation characterised by monoclonal NK or T cell expansions . Clonal neg B-CLL cells. This suggests recognition of a cellular rather than viral determinant, but TCR-dependence of this activity was not reported [Another manifestation of acute EBV infection is post-transplantation lymphoproliferative disorder (PTLD), a syndrome linked to immunosuppression and associated with the reactivation of EBV. In PTLD, the reactivation of EBV in B cells induces an outgrowth of infected B lymphocytes. In more advanced stages this can lead to B cell transformation and lymphomas . Cases oreported . A humanreported . Taken together, above studies reveal that EBV infection, symptomatic or not, can be detected by the Vδ2 and the Vδ1 subsets, which might contribute by recognising infected B cells at different stages of maturation/differentiation.HSV1 and HSV2 are reported to infect, respectively, 66% and 11% of the worldwide population . HSV1/2 It is estimated that 60 to 70% of the population harbour HHV-6 and HHV-7 in their salivary gland . Both viKSV is not ubiquitous. It’s prevalence in some Eastern or African areas may reach 50%; however, in Western countries, the prevalence varies from 3 to 23% and is tightly linked to HIV infection, due to frequent co-transmission ,166. SimIn temperate countries, 90% of the population is infected with VZV by adolescence, while inhabitants of tropical countries are infected later in life . The reaIn the context of immune suppression and transplantation, the infection/reactivation of multiple HHVs is frequent, CMV-HHV-6 being the most frequent co-infection ,174. In In spite of these multiple interactions, how co-infections with multiple HHVs affect γδ compartments is not known. Analysis of γδ T cells’ cross-reactivity against different HHVs is reported in few instances ,168,188.The possible cross-reactivity between HHV-1/2-reactive and EBV-reactive γδ is another open question. Vδ2 cells are possibly targeted in both contexts, and PhAg-reactive cells are involved in both cases. To our knowledge, an analysis of possible cross-reactivity of Vδ2 cells against cells infected by these viruses is not reported.pos/Vδ2neg ratio. This may precede inversion of the CD4/CD8 ratio [The effects of HIV on γδ have been reviewed recently ,191. AloD8 ratio . ImportaD8 ratio : it is rD8 ratio ,195. TheD8 ratio , and PhAD8 ratio . Vδ2 areD8 ratio . HoweverD8 ratio . It is nD8 ratio ,119,120.D8 ratio ,163,164.neg cells in HIV patients did not show clear differences with healthy controls, arguing for an unbiased polyclonal or oligoclonal amplification [neg counts in some ECs do not correlate with the presence of HIV in blood, but parallel increased HIV replication in gut [Paralleling Vδ2 depletion, a persistent increase in Vδ1 T cells occurs in the blood of HIV-viraemic patients, and may start prior to seroconversion ,200. Thification ,203. Indfication ,204; andSeveral factors possibly contributing to Vδ1 expansion have been identified. (a) A stimulation by opportunistic pathogens is possible, as the highest Vδ1 counts were observed in the context of co-infection by Candida albicans ; in vitr+ NK cells expand while NKG2A+ NK cells are lost in transplant patients, but also in healthy CMV+ children [+ NK cell expansion [+ Vδ1 cells decreases, while NKG2C+ Vδ1 cells increase in number and display higher cytolytic activity on HLA-E-expressing targets [pos patients had mainly NKG2Aneg Vδ1 cells, this was also the case in control CMVneg transplant patients [Most HIV patients carry HHVs subclinically. One study followed viral shedding in semen by HIV-infected men who have sex with men (MSM), in a 48-week follow-up. HHV secretion was found in 94% of cases, with the following percentages for different viruses: 85% (CMV), 81%(EBV), 35% (HHV-7), 29% (HHV-6) and 26% (HSVs) . CMV waschildren ,219,220.children ,222,223.children . In compxpansion ,226. NKGxpansion . In the  targets ,214,228.patients ,229; in patients ; and in patients . Thus, bThe effect of CMV in HIV infection seems detrimental, as CMV-seronegative individuals recover better after ART . In longneg cells. Vδ3 cells, which are almost absent in blood, represent a significant population in liver. Whereas one study reported the predominance of Vδ2 cells [lo/CD45RAhi/Vδ2neg cells (Vδ1+ or Vδ3+) which express the markers of tissue-resident CD69, CXCR3 and CXCR6, which are absent from blood. These cells represent liver-restricted clonotypes, and are possibly involved in liver tissue immunosurveillance [lo/CD45RAlo population contains highly expanded clonotypes which are present in blood, and thus circulate between both compartments. Very little sharing of clonotypes between donors was observed, and most sequences were essentially private [γδ T cells are enriched in liver tissue compared to peripheral blood ,236,237,Hepatitis B and C viruses  infect approximately 250 and 70 million persons worldwide, respectively, and are known to provoke liver cirrhosis, liver failure and hepatocellular carcinoma. HBV infection is cleared in 95% of healthy adults. In contrast, 60 to 80% of HCV-infected patients progress toward a chronic liver disease . Spectratyping of the Vδ1 TCR CDR3 regions of liver-infiltrating cells did not reveal oligoclonality [neg compartment may not be specifically due to HCV infection, and it has been suggested that it may rather reflect a homeostatic response to liver inflammation [An increase in blood γδ cells occurs in chronic HCV patients ,240,241,lonality ,244. A clonality . These rThe Vδ2 cell compartment may also be altered in chronic HCV patients. Two studies indicate a decreased percentage and count of Vδ2 cells in blood ,247. Theneg subsets are reported in asymptomatic HBV carriers compared to healthy controls [During HBV infection, γδ T cell subsets frequencies are not dramatically altered in blood. Increased levels of both Vδ2 and Vδ2controls . In a stcontrols . In chrocontrols ,251 or tcontrols . A decrecontrols ,250,252,+ or HBV+ liver transplant patients, the predominance of the Vδ1 subset in blood γδ T cells has been observed in CMV+ but not EBV+ patients (on a serologic status basis) [+ patients Vδ1 cells displayed mostly a terminally differentiated phenotype, as reported in persistent CMV infections. However, a link with CMV carriage was also seen in immunocompetent patients, in whom the presence of CMV-DNA has been examined in liver tissue by PCR. CMV infection of the liver was frequent in HBV+ (52%) and HCV+ (36%) patients. Interestingly, the presence of CMV-DNA correlated differently with liver damage in HCV+ or HBV+ livers, estimated from ALT and bilirubin levels: while CMV-DNA was associated with decreased ALT and bilirubin in the case of HBV, the reverse was observed in HCV patients, arguing for a detrimental effect of CMV in HCV infection, but not in the case of HBV [In HCVs basis) . In HCV+e of HBV . Thus, CAs with HIV infections, NK responses provide evidence that CMV modulates the responses to HBV/HCV. In co-infections, CMV seropositivity determines the expansion of highly differentiated NK cells expressing NKG2C and cytotoxic markers, and is not associated with adverse clinical outcomes, suggesting a role in limiting immunopathology . neg cells compared to age- and sex-matched healthy controls. Segregation of patients and controls according to CMV serology revealed that high Vδ1 frequency was associated with combined HEV infection and CMV seropositivity. Again, high Vδ1 levels were associated with a higher proportion of Vδ1 cells with a TEMRA phenotype. In vitro, γδ T cells from patients were found to upregulate CD69 activation markers when stimulated with HEV-infected hepatocarcinoma cells, and this was selective for CMV carriers. This concerned Vδ2neg and Vδ2pos cells, although the response of the two subsets correlated negatively. HEV infection in CMV carriers was also associated with an increased concentration of IL-10 in the serum. This indicates that CMV carriage is determinant for the expansion of Vδ2neg cells, although both Vδ2neg and Vδ2pos subsets may be involved in the response to HEV. The data also suggest a possible role in limiting immunopathology through IL-10 production by uncharacterised cells. HEV-infected hepatocarcinoma cells also induce the upregulation of IL-10 production by co-cultivated γδ cells, suggesting alternatively that this production could provide an immune escape mechanism for HEV by dampening the activity of antiviral effectors. Strikingly, a skewing of Vδ2pos/Vδ2neg proportions was not observed in immunosuppressed transplant patients at the acute or chronic phase [pos (CD27−CD45RA−) and Vδ2neg cells  was observed, suggesting their recruitment in tissues, as reported in the case of chronic HCV patients [HEV infection is usually benign and asymptomatic, and the infection is naturally cleared in immunocompetent individuals. It becomes a concern in the context of solid organ transplantation, as about 2/3 of patients progress towards chronic infection . Our groic phase . Neverthpatients .The mobilisation of γδ subsets in acute viral infections in humans and animal models has been extensively reviewed, and we will discuss here the findings relevant to HHV co-infection and cross-protection conferred by different microbes ,17,257.Following influenza virus infection in mice (reviewed in ), succesnegVδ1neg γδ cells. Remaining Vδ2 cells displayed markers of activation, suggesting activation and traffic to the lung, similar to what was observed in influenza infection in mice. Again, the reactivation of EBV in severe COVID-19 disease is a factor of morbidity and complications, but the reactivation of other HSVs can also affect the course of disease [The contribution of HHV carriage is also questionable in the context of SARS-CoV and SARS-Cov-2 infections. These viruses cause an acute respiratory infection of variable severity, and Vγ9Vδ2 T cells are mobilised in both cases ,263. In  disease ,267,268.Alterations of γδ subsets in malaria have been reviewed recently ,270,271.-DR-), a TEMRA phenotype (CD45RA+) and frequent expression of CD8. Vδ1 cells in malaria thus partly phenocopy Vδ2neg γδ T cells that expand during CMV expansions [neg cells proliferated in vitro against crude Pf antigens in the presence of IL-2, produced IFNγ as well as IL-10 and were associated with high concentrations of IL-10 in plasma, suggesting anti-inflammatory potential and a role in the control of chronic inflammation. Vδ1 cells from infected donors also expanded and secreted IL-10 in the absence of Pf antigens when IL-2 was present, presumably primed, in vivo, by an unknown stimulus [In highly endemic areas, the main observable perturbation in the γδ T cell compartment is an expansion of Vδ1 T cells displaying resting  or if they exclusively respond to CMV. It will be important to study whether different HHVs induce or modulate alternative γδ responses and whether these are beneficial or not to the host, with the ultimate goal to determine whether HHVs should be considered symbiotic microbes.


Objects: To analyze the characteristics of apparent diffusion coefficient (ADC) and R2* map. DW imaging acquired ADC values by combining a single-shot spin-echo echo-planar imaging pulse sequence and the additional motion probing gradient pulses along the x,y, z-axes with two b values:0 and 200, as well as 0 and 800 s/mm2. ATIN patients performed DW and BOLD magnetic resonance at renal biopsy(T0) and the third month(T3). We assessed the pathological changes semiquantitatively, and conducted correlation analyses within functional MR, pathological and clinical indexes.Four acute TIN, thirteen chronic TIN patients, and four controls were enrolled. We used multiple gradient-echo sequences to acquire 12 T2*-weighted images to calculate the Rb was 0,200 s/mm2, 2.86 ± 0.19 vs. 3.39 ± 0.11, b was 0,800 s/mm2, 1.76 ± 0.12 vs. 2.16 ± 0.08, P < 0.05) than controls, showing an obvious remission at T3. Cortical and medullary R2* values  were decreased, significant difference was only observed in MR2*. No relationship was found between functional MR and histopathological indexes.MR2* had a close relationship with eGFR  and serum creatinine. Patients with lower ADC when b was 0,200 s/mm2 showed more increase of ADC and MR2* after therapy. In CTIN group, lowered MR2* and MR2*/CR2* provided evidence of intrarenal ischemia. CTIN with advanced CKD (eGFR< 45) had significantly lower ADCb200 value.In ATIN, ADCs were significantly lower(2* values in ATIN case series. ATIN patients had concurrently decreased ADCb800 and MR2*. The pseudo normalization of CR2* with persistently low MR2* in CTIN suggested intrarenal hypoxia.We observed the reduction and remission of ADC and R Functional magnetic resonance (fMR) imaging has recently grown to be a useful tool to evaluate real-time renal function . The funb-value) range. It is regarded as a marker sensitive to alterations in the interstitium, for example, interstitial edema with inflammatory cells infiltration or fibrosis, perfusion, and water handling in the tubular compartment. DW magnetic resonance imaging (MRI) helps diagnose acute renal transplant dysfunction . Thus, the ratio of MR2* to CR2* was 1.68 ± 0.03.In control kidneys Fig. , the out3 mm3. ADC values were obtained in DWI both when b value was 0, 200 s/mm2 and 0, 800 s/mm2, and all were found to have significant decreases as 18.5, 15.6% respectively than the control group . The MR2* values firstly went back to a level similar to control, while the CR2* values had not yet fully recovered (92.9% of control). The MR2* to CR2* ratio (M/CR2*) was 1.39 ± 0.13, 1.8 ± 0.52 respectively, in the acute and remission stage. For CTIN patients  and interstitial changes  when compared separately. We also found no relationship within ADC values, R2* values, AI and CI. It seems that ADC and R2* values changed along with that of renal function , and Scr . The association was similar in CTIN patients  or all the TIN patients , but differences were not significant within subgroups  in ATIN patients, while had no such relationship in CTIN. We noticed that significantly lowered ADCb200 values occurred in advanced CKD patients with eGFR< 45 ml/min . We could figure out from Fig. b800 and MR2* (green dots), distinguished from other diseases. Further analysis revealed that ATIN Patients with lower ADCb200 were correlated with more ΔADCb200  and ΔMR2*  after therapy. There was also a relationship between ΔADCb200 and ΔMR2* . However, renal prognosis analysis among candidate predictive markers showed no relationship between time-point ADC or R2* values and eGFR. But as MR2* has a significant correlation to eGFR and Scr levels, it was speculated that a lower ADCb200 value could predict the degree of ADC and MR2* recovery after therapy, which meant a better restoration of renal function.Correlation analysis disclosed that neither ADC values nor Ron Table  in ATIN ups Fig. . ADCb800With MR techniques and clinical applications’ developments, it is possible to make a noninvasive assessment of renal pathology types by fMR imaging in patients with kidney diseases. However, at present, there is little evidence directly from kidney histopathology –31. In tb values) in ATIN patients self-pre-and post-therapy. The delayed recovery of ADC, especially when b was 0, 800 s/mm2 in the remission stage, meant that kidney injury was still in repair or left behind chronicity. The decrease of ADC  in CTIN patients was mild, suggesting its insensitivity to fibrosis. The finding relationship of albuminuria with ADCb800 shares the same view of previous studies [b200 served as a factor determined by perfusion and interstitial changes, showed better recognition between mild and severe CKD (eGFR< 45) in our CTIN patients. This result was in line with a recently published study by Buchanan CE et al. [Diffusion-weighted MR imaging generates the ADC value as an index reflecting the microenvironment of diffusing water molecules. It is considered a simple marker to reflect tissue microstructure. In renal dysfunction, tubular injuries lead to a reduced water reabsorption process resulting in decreased diffusion . Factors studies , 35. OnlE et al.  that ADC2* value of BOLD MR imaging has been regarded as a factor reflecting tissue oxygenation [2* was decreased while MR2* remained constant [2* and eGFR. CR2* and MR2* were decreased and in remission simultaneously in ATIN, demonstrating that inflammation-relating tubular injury was diffusely distributed in the interstitium. The reduction of MR2* to CR2* ratio detected in ATIN and CTIN declared the involvement of ischemic factors leading to tubular injury. Interestingly, MR2* was persistently low against the “pseudo normalization” of CR2* with a declining M/CR2* ratio, indicated the existence of chronic intrarenal ischemia and hypoxia despite a relatively sufficient oxygen supply than consumption. Intrarenal hypoxia was later emphasized by Sugiyama K et al. that reduced oxygenation but not fibrosis defined by functional MRI predicts the long-term progression of CKD [2* with ADCb800 in ATIN patients [As we have known, the Rgenation . Intraregenation . The chaconstant . This isconstant . Renal hn of CKD . From thpatients .Our study’s limitation was the disability to get direct information on intrarenal perfusion. DW and BOLD imaging were the most frequently used fMR imaging techniques in human diseases. Combining these data and developing new strategies would provide more accurate methods to diagnose, assess, and evaluate the mechanism of diseases , 29. A r2* in the ATIN case series. The ADCb800 and MR2* were concurrently decreased. Thus, further sample recruitment is essential to verify the diagnosis significance of these functional MR parameters and test sensitivity. The pseudo normalization of CR2* with persistently lowered MR2* in CTIN provided evidence of intrarenal hypoxia.This study observed significant reduction and remission of ADC values and R


Thirty-six male Sprague–Dawley rats were randomised into sham operation, SAP and rhein groups. The SAP model was induced by retrograde pancreatic bile duct injection of sodium taurocholate. Serum TNF-α and interleukin (IL)-6 levels were determined by ELISA, whereas serum amylase and lipase concentrations were measured using test kits. Western blot and/or immunohistochemistry quantified JAK2 and STAT3 expression. Furthermore, histopathological pancreatic changes were detected by haematoxylin and eosin staining. AR42J cells were randomly divided into the control, cerulein and rhein groups. Amylase activity was assessed using an amylase test kit; the tumour necrosis factor-α (TNF-α) expression was determined by enzyme-linked immunosorbent assay (ELISA). JAK2 and STAT3 protein expression were evaluated by western blot. SAP was concomitant with increased JAK2 and STAT3 expressions in vivo. Pre-treatment with rhein attenuated serum TNF–α and IL-6 levels effectively, and notably reduced p-JAK2, p-STAT3, JAK2 and STAT3 protein expression. Rhein significantly alleviated pancreatic histopathology. Compared to untreated groups, rhein significantly reduced amylase activity in supernatants of AR42J cells induced by cerulein in vitro. Furthermore, rhein altered JAK2 and STAT3 protein levels in AR42J cells after cerulein induction. Overall, rhein exerted protective effect on SAP in vitro and in vivo, possibly through the JAK2/STAT3 signalling pathway.Rhein is widely used in inflammation treatment in China, but its effects on severe acute pancreatitis (SAP) have not been studied closely. This study investigated rhein’s protective effects against SAP using  Acute pancreatitis is an acute inflammatory disease that severely affects health and threatens life, and about 30% of patients with acute pancreatitis progress to severe acute pancreatitis (SAP) . About 1α (TNF-α) and interleukin (IL)-6 are important transduction factors in the JAK2/STAT3 signalling pathway that can induce the inflammatory cascade, enhance the inflammatory effect and even lead to organ damage and multiple organ dysfunction syndrome in SAP  was purchased from the National Institute for Food and Drug Control , the purity by authorization >98%. Sodium taurocholate (Cat. No. T4009), cerulein (Cat. No. C9026) and 5% carboxymethylcellulose sodium (CMCS) (CAS number:9004-32-4) were purchased from Sigma-Aldrich Merck KGaA . Amylase kits (Cat. No. C016-1-1) were purchased from Nanjing Jiancheng Institute of Bioengineering . Foetal bovine serum (Cat. No.10099-141), F12K medium (Cat. No. C11330500BT) and trypsin (Cat. No. 25200-056) were purchased from Gibco Ltd. (United States). PCR kits (Cat. No. RR820Q) were purchased from TaKaRa Corporation (Japan). GoScript Reverse Transcription Kits (Cat. No. A5001) were purchased from Promega (United States). Phosphatase inhibitors (Cat. No. P1081), SDS–PAGE gel preparation kits (Cat. No. P0012A) and ECL hypersensitive colour development kits (Cat. No. P0018AS) were purchased from Shanghai Biyuntian Biotechnology .2. The AR42J cells were divided into three groups: control group, cerulein group (cerulein 10–8 mol/L)  supplemented with 20% Australian foetal bovine serum , 100 U/ml penicillin and 100 mg/l streptomycin  at 37°C and 5% CO8 mol/L)  and rhein = 12), rhein (dissolved in 0.5% CMCS) treatment group (n = 12). All rats were maintained under standardised conditions on a 12 h light/dark cycle and allowed free access to food and water. The rats fasted for 12 h before the operation, however, drinking water was available ad libitum. The SAP model was induced via a standardised pressure-controlled infusion of 3.5% sodium taurocholate  into the bile–pancreatic duct, and the common hepatic duct was closed with a small clamp for 5 min as previously described  were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd. SD rats were randomly divided into three groups: sham operation group (n = 12), SAP model group , lipase (Cat. No. A054-2-1) and myeloperoxidase (Cat. No. A044-1-1) were purchased from Nanjing Jiancheng Institute of Bioengineering . Serum amylase, lipase and myeloperoxidase concentrations were measured using test kits according to the manufacturer’s instructions. The amounts of TNF-α (Cat. No. E-EL-M0049c) and IL-6 (Cat. No. E-EL-R0015c) were determined using ELISA kits  according to the manufacturer’s instructions.g at 4°C for 15 min. Supernatants were harvested, and the BCA protein kit was used to determine each protein sample’s protein content. SDS–PAGE gels  were prepared. After electrophoresis, the protein was transferred to a PVDF membrane, which was then sealed using 5% BSA for 2 h at room temperature. The following primary antibodies were added and incubated at 4°C overnight: JAK2 , STAT3 , p-JAK2 , p-STAT3  and GAPDH . The membranes were then washed with Tris-Buffered Saline Tween buffer three times for 10 min and incubated with horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG secondary antibody  for 2 h at room temperature. Afterwards, the membranes were washed with Tris-Buffered Saline Tween buffer three times for 10 min. Ultra-sensitive ECL luminescence solution was then added to detect the proteins, and a Fluorchem FC3 gel imager was used for detection. ImageJ 1.52a software was used to determine the gray value of the band. .RIPA lysis buffer was added to pancreatic tissues and centrifuged at 13,700× The rat pancreatic tissues were fixed in 4% paraformaldehyde and embedded in paraffin wax for routine sections. The sample was cut into 5 µm thick sections. After staining with haematoxylin and eosin, the sections were photographed under the microscope  at ×200 magnification for image processing and analysis. Pancreatic histopathology was scored by the double-blind method . The meaThe methods of histological score of pancreatic injury were as follows: Edema: 0, absent; 1, focal expansion of the interlobar septa; and 2, diffuse expansion of the interlobar septa. vascular: 0, absent; 1, congestion; 2, congestion plus local interlobar or intralobar hemorrhage; 3, multifocal diffuse hemorrhage; and 4, vascular fibrinoid necrosis or thrombosis. Fat necrosis: 0, absent; 1, focal dissolution of the interlobular or peripancreatic fat; and 2, diffuse dissolution of the interlobular or peripancreatic fat. Acinar necrosis: 0, absent; 1, <10% patchy necrosis of the edges of the lobules; 2, 10–30% patchy, peripheral necrosis of the lobules; 3, <30% confluent lobules necrosis; 4, <50% confluent lobular necrosis; and 5, formation of microabscesses. Calcification: 0, absent; 1, focal in fat or acinar necrosis; and 2, diffuse in fat or acinar necrosis.The rat pancreatic tissues slices were dewaxed with xylene, dehydrated with an ethanol gradient, and then placed in a sodium citrate buffer solution for 15 min. The slices were cooled for 30 min and then treated with 3% hydrogen peroxide for 10 min at room temperature. After three washing with PBS , shook off the PBS before adding antibody to each slice. The following antibodies were added and incubated at 4°C overnight: p-JAK2 , p-STAT3 , After three washings with PBS, the HRP-labelled goat anti-rabbit IgG secondary antibody was added and incubated at room temperature for 1 h. The tissue sections were then covered with DAB solution. After colour development, the sections were rinsed with water. Hematoxylin stained for 1 min 20 s, then soaked in tap water, alcohol fractionated in 1% hydrochloric acid for 1 s, soaked in tap water and rinsed under running water for 7–8 min, dried at 65° for 30 min, then taken out and cooled to room temperature and sealed in neutral gum. Image processing and photography, two different observers calculated the positive areas.Rhein was preprocessed by the Ligprep 3.6 program  applying OPLS_2005 force field before molecular docking, with Epik 3.4  to generate the proper protonation states at pH 7.0 ± 2.0. A restrained minimization of the crystal structure was performed to reorient side-chain hydroxyl groups before further processing. The DQX pocket of 3KRR was selected to define and generate the receptor grid. In silico docking was performed by Glide 6.9  in standard precision (SP) with default values for other parameters.p < 0.05 was considered statistically significant.Statistical Analysis Statistical analyses of the obtained experimental data was carried out using SPSS version 21.0. ImageJ 1.52a software was used to calculate the greyscale value. Significance between groups was evaluated by one way analysis of variance (ANOVA) followed by a Tukey post hoc test. Data are expressed as mean ± SD, and The network pharmacology results of rhein with JAK2 molecule.3KRR is a Crystal Structure of JAK2 complexed with a potent quinoxaline ATP site inhibitor . The pocp < 0.001). After rhein treatment, the levels of serum amylase, serum TNF-α, serum IL-6, myeloperoxidase and serum lipase were significantly decreased in the rhein group compared with the SAP group (p < 0.001).p < 0.01). Compared with the SAP group, the pathological scores were significantly decreased in the rhein group (p < 0.05).p < 0.001). After rhein treatment, the activity of p-JAK2 and p-STAT3 were significantly decreased compared with the SAP group (p < 0.001). p < 0.001). This increasing trend was significantly reversed by treatment with rhein (p < 0.001). These phenomena suggest that four proteins closely related to the JAK2/STAT3 signalling pathway are activated in the pancreatic tissue of SAP rats.in vitro cell model mimicking SAP by treating the AR42J cells line with cerulein to stimulate pro-inflammatory cytokine expression. p < 0.001), to gradually decrease at 6, 12 and 24 h. Therefore, we chose 4 h as the checkpoint for sample collection of cells in the succeeding experiments. These results suggested that the AR42J cell injury model was successfully established.We constructed an p < 0.001). When rhein was added to the supernatants of cells at concentrations of 0.25, 0.5 and 1 μg/ml 20 min before adding cerulein, the amylase level downregulated in a concentration-dependent manner (p < 0.001). p < 0.001). This increasing trend was significantly reversed by treatment with rhein in a concentration-dependent manner (p < 0.001). These results suggested that rhein protected the injured AR42J cells.p < 0.001). After rhein treatment, the activity of p-JAK2, JAK2, p-STAT3 and STAT3 were significantly decreased compared with the cerulein group (p < 0.001). The expression of p-JAK2, JAK2, p-STAT3and STAT3 proteins at the middle and lower doses in the rhein group differed significantly from those at the high dose. These results suggest that rhein could inhibit the expression of protein p-JAK2, JAK2, p-STAT3and STAT3, which are important components in the JAK2/STAT3 signalling transduction pathway induced by cerulein in AR42J cells.Effect Of Rhein On JAK2 and STAT3 Expressions In The AR42J Cell Injury Model In the current study, we found that rhein significantly alleviated pancreatic histopathology, attenuated proinflammation factors, and inhibited JAK2/STAT3 signalling pathway in SAP rat model. In SAP cell model, rhein significantly decreased the expression of amylase and TNF-α induced by cerulein, and inhibited JAK2/STAT3 signalling pathway.The pathogenesis of SAP seems to be related to multifaceted pathological processes, involving inflammation, parenchymal acinar cell death by necrosis, and cellular damage in the pancreas. We established SAP rat models by retrograde pancreatic bile duct injection of sodium taurocholate . HoweverTraditional Chinese medicine has rich experience in the prevention and treatment of SAP. At present, it has been reported that Dachengqi Decoction plays an important role in reducing the complications of SAP in patients . In SAP Sennae folium, Semen cassiae, and Polygonum multiflorum, and it is widely used in clinical practice (At present, natural products with low side effects and efficacy have been considered for the treatment of SAP. In SAP model, huperzine II treatment was able to increase antioxidant, anti-inflammatory and anti-apoptotic activity , possiblpractice . These mNowadays, rhein is widely used in inflammation treatment in China . In thisAfter rhein treatment, inflammatory cytokines were decreased, and JAK2/STAT3 signalling pathway related proteins were also downregulated, suggesting that rhein plays an anti-inflammatory role by inhibiting JAK2/STAT3 signaling pathway. In addition, HE staining is another method frequently used in evaluation of pancreatic injury . In the −8 mol/L of cerulein for 24 h. Then the model was tested by inflammatory cytokines and JAK2/STAT3 signalling pathway related proteins at 4 h. These alterations can be attributed to the action of cerulein. In this work, amylase and TNF-α activity in the SAP group were significantly upregulated compared with the control group, and p-JAK2, JAK2, p-STAT3 and STAT3 proteins expression were significantly increased in the SAP group compared with the control group. The above-mentioned findings indicated that SAP cell model was successfully established. After rhein treatment, inflammatory cytokines were downregulated, and JAK2/STAT3 signalling pathway related proteins were also decreased, suggesting that rhein plays an anti-inflammatory role by regulating JAK2/STAT3 signaling pathway.According to published many literatures about rhein anti-inflammatory mechanism, such as the interactive relationships of rhein on multiple inflammatory signaling pathways and cellular processes  The SAP in vivo and in vitro. Our findings indicate the potential therapeutic effects of rhein on SAP.In summary, rhein not only inhibits the release of inflammatory factors but also alters the expression of JAK2/STAT3 signal pathway-related proteins in the SAP model in vivo and in vitro. This effect may be related to the regulation of JAK2/STAT3 signal pathway. This study will be helpful for the treatment of SAP.Taken together, our data provided evidence that rhein plays therapeutic role by inhibiting therelease of inflammatory factors in the SAP model


Lipolytic bacteria in this study tended to grow and achieved a high lipase activity at temperatures of 35–40 °C and at pH 6–7, reaching a peak of 480 U/mL and 420 U/mL produced by Lysinibacillus PL33 and Lysinibacillus PL35, respectively. These potential lipase-producing bacteria are excellent candidates for large-scale applications in the future.Compared to lipases from plants or animals, microbial lipases play a vital role in different industrial applications and biotechnological perspectives due to their high stability and cost-effectiveness. Therefore, numerous lipase producers have been investigated in a variety of environments in the presence of lipidic carbon and organic nitrogen sources. As a step in the development of cultivating the unculturable functional bacteria in this study, the forest soil collected from the surrounding plant roots was used to create an artificially contaminated environment for lipase-producing bacterial isolation. The ten strongest active bacterial strains were tested in an enzyme assay supplemented with metal ions such as Ca Lipases, which belong to the family of triacylglycerol ester hydrolase, can catalyze the hydrolysis and synthesis of esters formed from glycerol and long-chain fatty acids without the addition of cofactors . Due to Bacillus genus are of interest in biotechnology [Compared to lipases from plants and animals, microbial lipases are increasingly valuable and preferable due to the diverse substrates used, high yield production, greater stability, shorter generation time, simple genetic manipulations, and lower production costs. Moreover, microbial lipases as multipurpose biological enzymes are of significant interest for various applications in biological and industrial processes including food and drink, leather, detergents, cosmetic, textile, agrochemicals, the pharmaceutical industries, and waste treatment ,6,7,8,9.chnology ,11. The chnology ,13.Achromobacter, Alcaligenes, Arthrobacter, Bacillus, Burkholderia, and Pseudomonas [Numerous bacteria have been investigated as lipase producers, which were isolated from diverse habitats such as oil-contaminated soil, industrial waste, dairy plants, oil-processing factories , and froudomonas . Howeverudomonas . In the udomonas .In the present study, lipolytic bacteria were identified using a modified dependent-culture method to attract multifunctional bacteria from soil. The growth and lipase production of the strong multi-enzyme-producing bacterial strains were optimized prior to its evaluation at extreme conditions. The most promising candidates will be applied to the treatment process of food waste leachate.2+ 0.005; Cu2+ 0.01, Fe2+ 0.01, Mn2+ 0.01, zipper bag 0.5 (Low Density Polyethylene- LDPE), and olive oil (10 mL), and was stirred at 120 rpm and ambient temperature for one month. One milliliter of the surviving bacteria in the culture was transferred to fresh LB medium containing autoclaved extracted soil (AES) prepared by adding 100 g soil to 1000 mL distilled water, which was adjusted to a pH of 7 before autoclaving [Soil samples were collected from the root-surrounding area of the forest soil at Kyonggi University, Suwon, South Korea. One of the targets in the isolation steps was to encourage bacteria that are capable of tolerating extreme and contaminated environmental conditions such as heavy metal-contaminated, oil-contaminated, and plastic-contaminated soil and water. Therefore, a modified cultivation method was artificially designed to mimic polluted environmental conditions. Ten grams of soil was added to 100 mL of artificial medium containing (g/L): NaCl 10; Cdoclaving . Pure co2·2H2O (0.1), and agar (20) in distilled water was sterilized by autoclaving for 15 min . After cooling the medium to 45 °C, 10 mL of Tween-80 was added as the final step. All microbial agar plates and the bacteria on tween-agar plates were incubated at 37 °C for 48 h. The enzyme activity was then determined based on the visual precipitation of calcium salts caused by the fatty acids released from the hydrolysis reaction [The culture medium containing (g/L): peptone 10, NaCl 5, CaCland agar  in disti2 1, and agar 15 in distilled water. The medium was adjusted to a pH of 7.4 using 0.1 M NaOH. After autoclaving for 15 min  and cooling to 60 °C, 10 mL of olive oil and 10 mL of phenol red (1 mg/mL) were added. Bacteria on phenol red plates were incubated at 37 °C for 48 h. Because of the presence of fatty acids, a slight decrease in pH changes the phenol red color from pink to yellow, which is an indicator of the bacterial lipase activity [Phenol Red agar (pH 7.4) containing olive oil as a substrate for lipase-producing bacteria was prepared by dissolving (g/L) peptone 5, yeast extract 3, CaCl 15 lb, 1 °C and cTo optimize the pH value and temperature, bacterial culture medium containing (g/L): peptone (5) and yeast extract (5) was dissolved in distilled water and autoclaved for 15 min , and 10 mL of olive oil was added after cooling the medium to 60 °C.For optimal pH screening, bacterial cultures were exposed to different pH values  and incubated at 30 °C. For optimal temperature screening, samples were incubated at different temperatures ranging from 5 to 75 °C  at pH 7. Produced enzymes were withdrawn at 8 h intervals over a period of 72 h.8 CFU/mL in a medium (pH 7) containing 1% of each type of carbon source, 5 g yeast extract, and 5 g NaCl. Lipase, considered as an inducible enzyme, is affected by various carbon sources . In addiDifferent nitrogen sources (1%) such as yeast extract, casein, peptone, ammonium nitrate, and potassium nitrate were added to the medium (pH 7) containing 5 g lactose and 5 g NaCl. All bacterial cultures were incubated at 30 °C and 120 rpm for 72 h. 2+, Zn2+, Cu2+, Fe2+, Mg2+, K+, Co2+, Mn2+, and Sn2+ at a concentration of 1mM were added to the enzyme solution to determine their effect on lipase activity [Metal ions such as Caactivity . After 1Bacterial cultures were established to study the effect of agitation: samples were incubated in a shaking incubator at 120 rpm, 150 rpm, and 160 rpm, and the other was maintained under static conditions at the same temperature of 30 °C.After optimizing the pH, temperature, carbon and nitrogen sources, bacterial strains were cultured in a fermentation medium that contained (g/L): yeast extract 5, NaCl 5, and glucose 5 at 120 rpm for 72 h. After 72 h, the bacterial cultures were centrifuged at 10,000 rpm for 10 min at 4 °C. Extracellular enzymes in the supernatant were used for enzymatic assays. Lipase activity was determined spectrophotometrically using p-nitrophenol palmitate (pNPP) as a substrate. The reaction mixture contained 700 μL pNPP solution and 300 μL lipase enzyme solution. The pNPP solution was prepared by mixing solution A (0.001 g pNPP in 1 mL of isopropanol) and solution B . After incubating at 30 °C for 2 min, the absorbance of the mixture was immediately measured at 410 nm. One unit of lipase activity was defined as the release of 1 µmol of free p-nitrophenol per minute under the test conditions . The enzhttp://ezbiocloud.net/ (accessed on 13 April 2021).) [The 16S rRNA gene sequencing was performed on the genomic DNA extracted from the strong lipase-positive bacterial strains using the InstaGene Matrix kit  according to the manufacturer’s instructions. As described by Frank et al. , amplifi 2021).) .Bacillus, Lysinibacillus, Paenibacillus, Brevibacillus, Solibacillus Stenotrophomonas, Enterobacter, Klebsiella, Acinetobacter, Sporosarcina, Viridibacillus, Burkholderia, Exiguobacterium, Vagococcus, Bhargavaea, and Leclercia compared to that of the popular Bacillus and Paenibacillus genera investigated from the traditional medium , Paenibacillus PL2 (19 mm), Bacillus L8 (18 mm), and 17 mm for Bacillus FW2, whereas Stenotrophomonas N-PL7 displayed the lowest lipolytic activity (10 mm)  .Lysinibacillus PL33, reaching 480 U/mL, followed by C with 420 U/mL, and Bacillus L8 with 390 U/mL at 35 °C. The strain Paenibacillus PL2 secreted a maximum of 400 U/mL, followed by strain Bacillus FW2, which produced 380 U/mL at 40 °C. Optimal lipase production by strains Paenibacillus L2 and Stenotrophomonas N-PL7 recorded at 30 °C reached 290 U/mL and 170 U/mL, respectively. Most of these bacterial strains grew and survived at 10 °C, except for strains Enterobacter PL4, Paenibacillus L2, and Enterobacter L7. Lysinibacillus PL33 and Bacillus FW2 were the strongest bacterial strains with lipase activity at 10 °C compared to the other strains and produced 120 U/mL. Enterobacter L7 and Stenotrophomonas N-PL7 did not grow at 40 °C and 45 °C, respectively. Other bacterial strains produced inadequate enzymes at 45 °C , followed by Bacillus L8 (240 U/mL), and the same amount of 190 U/mL for both strains Bacillus FW2 and Enterobacter L7. However, the supplementation of potassium nitrate and ammonium nitrate in the culture medium did not significantly affect lipase activity compared to the other nitrogen sources  lipase activity compared to that of the control in strains Bacillus FW2, Lysinibacillus PL35, Lysinibacillus PL33, and Enterobacter L7. Mg2+ stimulated the lipase effect on Bacillus L8, Paenibacillus L2, and Bacillus N-PL4. Mn2+ and Fe2+ improved lipase generation of Lysinibacillus PL33, Lysinibacillus PL35, Bacillus FW2, Bacillus L8, and slightly accelerated the enzymatic activity of Bacillus N-PL4 and Enterobacter L7. The results illustrated that Cu2+, Zn2+, Co2+, and Sn2+ inhibited the enzyme activity of all bacterial strains in this study, which demonstrated competitive inhibition with the enzyme, thereby reducing their catalytic activity.Lipase production, under shaking conditions in all bacterial strains, observed a 1.3–1.7 folds increase compared to static conditions by improving the rate of oxygen transfer, leading to easy dispersal of oil micelles into the microbial cell. The result of this study showed the similar trend to the previous studies .Lysinibacillus and Paenibacillus so far [The published closest bacterial strains of ten strains in this study were investigated as non-lipase-producing bacteria. Moreover, there were limited studies working on the ability of lipase production of the members from the genus s so far ,30,31,32Lysinibacillus PL33 and Bacillus FW2 were investigated as strong candidates that were capable of producing lipase at 10 °C that reached the goal of the research. In other studies, temperatures of 50 °C and 55 °C were considered optimum for effective lipase activity [Bacillus sp. produced the highest lipase at 60–80 °C [Janibacter sp. R02 has an optimal point at 90 °C [In general, the temperature for optimal lipase production rate is approximately 30–35 °C. However, other studies have demonstrated that bacteria may secrete the highest lipase enzyme at either low or high temperatures, such as at 10 °C or 40 °C ,36. One activity ,38,39. H60–80 °C ,42,43. Wat 90 °C .Bacillus species in this study were able to produce lipase at pH 5. Another Bacillus sp. that exhibited maximum lipase activity at pH 5.6 was also investigated in another study [Pseudomonas and Enterococcus sp. produced lipase at acidic conditions of pH 3.5 and 4.6, respectively [Bacillus species [er study . In partectively ,47. In t species ,49,50,51It was demonstrated that carbon and nitrogen sources have a significant effect on lipase-producing efficiency. The remarkable point in most studies was the fact that the presence of lipidic carbon sources such as oils and fatty acids enhanced the lipase activities . Such Tw2+ and Ca2+ have been explored as the positive effect on lipase activity. It was explained that their contribution as the requirement of metalloprotein and the increase of thermal stability with the supplement of Ca2+ due to the presence of more binding sites [2+ partially reduced lipase activity in other studies [+ had no significant effect on the enzyme activity of Bacillus sp. [2+ can stimulate lipase activity [2+ was an inhibitor of lipase in Bacillus sp. [2+, Co2+, Zn2+ inhibition [Metal ions play a critical role in maintaining the enzyme structure and activity . As explng sites . However studies ,61,62. Nllus sp. . It was activity  but in cllus sp. ,65. The hibition ,66.An agitation speed of 150 rpm was found to be optimal for lipase activity due to enhanced oxygenation in the culture medium . HoweverConclusively, a modified culture method was introduced in this study investigating functional bacterial candidates that can tolerate various contaminants. By concocting an artificial mixture of metal ions and plastic-supplemented nutrient molecules from soil extract under shaking conditions, a number of lipolytic bacteria were explored, and their adaptability under harsh conditions was improved. A high lipase yield from these candidates may be of a potential value in other applications. Particularly, in the treatment of oily wastewater and oil-contaminated soil, the use of lipolytic bacteria may represent a green and economic alternative approach in the future. Therefore, further experiments in certain applications need to be carried out in lab-scale and large-scale as the next steps after the screening experiments in this study.


Topical application of β-blocker eye drops induces damage to the ocular surface in clinical. However, the mechanism involved remains incompletely understood. The purpose of this study was to investigate the influence and mechanism of β-blocker eye drops on corneal epithelial wound healing.Corneal epithelial wound healing models were constructed by epithelial scraping including in the limbal region and unceasingly received eye drops containing 5 mg/mL β-blocker levobunolol, β1-adrenoceptor (β1AR)-specific antagonist atenolol or β2-adrenoceptor (β2AR)-specific antagonist ICI 118, 551. For the migration assay, the murine corneal epithelial stem/progenitor cells (TKE2) were wounded and subsequently incubated with levobunolol, atenolol, or ICI 118, 551. The proliferation and colony formation abilities of TKE2 cells treated with levobunolol, atenolol, or ICI 118, 551 were investigated by CCK-8 kit and crystal violet staining. The differentiation marker Cytokeratin 3 (CK3), the stem cell markers-Cytokeratin 14 (CK14) and Cytokeratin 19 (CK19), and corneal epithelium regeneration-related signaling including in Ki67 and the phosphorylated epithelial growth factor receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) were assessed by immunofluorescence staining.Levobunolol and ICI 118, 551 impaired corneal wound healing, decreased the expressions of CK3, CK14, and CK19 after limbal region scraping in vivo and reduced the migration and proliferation of TKE2 in vitro, whereas atenolol had no significant effect. Moreover, levobunolol and ICI 118, 551 inhibited corneal wound healing by mediating the expression of Ki67, and the phosphorylation of EGFR and ERK1/2 in the limbal and regenerated corneal epithelium.β-blocker eye drops impaired corneal wound healing by inhibiting the β2AR of limbal stem cells, which decreased corneal epithelial regeneration-related signaling. Therefore, a selective β1AR antagonist might be a good choice for glaucoma treatment to avoid ocular surface damage. Toxic keratitis involves complex clinical manifestations, which is difficult to differentiate from other ocular surface diseases, and requires a careful medical history and clinical examination. The characteristic features of toxic keratitis mainly include superficial punctuate or epithelial defects, ulceration, scarring, pannus, neovascularization, or perforation . Common Adrenoceptors (ARs), which belong to the G protein-coupled receptor superfamily, mediate diverse effects at all sites throughout the body . βARs, wIn the present study, we investigated the effects of the anti-glaucoma agent levobunolol on corneal limbal stem cells by establishing corneal wound healing models with the entire corneal epithelium, including limbal region scrape. Atenolol is a selective β1AR blocker, and ICI 118,551 is a highly selective β2AR antagonist, which is used widely for hypertension, angina pectoris, and for the regulation of pulmonary systolic pressure , 25.Furthermore, the primary AR inhibited by levobunolol has been identified using β1AR-specific antagonist atenolol and β2AR-specific antagonist ICI 118, 551 in the corneal wound healing model. Kawakita et al. separated a mouse corneal epithelial progenitor cell line (TKE2) from CD-1 albino mice and successfully constructed a tissue engineered corneal epithelium . Qu et aThe experimental study included 141 C57BL/6 mice with an age of 8 weeks and a body weight of 20-25 g at baseline. The Medical Ethics Committee of Shandong Eye Institute approved the study, and the ARVO Statement and the ARRIVE Guidelines for the use of animals in ophthalmic and vision research were employed. The animals were purchased from Beijing Vital River Laboratory Animal Technologies Co. Ltd. in Beijing, China. All animals were sacrificed by cervical dislocation, and the eyes were extirpated immediately for further research.The mice were systemically anesthetized with pentobarbital sodium  and topically anesthetized with 2% xylocaine. Then, the corneal epithelium was marked with 2.5 mm or 3.0 mm trephine and scraped with Alger Brush II corneal rust ring remover . Ofloxacin was used to prevent postoperative infection. The experimental groups unceasingly received eye drops containing 5 mg/mL of levobunolol , atenolol (MedChemExpress) or ICI 118, 551 (MedChemExpress) for 4 times a day and mice received saline as vehicle control. The defects of corneal epithelium were visualized every 12 h by staining with fluorescein sodium and photographed with BQ 900 slit lamp . The experiment was carried out including five mice for each group and repeated at least three times. The defect area was analyzed by Image J software .The TKE2 cells, which were kindly presented by Dr. Tetsuya Kawakita , were cultured in keratinocyte serum-free medium  and supplemented with human keratinocyte growth supplement  and recombinant epidermal growth factor .The TKE2 cells were trypsinized and plated into the 12-well plates until confluence. Then the TKE2 cells were starved with KSFM in the presence of EGF (5 ng/mL). After starvation for 24 h, the TKE2 cells were wounded with a micropipette tip and subsequently incubated with 50 μM levobunolol, atenolol, or ICI 118, 551 for another 24 h, and PBS was used as control. The photographs of wound closure were used for migration quantification.The TKE2 cells were trypsinized and plated into the 96-well plates (2000 per well) and the TKE2 cells were incubated with 50 μM levobunolol, atenolol, or ICI 118, 551 for 48 h, and PBS was used as control. Then, the proliferation abilities of TKE2 cells treated by levobunolol, atenolol, or ICI 118, 551 were investigated by cell counting kit-8  and the absorbance of each well was measured at 450 nm by using a microplate reader .The TKE2 cells were trypsinized and plated into the 12-well plates (800 per well) and then the TKE2 cells were incubated with levobunolol (50 μM), atenolol (10 μM), or ICI 118, 551 (10 μM) for 7 days, and PBS was used as control. Then, the Colony formation abilities of TKE2 cells treated by levobunolol, atenolol, or ICI 118, 551 were investigated by crystal violet staining solution  and the images were observed using an Echo microscope .Mouse eyeballs were excised and snap-frozen in Tissue-Tek OCT compound at 2 or 3 days after levobunolol, atenolol, or ICI 118, 551 treatment. The frozen sections (7 μm) were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and then blocked with 5% BSA for 1 h at room temperature. The sections were then incubated with anti-CK3 , anti-CK14 (Abcam), anti-CK19 , anti-Ki67 (Abcam), anti- phosphorylated epithelial growth factor receptor , or anti-phosphorylated extracellular signal-regulated kinase 1/2  overnight at 4 °C and subsequently incubated with fluorescein-conjugated secondary antibody  for 1 h, followed by 4′, 6-diamidino-2-Phenylindole  staining for 5 min. All stainings were observed through an Eclipse TE2000-U microscope  and were quantified by Image J software. Firstly, the images were put on, then, which were conversed to 8 bit and adjusted to gray scale followed by measuring parameters including the area, mean gray value (Mean), integral optical density. Finally, we got average gray scale of these images which were used to statistically analyze.P value less than 0.05 was considered statistically significant, a P value less than 0.01 was considered extremely significant.The statistical analysis was performed with GraphPad Prism 8.0 and SPSS 24.0. The statistical significance was analyzed by Student’s t-test and one-way ANOVA. A To assess the influence of β-blocker eye drops on corneal wound healing, we removed the central or the entire corneal epithelium including the limbal region by marking with 2.5 or 3 mm trephine. Approximately 5 mg/mL β-blocker eye drops levobunolol, which was equivalent to the concentration used clinically, was then applied topically to the mice, and wound healing was observed using fluorescein sodium staining. After central region debridement, the corneal epithelial healing rate of the levobunolol-treated group exhibited a marginally significant difference compared with the control group Fig. A, B. HowTo delineate the primary AR antagonized by the non-selective β1 and β2AR inhibitor levobunolol during corneal wound healing, the β1AR specific antagonist atenolol or β2AR specific antagonist ICI 118, 551 was applied topically to the mice after the entire corneal epithelium, including the limbal region, was removed. The results showed that the wounded area of mice treated with ICI 118, 551 was significantly larger than that in the control and atenolol groups Fig.A, B. TheIt was revealed that levobunolol had more influence on corneal limbal stem cells, which resulted in the delay of corneal wound healing. To investigate the effects of β-blocker eye drops in regulating the corneal wound healing in vitro, TKE2 cells, which shared a similar characteristic to the murine limbal stem cells, were chosen to conduct further study . FirstlyThe colony forming of TKE2 treated with levobunolol, atenolol, or ICI 118, 551 for 7 days were compared by crystal violet staining and the results showed that there was no significant difference in the clone number of the control group and levobunolol, atenolol, or ICI 118, 551 groups Fig. D, E. HowTo elucidate the critical signaling pathways mediated by the β-blocker eye drops during corneal wound healing, we investigated the proliferation marker Ki67, and the phosphorylation of EGFR and ERK1/2 in the limbal and regenerated corneal epithelium. As shown in Fig. βAR, which is expressed by trabecular meshwork cells, ciliary bodies, microvessels, and optic nerve tissue, is involved in glaucoma pathogenesis –6. MoreoβAR regulates multiple cellular processes, such as cell proliferation, cell motility, inflammation, angiogenesis, and apoptosis . Recent Ki67, localized in the nucleus, has been found negative in the G0 phase, whereas it increased during the G1 phase, and slight variation resulted in variable Ki67 levels in the G1 phase after cell cycle re-entry –37. TherIn clinical trials, both the β-blocker eye drops levobunolol (0.5%) and timolol (0.5%) applied twice daily for 4 weeks significantly decreased tear volume and corneal epithelial barrier function , 40. In β-blocker eye drops impaired corneal wound healing through inhibition the β2AR of limbal stem cells and resulted in the decrease of corneal epithelial regeneration-related signaling. Therefore, a selective β1AR antagonist might be a good choice for glaucoma treatment to avoid the ocular surface damage.


Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that participates in various cellular events, such as DNA repair and apoptosis. The functional diversity of GAPDH depends on its intracellular localization. Because AMP-activated protein kinase (AMPK) regulates the nuclear translocation of GAPDH in young cells and AMPK activity significantly increases during aging, we investigated whether altered AMPK activity is involved in the nuclear localization of GAPDH in senescent cells. Age-dependent nuclear translocation of GAPDH was confirmed by confocal laser scanning microscopy in human diploid fibroblasts (HDFs) and by immunohistochemical analysis in aged rat skin cells. Senescence-induced nuclear localization was reversed by lysophosphatidic acid but not by platelet-derived growth factor. The extracellular matrix from young cells also induced the nuclear export of GAPDH in senescent HDFs. An activator of AMPK, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), increased the level of nuclear GAPDH, whereas an inhibitor of AMPK, Compound C, decreased the level of nuclear GAPDH in senescent HDFs. Transfection with AMPKα siRNA prevented nuclear translocation of GAPDH in senescent HDFs. The stimulatory effect of AICAR and serum depletion on GAPDH nuclear translocation was reduced in AMPKα1/α2-knockout mouse embryonic fibroblasts. Overall, increased AMPK activity may play a role in the senescence-associated nuclear translocation of GAPDH. Cellular senescence is a complex and heterogeneous process characterized by cell cycle arrest after a limited number of cell divisions. Telomeres shorten with each mitotic division in cells, and a DNA damage response terminally arrests the cell cycle as a protective mechanism . The graAmong the events associated with cellular senescence, changes in the expression and secretion of extracellular matrix (ECM) components, such as collagens, glycoproteins, proteoglycans, and ECM-associated proteins, and ECM remodeling enzymes such as matrix metalloproteinases are included . AlthougGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that catalyzes the conversion of glyceraldehyde-3-phosphate into 1,3-biphosphoglycerate, producing ATP and pyruvate through anaerobic glycolysis in the cytoplasm. GAPDH also participates in various cellular events, such as DNA replication and repair, transcriptional and post-transcriptional regulation of gene expression, membrane fusion and transport, autophagy, apoptosis and cellular senescence –16. The 172 via upstream AMPK kinases, such as liver kinase B1 (LKB1) and Ca2+-calmodulin-dependent protein kinase kinase (CaMKK) β. In contrast, AMPK activity is inhibited by dephosphorylation at Thr172 by protein phosphatases 2A/2C and phosphorylation at Ser485/491 of the α1/α2 subunits by other kinases, such as Akt, protein kinase A, and ERK -3-pyridin-4-yl-pyrazolo  pyrimidine (Compound C: abbreviated to CompC) is a potent AMPK blocker via ATP-competitive inhibition. In this study, we used these AMPK activity modifiers. To determine the effects of AICAR and CompC on AMPK activation and GAPDH distribution, subconfluent senescent HDFs were serum-deprived for 2 days and were treated with vehicle, 1 mM AICAR. and/or 10 μM CompC for 2 days. Following treatment with AICAR and CompC, the cells were assessed by western blot analysis. The addition of AICAR to senescent HDFs increased AMPK activity, and the activation was negated by the addition of CompC  = 220.508, p < 10–3) followed by Dunnett’s T3 post hoc test. Compared with HDFs without treatment with AICAR or CompC (−/−), the number of HDFs with cytosolic GAPDH only decreased significantly (***p < 0.001) from 20.62 to 7.47% with the addition of AICAR, but increased significantly (***p < 0.001) to 43.60% with the addition of CompC. As expected, the number of HDFs with nuclear GAPDH with or without cytosolic GAPDH increased significantly (###p < 0.001) from 79.38 to 92.53% with the addition of AICAR, but decreased to 56.40% with the addition of CompC (###p < 0.001). When AICAR and CompC were both added to the medium, the number of HDFs with nuclear GAPDH was significantly reduced, similar to when only CompC was added. These data suggest that AMPK activation might be required for the nuclear translocation of GAPDH in senescent HDFs.The effects of AICAR and CompC were examined using the number of subconfluent senescent HDFs with GAPDH in the cytosol alone (cytosol) or the nucleus, regardless of cytosolic GAPDH (nuclear +/− cytosol). Following treatment with AICAR and CompC, senescent cells were analyzed by immunofluorescence staining and confocal laser scanning microscopy. The number of cells having cytosolic GAPDH only (cytosol) and cells having nuclear GAPDH with or without cytosolic GAPDH (nuclear +/− cytosol) was counted, and the percentage distributions were plotted . Statist1 and AMPKα2, we used a mixture of siRNAs against AMPKα1 and AMPKα2 to block the expression of both isoforms. As depicted in 1/2 protein expression. Moreover, AMPKα siRNA-transfected HDFs presented with increased amounts of cytosolic GAPDH when compared with the control scrambled siRNA duplex-transfected HDFs  = 239.297, p < 10–3) followed by Dunnett’s T3 post hoc test. The percentage distributions of HDFs with cytosolic GAPDH only for the mock control group were 20.44 and 19.21% at 48 and 72 h, respectively. In the AMPKα siRNA-transfected cells, these were increased significantly (***p < 0.001) to 32.55 and 46.68% at 48 and 72 h, respectively. In contrast, the percentage distribution of HDFs with nuclear GAPDH decreased from 79.56 to 67.45% at 48 h and from 80.79 to 53.32% at 72 h (###p < 0.001). These data support the idea that AMPK activation is responsible for the nuclear accumulation of GAPDH in senescent HDFs.We applied an AMPKα-specific siRNA approach to support the effect of AMPKα isoforms on the nuclear accumulation of GAPDH in senescent HDFs. Because the major isoforms of the AMPKα subunit in HDFs are AMPKαted HDFs . AMPKα1/1 null (α1−/−), AMPKα2 null (α2−/−), and AMPKα1 and α2 null (α1−/−α2−/−), the effects of the catalytic α subunit of AMPK on GAPDH translocation were assessed. The baseline level of nuclear GAPDH and the level of its nuclear translocation upon addition of 1 mM AICAR or culturing in SFM for 48 h were compared in control MEFs and AMPKα-knockout MEFs. The statistically significant difference between groups was determined by one-way ANOVA  = 242.501, p < 10–3) followed by Dunnett’s T3 post hoc test. The control MEFs showed a significant increase in the level of nuclear GAPDH upon AICAR addition to 78.74% (5.84-fold increase), compared to the basal level of 13.49% (***p < 0.001) in AMPKα1−/− and AMPKα1−/−/α2−/− MEFs, respectively. The rates of nuclear GAPDH upon AICAR addition significantly (###p < 0.001) decreased to 25.65, 60.06, and 17.15% in AMPKα1−/−, AMPKα2−/−, and AMPKα1−/−/α2−/− MEFs, respectively, compared to 78.74% in control MEFs. These data indicate the critical role of AMPKα1 and the partial role of AMPKα2 in the nuclear translocation of GAPDH in MEFs.Using three types of AMPKα-knockout MEFs, AMPKαf 13.49% . CompareF = 217.107, p < 10–3) followed by Dunnett’s T3 post hoc test. Culturing in SFM for 48 h significantly (###p < 0.001) induced nuclear GAPDH from the baseline level of 13.86 to 50.38% (3.63-fold increase) in control MEFs , compared to 13.86% of the control MEFs. Furthermore, the levels of nuclear GAPDH in SFM culture significantly (***p < 0.001) decreased to 5.78, 8.73, and 10.55% in AMPKα1−/−, AMPKα2−/−, and AMPKα1−/−/α2−/− MEFs, respectively, compared to 50.38% in control MEFs  and AMPK signaling pathways, in HDFs  and in pFurthermore, we found that age-related GAPDH translocation might be reversed by the addition of young ECM . This isThe dysregulation of ECM composition and structure contributes to several pathological conditions such as fibrosis and invasive cancer . Fibrosi1 and AMPKα2 activities were unaltered during aging at rest, but activation of AMPKα1 was enhanced, while activation of AMPKα2 was suppressed immediately after endurance-type muscle contractions in aged rats, resulting in inactive heterotrimer composition of AMPK and consequently increasing skeletal muscle atrophy sarcopenia [172 , AMPKα2 null (α2−/−), and AMPKα1/α2 null (α1−/−α2−/−). AMPKα depletion by AMPKα1/2 siRNA transfection reduced AMPKα1/2 protein levels , and rabbit monoclonal anti-GAPDH IgG (#2118S) for immunohistochemistry were purchased from Cell Signaling Technology . CompC and AICAR were purchased from Calbiochem  and mouse anti-GAPDH monoclonal antibody (#MAB374) was from Chemicon . Horseradish peroxidase-conjugated anti-rabbit (#PI-1000) and anti-mouse (#PI-2000) secondary antibodies were obtained from Vector Laboratories  and protein assay kit was from Bio-Rad Laboratories . Trypsin and Zymed Picture Plus Kit was purchased from Zymed , and predesigned human AMPKα1/2-specific and control scrambled siRNA duplexes were purchased from Santa Cruz Biotechnology . Lipofectamine™, RNAiMAX, and Permount mounting solution including 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Invitrogen Life Technologies  and enhanced chemiluminescence (ECL) system was from GE Healthcare .PDGF-BB, LPA, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (#F0257), and mouse anti-β-actin monoclonal antibody (#A5441) were purchased from Sigma-Aldrich . Dulbecco’s modified Eagle’s medium (DMEM), FBS, opti-MEM I, penicillin, and streptomycin were obtained from Gibco/BRL Life Technologies, Inc . Rabbit monoclonal antibodies against AMPKα (#2532S) and phospho-Thr2 incubator at 37°C. The cells were divided into three groups depending on the duration of growth: young, middle, and senescent. Young HDFs were cultured until PD was less than 25, middle HDFs until PD between 45 and 65, and senescent HDFs until PD greater than 65. Senescent HDFs were identified by their characteristic morphological changes, enhanced beta-galactosidase activity, and cell growth arrest. Before serum depletion, regardless of their classification, HDFs were grown for 2 days to reach 60–70% confluency in a culture medium containing 10% FBS. To determine the effect of serum or growth factor re-addition on the nuclear localization of GAPDH, subconfluent senescent HDFs were serum-deprived via a 5-day-incubation in SFM containing 0.1% bovine serum albumin (BSA).HDFs were isolated from the foreskin of neonates, as described previously . HDFs we5 HDFs were cultured on coverslips in 6-well culture plates for 1 day and treated with the indicated medium or reagents for the indicated times. The cells on coverslips were washed twice with ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 10 min, and washed with PBS. Subsequently, blocking solution with 2% BSA in Tris-buffered saline with Tween 20  was used to saturate non-specific protein binding sites for 1 h with gentle shaking. The cells were incubated with primary monoclonal anti-GAPDH antibody  for 1 h at 25°C, and were washed three times with ice-cold TBST for 10 min each. Cells were incubated with FITC-conjugated anti-mouse secondary antibody (1:500) in blocking solution for 30 min. Cells were washed three times with TBST for 10 min each, and were dehydrated in three washes with 100% methanol for 1 h. The coverslips were mounted on glass slides with a mounting solution containing DAPI for nuclear staining, and fluorescence images were captured with a confocal laser scanning microscope . GAPDH was indicated by FITC fluorescence, which glows in green. Blue DAPI-stained nuclei and merged images are also visible in confocal microscopic pictures. Each sample was counted in 10–20 different fields at 100× magnification. The data were combined to calculate the total cell number of approximately 100 and were converted to percentage distribution. All experiments were repeated at least three times to obtain reliable statistical inferences.For immunofluorescence staining, 1.0 × 101/2-specific or control scrambled siRNA duplexes, as described previously [4 senescent HDFs per well were seeded in 6-well plates in 2 mL of antibiotic-free culture medium supplemented with FBS. Cells were incubated at 37°C in a CO2 incubator overnight until the cells were 30–50% confluent. The mixture of 50 pmol siRNA duplex in 100 μL of Opti-MEM I reduced serum medium and 2 μL of Lipofectamine™ RNAiMAX transfection reagent were preincubated at room temperature for 15 min and then diluted in 1 mL of transfection medium. The mixtures were overlaid onto the washed cells. After 6 h of transfection at 37°C in a CO2 incubator, 1 ml of DMEM containing 20% FBS and 2× antibiotics was added to each well, and cells were incubated for an additional 24 h at 37°C. After refreshing the culture medium to DMEM containing 10% FBS and 1× antibiotics, cells were incubated for 48–72 h and assayed for AMPKα1/2 levels using western blot analysis and for GAPDH localization using immunofluorescence staining and confocal microscopy.Senescent HDFs were transfected with 50 nM human AMPKα6 HDFs grown in 100-mm culture dish were lysed in lysis buffer  at 4°C for 30 min on a rocker. The lysates were centrifuged at 14,000 × g for 15 min in an Eppendorf centrifuge to remove cell debris, and the protein concentration of each lysate was determined using a Bio-Rad protein assay kit, according to the manufacturer’s protocol. Cell lysates of equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred electrophoretically onto Immobilon PVDF membranes. A solution containing 5% non-fat dried milk or 1% BSA in TBST buffer was used to block the blots for 1 h at 25°C. Moreover, proteins were incubated overnight with monoclonal antibody (1:200 to 1: 1000) in the blocking solution at 4°C, washed three times with TBST, and further probed with horseradish peroxidase-conjugated anti-rabbit secondary IgG (1:5000) for 30 min. An ECL detection system was used to visualize the immune complexes on the blots.Protein expression levels were examined by western blot analysis, as described previously . A totalAdult male Fisher 344 rats were purchased from Samtaco BioKorea . The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Basic Science Institute (KBSI) (KBSI-ACE1913). The animals were housed two per cage in a room with controlled temperature and humidity with a 12-h light/12-h dark cycle. They were maintained on a standard diet with food and water ad libitum in an animal facility approved by the Korean Association for Assessment and Accreditation of Laboratory Animal Care. The back skin sections of young (6 months) and aged (24 months) rats were obtained as described previously . Rats weP values of less than 0.05 were considered statistically significant.For statistical analyses of the data, one-way analysis of variance (ANOVA) followed by Dunnett T3 post hoc test  was performed to assess significant differences between groups. Data are presented as the mean ± standard deviation of at least three experiments. Supplementary Figures