The direct interaction of miR‐140‐5p with Pin1 mRNA and its inhibitory role in protein translation has been identified. The main purpose of this study was to investigate the role of miRNA‐140‐5p inhibition in promoting Pin1 expression and the therapeutic potential of the AntimiR‐140‐5p in the Aß oligomer (AßO)‐induced AD rat model.The peptidyl‐prolyl Spatial learning and memory were assessed in the Morris water maze. RT‐PCR, western blot, and histological assays were performed on hippocampal samples at various time points after treatments. miRNA‐140‐5p inhibition enhanced Pin1 and ADAM10 mRNA expressions but has little effect on Pin1 protein level.The miRNA‐140‐5p inhibitor markedly ameliorated spatial learning and memory deficits induced by AßO, and concomitantly suppressed the mRNA expression of inflammatory mediators TNFα and IL‐1β, and phosphorylation of tau at three key sites  as well as increased phosphorylated Ser473‐Akt.According to our results, Antimir‐140‐mediated improvement of AβO‐induced neuronal injury and memory impairment in rats may provide an appropriate rationale for evaluating miR‐140‐5p inhibitors as a promising agent for the treatment of AD. miRNA‐140‐5p inhibitor ameliorated cognitive deficits induced by AßO. Further, miR‐140‐5p downregulation was associated with suppressing neuroinflammation and abnormal tau phosphorylation. This study exhibits a crucial role for miR‐140‐5p in the key events of oligomeric amyloid‐beta induced neurotoxicity, which may serve as a potential therapeutic target for AD. The mature miRNA directs translational attenuation through binding to an mRNA target site for its degradation or translational suppression.Over the last few years, miR‐140‐5p has attracted interest as a diagnostic and therapeutic target for several diseases including neurodegeneration and oncogenesis. The association between miR‐140‐5p downregulation and various kinds of tumor tissues and cell lines, and the antitumor activity of the miRNA through targeting certain oncogenic genes have been widely documented.22.12 inhalation. All experimental protocols involving rats were approved by the Ethical Committee of Neuroscience Research Center, Shahid Beheshti University of Medical Sciences  in compliance with the standards of the European Communities Council Directive (86/609/EEC). All efforts were made to reduce animal suffering and the number of rats needed for the study.A total of 64 adult male Wistar rats with an average age of 12 weeks weighing approximately 250–280 g were procured from the breeding colony of Neuroscience Research Center, Shahid Beheshti University of Medical Sciences. The rats were held four per cage under standard animal room conditions at 21 ± 2°C and a 12 h light/dark cycle with free access to food and water ad libitum. In the present study, the rats were sacrificed by CO2.2Anti‐miR‐140‐5p (AM‐140) was synthesized by Metabion . AM‐140 contained 2’‐O‐methyl modifications (5′‐(2’OMe‐C) *(2’OMe‐U) *a* cca uag ggu aaa acc *(2’OMe‐A)* (2’OMe‐C)*(2’OMe‐U) *(2’OMe‐G)‐3′). Aβ (1–42) (CAS number: ab120301) were purchased from Abcam. Oligomeric Aβ1–42 was prepared as originally established by Klein et al.2.3The rats were randomly distributed into four equal groups with 16 rats in each one as follows: control group received AβO vehicle  and AM‐140 vehicle, AβO group received oligomeric Aβ and AM‐140 vehicle; AM group received Aβo vehicle and AM‐140 vehicle; AM+AβO group received oligomeric Aβ in combination with AM‐140. Stereotactic (Stoelting) surgery was carried out for the bilaterally intrahippocampal injections under anesthesia using a mixture of ketamine and xylazine . Oligomeric Aβ 1–42  and AM‐140  were injected into the CA1 region of the rat hippocampus  phase and a probe phase. The place navigation test (training) was carried out as four trials daily for three consecutive days to induce learning starting on day 18 following surgery. In each trial, the animal was inserted into the water facing the wall of the pool at one of the quadrants, allowed to swim, and expected to find the hidden platform within the 60 s. An animal that failed to find the platform was manually guided and let stay there for 20 s for reinforcement. On the 4th day of the experiment, 24 h after the last acquisition trial, the probe phase was conducted without the platform and each rat was left to swim within 60 s. The rats were expected to spend the longest time in the quadrant containing the platform (target quadrant). All behavioral parameters, including traveled distance, escape latency (time to find the platform), and swimming speed in the learning phase, as well as the time spent in the target quadrant, the number of crossings over the platform in the probe phase, were recorded by a video camera and analyzed through EthoVision program.2.5n = 3) from each group were deeply anesthetized and perfused transcardially using ice‐cold PBS , followed by 4% paraformaldehyde (PFA) in PBS. Then, the whole brains were isolated and post‐fixed in PFA for at least 72 h at 4 °C. Following the gradient dehydration, the tissues were embedded in paraffin wax at 59 °C and sectioned to a thickness of 5–7 μm.Brain and hippocampus samples were collected and preserved differently based on the following analysis methods. Three rats from each group were sacrificed at different time points  after the stereotactic injections, snap‐frozen, and kept at −80°C to be later used for molecular and biochemical assessments  method was employed to quantify the expression levels of miR‐140 and mRNAs encoding Pin1, ADAM10, IL‐1ß, and TNFα in hippocampus samples collected 24 h, 7, 14, and 21 days post‐surgery. Initially, the total RNA was extracted from the tissues by the means of a GeneAll Hybrid‐R RNA purification kit (GeneAll) based on the instruction included in the kit. The concentration of the purified RNAs was measured by Nanodrop at 260 nm (Thermo Fisher Scientific). The expression analysis of miR‐140 was quantified using stemloop RT‐qPCR. Complementary deoxyribonucleic acid (cDNA) and PCR products were synthesized by the SYBR green MicroRNA kits (AnaCell) for rno‐miR‐140 in accordance with the manufacturer's instructions. A primer set for the U6 gene was also included in the kit as the endogenous control. The reaction protocol contained the following steps: heating for 15 min at 95°C, followed by 40 cycles of amplification (15 s at 95°C and 1 min at 60°C). After the last amplification cycle, melting analysis was performed by one cycle of heating to 95°C.−ΔΔCt method.To analyze the mRNA expressions, a cDNA synthesis kit with OligodT primers  was employed for RNA reverse transcription to cDNA in a final volume of 20 μl. Transcript levels of Pin1, ADAM10, IL‐1ß, and TNF‐α were assessed by an ABI 7500 StepOnePlus™ Real‐Time PCR System (Applied Biosystems) with RealQ Plus 2x Master Mix Green, High ROX TM  and primers  listed in Table 2.7g and 4°C, and the supernatants were collected. The total protein concentration was determined through Bradford assay against BSA as standard. The lysates were then fractioned by 12.5% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and blotted onto polyvinylidene difluoride (PVDF) membranes with a pore size of 0.45 mm (Millipore). 5% non‐fat dry milk  dissolved in Tris‐buffered saline with 0.1% Tween® 80 detergent (TBST) was used as membrane blocking solution. After 75 min blocking, the blots were probed with primary antibodies by overnight incubation at 4°C with antibodies against Pin1 , p‐Tau (Thr231) , p‐Tau (Ser396) , p‐Tau (Ser404) , p‐GSK3ß(Ser9) , p‐Akt1/2/3 (Ser 473) , and β‐actin . Subsequently, the membranes were incubated with secondary horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit antibody  for 2 h at room temperature. The enhanced chemiluminescence reagent (ECL) (Bio‐Rad) was used to visualized protein bands on x‐ray films. The relative protein expression levels, normalized to β‐actin, were quantified using ImageJ program .Western blot analysis was performed based on previous protocols.2.8Coronal sections were stained as previously described.2.9p < 0.05 were considered to indicate statistical significance.Statistical analyses were completed using GraphPad Prism software version 6 , and all data are presented as mean ± standard error of the mean (SEM). Either one‐way analysis of variance (ANOVA) or two‐way repetitive measure ANOVA followed by Tukey's multiple comparisons post‐hoc tests were conducted where appropriate. Shapiro–Wilk normality test indicated that the data have normal distribution; therefore, comparisons were done using parametric tests. Data followed by 3In the present study, we used AM‐140 contained 2’‐O‐methyl modifications. Based on previous investigations, anti‐miRNA oligonucleotides with complementary sequences of the target miRNA bind miRNAs, form a duplex and inhibit miRNA functions in living cells.3.1 = 15.68, p < 0.0001) and treatment  = 12.05, p < 0.0001)  = 13.39, p < 0.0001) , while it was substantially reduced in rats received both AM140 and AßO (p < 0.0001 vs. AßO group). As indicated in Figure  = 15.17, p < 0.0001) and treatment  = 17.91, p < 0.0001) through RM‐ANOVA  = 25.95, p < 0.0001) and revealed that Aßo rats traveled longer distances relative to controls (p < 0.0001), whereas AM140 significantly limited the distances traveled by AßO received rats during 3 days of the acquisition phase (p < 0.0001) , however, rats with co‐injection of AM140 with AßO showed an increase in the duration F  = 3.399, p = 0.0320)  = 1.199, p = 0.3281) , and AM140 treatment did not make any significant difference in escape latency compared with AD‐like rats (p = 0.9535)  = 7.476, p = 0.0007)  = 1.450, p = 0.2495) and speed  = 2.082, p = 0.1252) were observed on day four of the MWM test  Figure . For fur) Figure . AUC calA Figure . AUC ana) Figure . In the ) Figure . In the ) Figure . The par) Figure . Toward 3.2 = 10.34, p = 0.0040) . Treatment of rats with mir‐140‐5p inhibitor considerably reduced the mir‐140 expression levels on day 7 in AßO + AM140 group vs. healthy rats  = 94.12, p < 0.0001)  = 4.156, p = 0.0476)  = 3.991, p = 0.0522)  after stereotactic injections using RT‐qPCR. 24 h post‐surgery, a marked increased expression was observed only in the AM+Aβo group compared with control and Aβo groups (F ) Figure . On the ) Figure . However) Figure  and 21  Figure  no signi3.3 = 11.18, p = 0.0031)  = 12.81, p = 0.0020)  and ADAM10 (p < 0.05) compared with healthy controls on day 7 post‐surgery. Mir‐140‐5p inhibitor alone did not make any significant change in both mRNA levels of Pin1 and ADAM10 in healthy rats, however, its administration following the AβO injection significantly increased the expression level of Pin1 (p < 0.01), and ADAM10 (p < 0.05) in comparison with AßO injected rats. As shown in Figure  = 0.8194, p = 0.5187), although a statistically insignificant elevated level of ADAM10 was detected in AßO group  = 2.382, p = 0.1452)  as well as to investigate the effects of the mir‐140‐5p inhibitor on the mRNA expression levels may occur, and for how long it might be maintained in the hippocampus. Statistical analysis revealed that there was a significant difference in Pin1 ((F ) Figure ) and ADA) Figure  mRNA exp) Figure .3.4 = 0.4047, p = 0.7538)  = 0.9260, p = 0.4712)  = 6.504, p = 0.0154) and  = 33.00, p < 0.0001), respectively). AßO administration markedly enhanced the mRNA expression of IL‐1ß (p < 0.05)   and TNF‐α (p < 0.05) versus AßO group.In order to obtain further evidence on the role of miR‐140‐5p in the AßO induced learning damage and memory decline, possible alterations in mRNA expression of the key inflammatory mediators (IL‐1ß and TNF‐α) were also evaluated in hippocampus samples on day 7 and 21 post‐surgery. No remarkable difference was found in fold change values of IL‐1ß (F ) Figure  and TNF‐) Figure  between ) Figure  and TNF‐) Figure  compared3.5 = 1.424, p = 0.3056)  = 1.980, p = 0.1956). However, one‐way ANOVA demonstrated a marked effect of treatment on pS473‐Akt protein expression on day 21 between groups  = 11.96, p = 0.0025). Although pS473‐Akt expression level had no statistically significant difference in the AßO group compared with control, AM140 administration caused a significant increase in the pS473‐Akt expression in healthy rats (p < 0.01) and was also associated with an elevated level of the protein in AßO rats (p = 0.0892)  Figure . The exp) Figure . = 10.27, p = 0.0041). As depicted in Figure p < 0.01). AM treatment caused a markedly lower level of pT231 tau in rats after exposure to AßO (p < 0.01). There was also a statistically significant difference between groups in the expression of tau phosphorylated in the Ser396 region  = 5.540, p = 0.0236) . Figure  = 7.477, p = 0.0104). A significantly elevated protein level of pT404 tau was found in rats that received only AßO (p < 0.05) versus healthy rats, and AM140 administration attenuated its expression in the hippocampus of AM140+ AßO rats in comparison with the AßO group (p < 0.05). All the original, uncropped blot images were provided in the Supplementary file In subsequent studies, phosphorylated tau proteins, including Thr231 (pT231), Ser396 (pS396), and Ser404 (pS404) were evaluated to determine whether the treatment with AM140 influenced the phosphorylation status of tau in AD‐like conditions. The results obtained 21 days post‐surgery indicated a significant difference in pT231 tau between groups (F ) Figure . A stati3.6The whole‐brain sections collected on day 21 were stained with cresyl violet to analyze the morphology of the hippocampus in the different groups Figure . Nissl s4In the present study, we addressed the question of whether miR‐140‐5p inhibition in rat hippocampus can be useful to target the main pathological processes of AD, namely, impaired cognitive function, neuroinflammation, hyperphosphorylation of tau in the brain, and defective brain insulin signaling. To our knowledge, up to now, no convincing data is showing that this strategy may buffer against AD cascades in vivo. Thus, we examined the preventive effect of AM140 against AβO and its pathological cascades. Previous reports have described soluble Aβ oligomers as the primary toxic agent that mediate early memory dysfunction and synaptic degeneration in AD rather than insoluble amyloid plaques.The primary conclusion of the current study is that i.h. injection of AM140 inhibitor results in an improvement of learning and memory deficits in AβO‐induced AD‐like conditions, which was examined by MWM test on day 21 following the agents' administration. Treatment of AD‐like animals with AM140 was associated with a significant improvement in the parameters studied in the learning phase, including escape latency and the path length. In the probe phase, positive changes were also observed in the time taken to reach the original platform position, although similar changes were not observed in other parameters for memory evaluation.A number of recent studies have found miR‐140‐5p upregulation to be involved in AD pathology.In this work, we presented evidence that AßO, as the leading neurotoxin in the AD progression, can increase miR‐140‐5p expression, leading to a significant decrease in Pin1 and ADAM10 mRNA levels, only on day 7 following the i.h. injection. Furthermore, AM140 treatment effectively suppressed the miRNA‐140 elevated level, which was simultaneously associated with improvements in Pin1 and ADAM10 mRNA expression levels. Nevertheless, the cause of the ambiguous expression pattern of miR‐140 on day one after the surgery and its upregulation only in AM140‐treated AD‐like rats remained questioned and more studies should be done on how the level of the miRNA changes in shorter time intervals. On the other hand, Pin1 expression on protein level was only analyzed on day 21 after the initial insult, which was not associated with a clear significant difference between treated and untreated animals. It should come as no surprise that this was the major limitation of this study and analysis of Pin1 protein level at a time‐point close to day 7 could therefore be more helpful.In the next part of the study, the association of miR‐140‐5p downregulation with some of the main neuropathological hallmarks of AD was examined. It is well known that neuroinflammation plays an important role in cognitive impairment and AD development. It is reported that AßO stimulates neuroinflammation through the direct activation of microglia, which in turn lead to the release of inflammatory cytokines, and ultimately to neuronal cell loss.The most apparent consequence of AM140 administration in AD‐like rats over 21 days was ameliorating the phosphorylation levels of tau at three studied sites  induced by AßO injection. Tau phosphorylation at the Thr231 region is thought to be an early crucial event prior to NFT formation.The indispensable role of aberrant neuronal Insulin signaling in AD brains has been confirmed through a huge number of studies.Our study only provides preliminary but informative evidence for our underlying hypothesis regarding the therapeutic potential of miR‐140‐5p modulation in AD patients and suffers from a number of limitations. Here, we conducted multi‐time‐point surveys to evaluate the effects of miR‐140‐5p inhibitor on AD's main pathological hallmarks. However, the exact prediction of the time‐course pharmacological properties of anti‐miRs is often complicated, since their pharmacological activities have been found to be related to the sum of pharmacological effects over time that leads to alterations in the disease phenotype.5This study exhibits a crucial role for miR‐140‐5p in the key events of oligomeric amyloid‐beta induced neurotoxicity, which may serve as a potential therapeutic target for AD. Our preclinical study also provided evidence that the local silencing of miR‐140‐5p within the hippocampus may attenuate memory impairment and prevents disease progression in patients suffering from AD.Pariya Khodabakhsh: investigation, acquisition, analysis, and interpretation of data, writing—original draft. Maryam Bazrgar: investigation, acquisition, and interpretation of data. FatemehMohagheghi: conceptualization and supervision. Siavash Parvardeh and AbolhassanAhmadiani: conceptualization, methodology, supervision, and funding acquisition. All authors revised and approved the final version of the manuscript. All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in the CNS Neuroscience & Therapeutics journal.This study was funded by Research Affairs (Grant No. 13.42) of Shahid Beheshti University of Medical Sciences, Tehran, Iran.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Appendix S1Click here for additional data file.


As one of the most important protein-degrading enzymes, ADAMTS-5 plays an important role in the regulation of cartilage homeostasis, while miRNA-140 is specifically expressed in cartilage, which can inhibit the expression of ADAMTS-5 and delay the progression of OA (osteoarthritis). SMAD3 is a key protein in the TGF-β signaling pathway, inhibiting the expression of miRNA-140 at the transcriptional and post-transcriptional levels, and studies have confirmed the high expression of SMAD3 in knee cartilage degeneration, but whether SMAD3 can mediate the expression of miRNA-140 to regulate ADAMTS-5 remains unknown.Sprague–Dawley (SD) rat chondrocytes were extracted in vitro and treated with a SMAD3 inhibitor (SIS3) and miRNA-140 mimics after IL-1 induction. The expression of ADAMTS-5 was detected at the protein and gene levels at 24 h, 48 h, and 72 h after treatment. The OA model of SD rats was created using the traditional Hulth method in vivo, with SIS3 and lentivirus packaged miRNA-140 mimics injected intra-articularly at 2 weeks, 6 weeks and 12 weeks after surgery. The expression of miRNA-140 and ADAMTS-5 in the knee cartilage tissue was observed at the protein and gene levels. Concurrently, knee joint specimens were fixed, decalcified, and embedded in paraffin prior to immunohistochemical, Safranin O/Fast Green staining, and HE staining analyses for ADAMTS-5 and SMAD3.P < 0.05). In vivo, it was found that ADAMTS-5 protein and gene were downregulated to varying degrees in the SIS3 and miRNA-140 mimic groups at three time points, with the most significant decrease at the early stage (2 weeks) (P < 0.05), and the expression of miRNA-140 in the SIS3 group was significantly upregulated, similar to the changes detected in vitro. Immunohistochemical results showed that the expression of ADAMTS-5 protein in the SIS3 and miRNA-140 groups was significantly downregulated compared to that in the blank group. The results of hematoxylin and eosin staining showed that in the early stage, there was no obvious change in cartilage structure in the SIS3 and miRNA-140 mock groups. The same was observed in the results of Safranin O/Fast Green staining; the number of chondrocytes was not significantly reduced, and the tide line was complete.In vitro, the expression of ADAMTS-5 protein and mRNA in the SIS3 group decreased to different degrees at each time point. Meanwhile, the expression of miRNA-140 in the SIS3 group was significantly increased, and the expression of ADAMTS-5 in the miRNA-140 mimics group was also significantly downregulated (The results of in vitro and in vivo experiments preliminarily showed that the inhibition of SMAD3 significantly reduced the expression of ADAMTS-5 in early OA cartilage, and this regulation might be accomplished indirectly through miRNA-140.The online version contains supplementary material available at 10.1186/s12891-022-05949-8. OA (osteoarthritis) is the leading cause of disability in the elderly over the age of 65 . The gloMechanical stress promotes the progression of osteoarthritis, with abnormal mechanical stress activating interleukin 1β, tumor necrosis factor α, nuclear factor-κB, Wnt, transforming growth factor β, microRNA pathways, and oxidative stress pathways that induce pathological progression in osteoarthritis . Data suThe regulatory relationship between SMAD3 and miRNA-140 has been reported, SMAD3 can inhibits the expression of miRNA-140 at the transcriptional and posttranscriptional levels , while tSD rats were purchased from the Animal Experimental Center. All experimental methods and procedures were approved by the Animal Experimental Ethics Inspection of First Affiliated Hospital, Shihezi University School of Medicine (A2017-130-01). SD rats were raised in a temperature-controlled dry environment (25 °C) and provided adequate food and water.Articular cartilage was extracted from the knee joints of the rats at 2 weeks of age. The tissue sample was digested with 0.2% type II collagenase  for 1 h. After centrifugation at 1000 g × 15 min, the supernatant was collected and digested with 0.25% trypsin  overnight (≤ 12 h). Centrifugation at 1000×g for 15 min was performed to remove the supernatant. The pellet containing chondrocytes was resuspended in DMEM  to a final volume of 6 mL, and 1 mL was aliquoted into each Petri dish. The rat chondrocytes were cultured in an incubator at 37 °C and passaged to the second generation when the cell density reached approximately 90% confluence.n = 90) were anesthetized by administering an intraperitoneal injection of 3% pentobarbital solution (30 mg/kg) and placed on a sterile operating table. A longitudinal incision about 2 cm long was cut in the medial knee joint, and the anterior and posterior cruciate ligament and medial collateral ligament were cut, and the medial meniscus was removed intact without damaging the articular cartilage surface. Postoperative injured limbs are not fixed, and antibiotics can be given to prevent infection. Osteoarthritis forms due to postoperative joint instability, increased friction of the articular surface, and the loss of the buffering effect of the meniscus.Male SD rats , miRNA-140 inhibitor, None-miRNA-140 inhibitor (N-miRNA-140 inhibitor). The in vivo experiments were conducted in two parts: the first part: blank, ADAMTS-5 inhibitor, SMAD3 protein and SIS3; the second part: blank, ADAMTS-5 inhibitor, miRNA-140 mimics, miRNA-140 inhibitor, random RNA sequence.Ultra-clean table sterilization, strict aseptic operation, and the addition of IL-1(interleukin)in the petri dish to induce normal chondrocytes into inflammatory cells, according to experimental groupings, were given corresponding treatment, ordering SMAD3-specific inhibitor SIS3 and miRNA-140 overexpression and inhibition fragments from reagent companies . Cell treatment was performed according to the concentration provided by the reagent company. The concentration of miRNA-140 mimics and miRNA-140 inhibitor was 60 nM, the concentration of SIS3 was 4 nM, the concentration of Smad3 protein was 500 ng, and the concentration of ADAMTS-5 inhibitor in the control group was 2.22 μM. Total RNA was extracted at 24 h, 48 h, and 72 h after treatment for qRT–PCR experiments, and total proteins were extracted at 72 h for Western blot experiments.After the model rats were grouped, they were intra-articularly injected 1 week after surgery, and the injection was repeated on the tenth day after the operation. Intra-articular injection was performed according to the concentration provided by the reagent company. The total volume of injection was 100 μl, the concentration of SIS3 and SMAD3 protein was 5 μg, miRNA-140 mimics and miRNA-140 inhibitor were lentiviral packaged, the total amount of injection was 1 × 107, and three collected knee specimens were sacrificed in each group at 2 W, 6 W and 12 W after surgery. Total RNA and protein were extracted, and the specimens were selected for fixation and decalcification at each time point in each group.The knee joint specimen was soaked in 4% paraformaldehyde at 25 °C for 3 d. After dissection, the muscle was removed, and 20% EDTA was used to decalcify the knee joint for 2 weeks. The knee joint was then embedded in paraffin. The paraffin-fixed tissue sections (5 μm) were baked, dewaxed, and hydrated for ADAMTS-5  and Smad3  protein immunohistochemistry as well as histological staining using Safranin O/Fast Green and H&E.-ΔΔCT method was used to calculate the relative gene expression. Internal reference: GAPDH, Gene sequences are as follows (5′-3′): miRNA-140 mimics, CAGTGGTTTTACCCTATGGTAG, miRNA-140 inhibitor, CTACCATAGGGTAAAACCACTG, ADAMTS-5, F: GGAGCGAGGCCATTTACAAC, R: CGTAGACAAGGTAGCCCACTTT.Total RNA was extracted using the TRIzol method at 24 h, 48 h, and 72 h after transfection of the cell samples. The articular cartilage was removed from the tissue samples and ground into a powder after adding liquid nitrogen. RNA was extracted using the TRIzol method, and all experimental steps were carried out on ice. A PCR assay  was performed using the TaqMan® miRNA reverse transcription kit to reverse transcribe the extracted RNA into complementary DNA (cDNA). For every sample, a final reaction volume of 20 μL, comprising 1 μL of each cDNA and specific primer, was used for the qRT–PCR assay. The thermal cycling conditions were as follows: initial denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and amplification at 65 °C for 1 min. The 2After culturing for 72 h, cell samples were washed twice with PBS, and cell lysates (RIPA: PMSF = 100:1) in approximately 200 μL were obtained. To obtain rat tissue samples from the joint cavity, the lower cartilage tissue was cut using a blade. After adding liquid nitrogen, the tissue samples were ground into powder, followed by the addition of 500 μL cell lysates (RIPA: PMSF = 100:1). The supernatant was centrifuged for 30 min . The protein concentration was measured by using the BCA method, in which 5× loading buffer was added (4:1) to the samples, which were then boiled at 100 °C for 8 min, aliquoted into tubes and frozen until further analysis. The protein samples were separated using SDS–PAGE and transferred onto a PVDF membrane. The membrane was blocked with 5% skimmed milk for 3 h and incubated overnight with ADAMTS-5 primary antibody  at 4 °C. Subsequently, the membrane was incubated for 2 h in a goat anti-rabbit IgG secondary antibody . The membrane was washed and examined under a chemiluminescence instrument. β-actin was used as an internal reference, and gray level analysis was performed using ImageJ software.P < 0.05 was considered as a statistically significant difference.The results of this study are all obtained from three or more independent experiments. Measurement data conforming to the normal distribution are described in the mean ± standard deviation, measurement data not conforming to the normal distribution are described in the median and interquartiles. Statistical analysis and the bar graph was made using Graphpad8.0, Adobe Illustrator 2020, Adobe Photoshop 2020 for picture editing, repeated measurement design between groups, multiple comparisons between multiple sample means were performed using the SNK-q test, non-parametric test was used between groups with uneven variance, P < 0.05; Fig. P < 0.05; Fig. P < 0.05). After miRNA-140 inhibitor treatment, ADAMTS-5 mRNA expression was significantly upregulated at 24 h (P < 0.05) and without any statistical significance at 48 and 72 h  and miRNA-140 mimics on ADAMTS-5 expression at the cellular level, qRT–PCR was performed    , while the expression of ADAMTS-5 protein in the miRNA-140 inhibitor group was significantly upregulated (P < 0.05).After 72 h of cell culturing, protein was extracted for western blot analysis Fig. . At the P < 0.05), and the difference at 6 weeks was not as profound as that of week 2, and there was almost no difference by 12 weeks  , positive cell rate in miRNA-140 mimics group is also larger than the blank group, 2 W difference is statistically significant (P < 0.05).The results of H&E staining for paraffin-fixed sections of specimens showed that there was no significant change in the cartilage structure of the early SIS3 and miRNA-140 groups compared to the other groups, but a rough cartilage surface and a reduced number of chondrocytes were observed after 6 weeks Fig. . QuantitP < 0.05).Immunohistochemical analyses for ADAMTS-5 and Smad3 proteins were performed on paraffin-fixed sections of specimens Fig. . CompareP < 0.05).The results for the severity of cartilage degeneration showed that in the early stage of the SIS3 and miRNA-140 mimic groups, the cartilage surface was smooth and without irregular cracks Fig. . The numOA is a complex disease characterized by deterioration in the structure, function, and metabolism of the entire joint and peri-joint tissue and is characterized by irreversible cartilage damage, inflammation, and chondrocyte phenotypic changes . NumerouGene silencing in OA chondrocytes by transient transfection of specific small interfering RNAs (siRNAs) has identified the binding site of the miRNA-140 regulatory sequence in OA chondrocytes, and SMAD3 can directly regulate the binding site of miR-140 [Aggrecanase  is a member of the newly cloned detangulating protein metalloproteinase family in recent years, with platelet thrombin-sensitive proteoid modal regions that bind to and participate in ECM metabolism , 43. TheUsing ADAMTS-4 and ADAMTS-5 single-gene knockout mice, it was found that compared with wild-type mice, gene knockout animal models showed a significant delay in the process of cartilage degeneration, of which ADAMTS-5 was the key enzyme for proteoglycan degradation in OA articular cartilage in mice , so ADAMLocal intra-articular injection of vascular endothelial growth factor has been reported to accelerate articular cartilage degeneration in rat osteoarthritis models , and cliIn our study, we found that in the early stage of OA, SIS3 and miRNA-140 mimics can significantly delay cartilage degeneration, the number of chondrocytes is not significantly reduced, and the tidal line is intact. SMAD3 is able to regulate ADAMTS-5 expression, and the two are a positive regulatory relationship.This study innovatively uses the method of intra-articular injection of SIS3 and miRNA-140 mimics, breaking the traditional method of administration through blood vessels and intraperitoneal injection, so that it acts directly on cartilage in the joint cavity, which can reduce the catabolism and ensure the high concentration of drugs. The limitation of this study is that it only pays attention to the changes of early OA, fails to repeatedly carry out intra-articular injection to observe the progress of mid- and late OA, because there will be metabolic inactivation and concentration reduction of intra-articular injection drugs and lentivirus. Effective utilization cannot be guaranteed, and future experiments need to be improved.Inhibition of SMAD3 can upregulate the expression of miRNA-140, which in turn inhibits the expression of ADAMTS-5. Concurrently, SMAD3 inhibition can reduce the in vivo and in vitro expression of ADAMTS-5, which has been confirmed at both the protein and gene levels. Our study demonstrated that the expression of target genes and proteins at both the protein and gene levels changed significantly at the early time point; hence, we inferred that SMAD3 could mediate miRNA-140 to regulate the expression of ADAMTS-5 in the early stages of OA, and in-depth studies are vital to reveal the specific regulatory mechanism of OA pathogenesis and provide therapeutic strategies for the clinic.Additional file 1.


Hair follicle stem cells (HFSCs) reside in the bulge region of the outer root sheath of the hair follicle. They are considered slow-cycling cells that are endowed with multilineage differentiation potential and superior proliferative capacity. The normal morphology and periodic growth of HFSCs play a significant role in normal skin functions, wound repair and skin regeneration. The HFSCs involved in these pathophysiological processes are regulated by a series of cell signal transduction pathways, such as lymphoid enhancer factor/T-cell factor, Wnt/β-catenin, transforming growth factor-β/bone morphogenetic protein, Notch and Hedgehog. The mechanisms of the interactions among these signaling pathways and their regulatory effects on HFSCs have been previously studied, but many mechanisms are still unclear. This article reviews the regulation of hair follicles, HFSCs and related signaling pathways, with the aims of summarizing previous research results, revealing the regulatory mechanisms of HFSC proliferation and differentiation and providing important references and new ideas for treating clinical diseases. Signaling pathways in HFSCs driving physiological/pathological changes in the HFSC niche have been confirmed.In view of HFSC signaling pathways, the mechanisms underlying therapeutic strategies for alopecia and other skin diseases are summarized.The crosstalk between signaling pathways in HFSCs and how this determines the fate of HFSCs is also briefly discussed.in vitro proliferation ability. HFSCs are adult stem cells and the main stem cell markers that they express are CD34, K15, Gli1, LIM homeobox 2 and SRY-related high mobility group-box gene 9 (SOX9) , which bind through cell-to-cell interactions . The NotHes-1 is constitutively expressed in the epidermis, eccrine glands and hair follicles and can promote cell cycle progression in the basal layer of the epidermis, thereby inducing the proliferation of keratinocytes . A reducNICD is an indicator of the activation of the Notch signaling pathway. Studies have found that the Notch signaling pathway inhibitor, 4,6-diamino-2-phenylindole, can specifically block the action of γ-secretase, thereby preventing the activation of Notch . The NotDuring hair follicle morphogenesis, the Notch signaling pathway plays a small role in determining cell fate, but it is essential for the maintenance of hair follicle structure . StudiesThe ligand of Notch is also a type I transmembrane protein; the extracellular domain also contains multiple EGF-like repeats and the intracellular domain is very short. When Notch receptors and ligands between adjacent cells interact through their EGF-like repeat sequences, the related proteases catalyze the proteolytic cleavage of Notch receptors in the transmembrane region so that the Notch intracellular region is released from the inside of the cell membrane and enters the nucleus. The Notch intracellular region entering the nucleus can interact with the transcription factor recombination signal binding protein J (RBP-J) through the regulation of amino acid metabolism (RAM) domain to activate the transcription of the promoter containing the RBP-J recognition site, thereby regulating the expression of cell differentiation-related genes such as Hes family molecules. RAM domain-mediated Notch intracellular segment activity is related to the binding of the transcription factor RBP-J, which activates the transcription of Notch intracellular segments. In transgenic mice that specifically block the expression of RBP-J, hair follicle bulge cells differentiate into epidermal cells, inhibiting the formation of hair. The Notch/RBP-J pathway can inhibit the differentiation of HFSCs into epidermal cells and promote their differentiation into hair follicle epithelial cells . The aboThe HH pathway is an important pathway for signal transduction between the epidermis and mesenchyme and is involved in intracellular regulation. The highly conserved HH pathway is one of the main signaling pathways in the human body. It plays an important role in controlling homeostasis and tissue development. It can promote the development of the epidermis and hair follicles, repair damage and maintain the characteristics of hair follicle bulge stem cells ; howeverThe HH protein is composed of an intracellular signal peptide, a highly conserved N-terminal region and a variable C-terminal domain. It can regulate the expression of N-Myc and cyclin D2 during embryonic hair follicle development and cooperate with the Wnt/β-catenin signaling pathway to participate in mature hair follicle development . The claIn vertebrates, Gli has three main members, Gli-1, Gli-2 and Gli-3. Relevant studies have shown that SHH-dependent Gli-1 and Gli-2 activation play a key role in the formation of hair follicles. During embryonic hair follicle development, SHH activates Gli-1 and increases the expression of Gli-1 mRNA, resulting in a faster hair growth cycle and increased hair . Gli-2 iStudies have shown that the SHH signaling pathway regulates the development of hair follicles by acting in the epithelium and mesenchyme, but the HH signaling pathway is not involved in the early stages of hair follicle development. In the mature stages of hair follicle development, a reduction in SHH selectively reduces the hair follicle epithelium. The proliferation of keratinocytes in the middle stages prevents epithelial cells from growing downstream ,112. NotHH signaling has recently been identified as an important signaling pathway that drives the proliferative activation of HFSCs. The detailed molecular mechanisms underlying this activity necessitate further investigation.et al. used a subtype-specific antisense oligonucleotide probe strategy to study the function of AKT and its three subtypes and found that among the three AKT subtypes, primarily AKT2 was involved in the activation of downstream substrates  ,117. Phoin vitro. After epidermal or HFSC injury, activated AKT phosphorylation is significantly increased and the hair follicle cell cycle is upregulated [et al. inhibited the PI3K/AKT signaling pathway with perifosine (krx-0401) and LY294002. They found that the inhibition of the PI3K/AKT signaling pathway could prevent hair follicles from transitioning from the telogen phase to the anagen phase. In addition, a PTEN inhibitor was used to enhance the activity of the PI3K/AKT signaling pathway. The PTEN inhibitor was found to induce the transformation of hair follicles from the telogen phase to the anagen phase. Relevant studies have confirmed that the PI3K/AKT signaling pathway can promote the transformation of hair follicles from telogen to early anagen, induce the proliferation of interfollicular epidermal stem cells and HFSCs, further activate the PI3K/AKT signaling pathway by releasing relevant molecules (such as cytokines and growth factors), promote hair follicle regeneration and accelerate wound healing [The PI3K/AKT signaling pathway is important for hair follicle growth and hair follicle reconstruction egulated ,120. To  healing . Recent  healing ,125. In HFSCs represent promising candidates for stem cell-based therapies for alopecia, skin cancer, skin inflammation and skin wound healing . SignaliPolygonum multiflorum extract could promote hair follicle cycling or accelerate the onset of anagen and delay hair follicle catagen via the activation of the Wnt/β-catenin signaling pathway in HFSCs [It is generally accepted that targeting the Wnt/β-catenin signaling pathway is a promising clinical treatment strategy for the regeneration of hair follicles and the downregulation of hair loss. Androgen receptor inhibitors, such as flutamide, bicalutamide, fluridil, cyproterone acetate and spironolactone, work with β-catenin to suppress Wnt signaling, thereby stimulating HFSC proliferation and differentiation and leading to hair follicle regeneration and partial hair regrowth . Tocotriin HFSCs ,133,134.in HFSCs . For wouin HFSCs .Thuja orientalis-extract and P. multiflorum-extract, which are traditionally used to treat baldness and hair loss, promoted hair growth by inducing the anagen phase in resting hair follicles. Additionally, early induction of β-catenin and Shh proteins in hair follicles of extract-treated patients was revealed [HH pathways are known to be involved in inducing the transition from the telogen phase to the anagen phase . Recent revealed ,139. Of revealed . These sCurrently, the food and drug administration (FDA)-approved medications for treating baldness are Finasteride® and Minoxidil®, in addition to surgical procedures such as hair transplantation. Minoxidil has a direct effect on promoting hair growth by stimulating HFSCs and epithelial cells . This coMany drug candidates targeting HFSCs have been shown to be effective in treating alopecia, healing wounds and addressing other skin diseases. However, the mechanisms underlying hair development induced by HFSC activation are still incompletely understood, which limits the clinical application of drug candidates. Future exploration will involve the precise regulation of niche-specific HFSCs by controlling their related signaling pathways during clinical treatment.The Wnt pathway plays an important role during hair follicle induction, SHH participates in morphology development and advanced differentiation, Notch signaling determines the fate of HFSCs and BMP is involved in hair follicle stem cell differentiation . The WntSome molecular agents have been found to modify the networks between signaling pathways in HFSCs and the hair follicle cycle. Eng is a key molecular component in the dynamic crosstalk between Wnt/β-catenin and TGF-β/BMP signaling in the hair follicle cycle. β-Catenin directly binds to the Eng promoter in a BMP signaling-dependent manner and interacts with Smad4 in a BMP/Eng-dependent context . Dlx3 isThere are also interactions between HFSCs and other cells, such as inflammatory cells and fibroblasts. Studies have found that the BMP-MAPK signaling pathway can protect the survival of HFSCs in the inflammatory environment by enhancing the expression of sirtuin-1 . In addiHFSCs are promising sources for wound healing. Upon injury, HFSCs near the wound site mobilize toward it for wound bed re-epithelialization and barrier restoration. Wnt/β-catenin signaling has been shown to play a central role during wound healing, and many transcription factors take part in or are downstream of this pathway ,160. Wouin vitro and multidirectional differentiation potential. With the development of various technologies such as gene knockout and transgenic technology, the use of HFSCs in skin self-renewal and tissue repair and regeneration will be further developed, and our understanding and research on HFSCs will also increase. This work will play a particularly important role in tissue engineering, regenerative medicine and other fields. For example, a recent study found that adding an activated Wnt signaling pathway to dermal papilla cells can stimulate the development of hair follicles, which is a key step in hair growth. These cells can be used in wound repair and skin regeneration [HFSCs are one of the most common adult stem cell types in the hair follicle bulge. They are feasible for use in promoting wound healing and are currently being used in regenerative medicine. HFSCs have advantages including a wide number of sources, large numbers, minimal damage to the body during isolation, strong expansion ability neration , in the neration , in the neration , all of In summary, hair follicles and their stem cells play an important role in promoting wound repair and maintaining normal skin function and the hair follicle cycle, during which they are regulated by LEF/TCF, Wnt/β-catenin, TGF-β/BMP, Notch, HH and PI3K/AKT signaling pathways, among others. The precise regulation of these large and complex signaling pathways effectively controls the proliferation and differentiation of HFSCs. Although the related signaling pathway network systems and specific clinical applications of HFSCs are still in the laboratory stage, current research results have indicated that there are wide range of application prospects, especially with regards to research on stem cells, and the use of tissue engineering may be valuable in solving clinical problems in the future.AKT: Protein kinase B; APC: Adenomatous polyposis coli; APM: Arrector pili muscle; Axin: Axis inhibition protein; BMP: Bone morphogenetic protein; CK1: Creatine kinase1; DKK3: Dickkopf3; EGF: Epidermal growth factor; Gli: Glioma; GPCR: G protein-coupled receptor; GSK-3β: Glycogen synthase kinase-3β; HES: Hairy and enhancer of split; HFSCs: Hair follicle stem cells; HH: Hedgehog; LEF: Lymphoid enhancer factor; LRP5/6: Lipoprotein receptor-related protein5/6; NICD: Notch intracellular domain; PDK1: PI3K dependent kinase 1; PDK2: PI3K dependent kinase 2; PI3K: Phosphoinositide 3-kinase; PTEN: Phosphatase and tensin homolog; RBP-J: Recombination signal binding protein J; Runx: Runt-related transcription factor; SHH: Sonic hedgehog; SOX9: SRY-related high mobility group-box gene9; TCF: T-cell factor; TGF-β: Transforming growth factor-β.XXW, RHY and XDC designed the experiments. XXW and YHL drafted the manuscript. XXW and RHY drew the figures and JH and JRW participated in modification of the paper. All the authors read and approved the final manuscript.This study was supported by the National Natural Science Foundation of China , the Medical Scientific Research Foundation of Guangdong Province (A2018113), the Guangdong Basic and Applied Basic Research Foundation , the Special Fund of Foshan Summit plan  and the Foundation of Foshan City .None declared.


Acinetobacter, Sphingomonas, and Pseudomonas were the advantage genera in SS. While, Lactococcus, Weissella, and Pediococcus were dominant in 3-day SSS and subsequently replaced by Lactobacillus in 60-day SSS. Spearman’s correlation heatmap showed that Pediococcus and Leuconostoc were negatively related to the pH value of SSS. There were great differences in the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional profiles of SS and SSS. Ensiling process downregulated the metabolism of amino acid, energy, cofactors, and vitamins, but upregulated the metabolism of nucleotides and carbohydrates. Overall, next-generation sequencing in conjunction with KEGG functional prediction revealed the distinct differences in the initial and late phases of ensiling in terms of both community succession and functional shifts. The knowledge regarding bacterial community dynamics and functional shifts of SS during ensiling is important for understanding the fermentation mechanism and may contribute to the production of high-quality sweet sorghum silage.The purpose of this study was to evaluate the fermentation quality, microbial community, and functional shifts of sweet sorghum during ensiling. The high-moisture sweet sorghum (SS) was naturally ensiled for 1, 3, 7, 15, 30, and 60 days. After 60 days of ensiling, sweet sorghum silage (SSS) showed homolactic fermentation with absent butyric acid, low pH value, acceptable concentrations of propionic acid, ethanol, and ammonia nitrogen and high lactic acid concentration.  Sorghum bicolor (L.) Moench) is a perennial economic or feed crop belonging to the genus Sorghum of Gramineae and native to eastern Africa. It has been extensively spread in more than 100 countries due to its characteristics in terms of high light, nitrogen and water use efficiency, and large biomass yield -driven anaerobic fermentation process involving complex microbial interactions and activities. On this basis, the microbial ecology involved in the silage process needs to be studied at great length . To dateBased on this, this study aimed to evaluate the fermentation quality, microbial community, and functional shifts of high-moisture sweet sorghum (SS) during ensiling.2) was equally separated into three blocks (replicates) and eight plots in each block were randomly chosen for silage making. After 12 weeks of growth, SS was harvested at the heading stage and chopped into 2 cm theoretical lengths by a feed cutter. After mixing thoroughly, the chopped SS was split into two parts for silage preparation and fresh material analysis, respectively. A total of 18 bags (6 ensiling time × 3 replicates per treatment) were prepared. Specifically, approximately 450 g of mixed material was packed into a pre-sterilized laboratory silo , sealed by an Aomitai DZD-400 vacuum sealer , and ensiled under ambient temperature (27.5 ± 2.5°C) for 1, 3, 7, 15, 30, and 60 days.Sweet sorghum was planted in the Jianghuai Watershed Experimental Station . The experimental field  was mixed thoroughly in an ethanol-sterilized container. About 300 g of SS or SSS was oven-dried at 105°C for 15 min (deactivation of enzymes) and 65°C for 48 h to determine DM content. Subsequently, the dried sample was ground by a laboratory knife mill equipped with a 1-mm screen. The water-soluble carbohydrate (WSC) content of SS or SSS was determined by the anthrone-sulfuric acid method . The neu3-N) concentration of SSS was determined by the phenol-hypochlorite method , as reported in our previous study . The ammonia nitrogen , and 60-day SSS (SSS-60) were sampled for the analyses of bacterial diversity and potential function. The bacterial DNA extraction and polymerase chain reaction (PCR) amplification were conducted according to the procedures reported in our previous study . The purThe functional profiles of the bacterial community in SS and SSS were predicted using the Tax4fun tool. First, the 16S rRNA profile based on the SILVA database was transformed into a taxonomic profile of prokaryotes based on the KEGG database. Then, the 16S rRNA copy number was applied to normalize the abundance of KEGG prokaryotes. Finally, the functional profiles from sequencing data were predicted by the linear combination of KEGG prokaryotes functional profiles and normalized taxonomic abundances .p < 0.05.One-way ANOVA was applied to investigate the effects of ensiling time on fermentation parameters, chemical composition, microbial number, and α-diversity indexes of silages based on the general linear model of SAS . Meanwhile, the analysis of similarity (ANOSIM) with 10,000 permutations was adopted to statistically compare the difference in β-diversity of bacterial community, and the Wilcoxon rank-sum test was applied to analyze the differences in KEGG functional profiles. The statistical differences were significant at 10 CFU/g FM, respectively.p < 0.05) affected the pH value, the concentrations of LA, AA, VFA, and NH3-N as well as the content of DM and WSC. The pH significantly (p < 0.05) decreased during the first 7 days of ensiling reaching the minimum of 3.96 on day 15 of ensiling, and then increased gradually (p > 0.05). The LA concentration exhibited the opposite trend to the pH value, with the highest concentration of 5.36% DM on day 30 of ensiling. The concentrations of AA, VFA, and NH3-N increased as ensiling proceed (p < 0.01). Low concentration of PA was detected throughout the ensiling process. No n-BA, IBA, or 1,2-propanediol was found in all SSS. As ensiling proceeded, the DM and WSC content significantly (p < 0.01) decreased.As shown in p < 0.001) effects on the number of LAB, aerobic bacteria, and yeasts (p < 0.001) increased to the maximum of 9.84 log10 CFU/g FM in the first 30 days of ensiling, then decreased significantly (p < 0.001). While the number of aerobic bacteria, yeast, molds, and enterobacteria constantly decreased to low or undetectable levels.Ensiling time had significant  increase in RA of Firmicutes was accompanied by the significant (p < 0.05) decrease in RA of Proteobacteria, Actinobacteriota, and Bacteroidota , followed by Sphingomonas (10.9%), Pseudomonas (9.15%), and Microbacterium (8.17%). As ensiling proceed, the RA of Acinetobacter, Sphingomonas, Microbacterium, and Enterobacter, etc. significantly (p < 0.05) decreased, while the RA of Lactobacillus, Lactococcus, and Pediococcus significantly (p < 0.05) increased (Lactococcus (62.2%), Weissella (18.2%), and Pediococcus (8.60%) were the advantage genera (Lactobacillus (89.9%) predominated its bacterial community (There were 21 genera with a RA greater than 1% in SS . The mosncreased . In SSS-e genera . And in ommunity .Lactobacillus was positively correlated with AA and VFA but negatively correlated with WSC content (p < 0.05). Also, there were negative correlations between RA of Lactobacillus and the number of aerobic bacteria, yeasts, molds, and enterobacteria. The RA of Pediococcus and Leuconostoc was negatively correlated with pH value and NH3-N concentrations while positively correlated with WSC content (p < 0.05). The concentrations of LA, AA, PA, and VFA were negatively correlated with the RA of all detected genera (top 30) except Lactobacillus and unclassified_f__Lactobacillaceae.The Spearman’s correlation analysis of fermentation parameters and bacterial community is visualized through a heatmap . The RA As shown in Under pathway level 3 , the car10 CFU/g FM) of SS far satisfied the requirement for well-preserved silage (10 CFU/g FM) might pose challenges to the production of high-quality silage.For ensiling, the WSC content and epiphytic LAB number are the key factors determining the quality of silage. In the present study, the WSC content (> 5% DM) and LAB number  was no more than the maximum limit (10% TN) for well-preserved silage  and the obvious proliferation of LAB . The ensiling process promoted the growth of Firmicutes because they are typical microbes in anaerobic and acid environments  is favorable for the establishment of an initial acidic environment and the growth of rod-shaped LAB (Lactobacillus), which is the prerequisite to ensuring the quality of silage  and most detected genera further confirmed that short-chain fatty acids have a broad spectrum of bacteriostatic properties  pathway, because heterofermentative LAB possesses the PPP (3-N during ensiling. And the acceptable NH3-N content (< 100 g/kg TN) in this study might be due to the suppression of valine, leucine, isoleucine, and lysine degradation.To further clarify the functional shifts of the bacterial community in SS and SSS, the carbohydrate metabolism and amino acid metabolism were specifically analyzed under pathway level 3. The metabolism of most carbohydrate components  was promoted with the process of ensiling, which confirmed that LAB can utilize a variety of carbohydrate sources for their proliferation. Among them, the high abundance of fructose, mannose, galactose, sucrose, amino sugar, and nucleotide sugar metabolism in SSS are primarily related to nditions . It is k the PPP . Amino aThe SS contains high WSC content and sufficient LAB number, which can be used as a promising alternative to silage maize for silage production. The ensiling process had remarkable effects on fermentation parameters, bacterial community, and functional profiles of sweet sorghum silage. The study could be a reference for providing comprehensive insights into the fermentative mechanism of silage through NGS and KEGG functional prediction analyses.The data presented in the study are deposited in the Sequence Read Archive (SRA), accession number PRJNA843380. We have released the above data.JZ and TS conceived and designed the research, contributed to manuscript revision, read, and approved the submitted version. JZ and XJ conducted experiments. JL and SW contributed analytical tools. JZ and ZD analyzed the data. JZ wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.This study was financially supported by the Joint Fund for Regional Innovation and Development of the National Natural Science Foundation of China (U20A2003).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Flaviviridae family are posing a significant threat to human health worldwide. Many flaviviruses are capable of inducing severe inflammation in humans. Flaviviridae nonstructural proteins, apart from their canonical roles in viral replication, have noncanonical functions strongly affecting antiviral innate immunity. Among these functions, antagonism of type I IFN is the most investigated; meanwhile, more data are accumulated on their role in the other pathways of innate response. This review systematizes the last known data on the role of Flaviviridae nonstructural proteins in molecular mechanisms of triggering inflammation, with an emphasis on their interactions with TLRs and RLRs, interference with NF-κB and cGAS-STING signaling, and activation of inflammasomes.Members of the  Flaviviridae is a family of enveloped ss(+RNA) viruses, consisting of four genera: Flavivirus, Hepacivirus, Pegivirus, and Pestivirus. Members of Flavivirus are mosquito-borne and tick-borne viruses, most of which infect humans, causing a spectrum of clinical symptoms from mild febrile illness to severe hemorrhagic fever or neuroinvasion. It includes Zika virus (ZIKV), West Nile virus (WNV), dengue virus (DENV), Yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and also non-mammal viruses such as Duck Tembusu virus (DTMUV). Hepacivirus includes bloodborne human Hepatitis C virus (HCV). Genus Pestivirus includes viruses that infect nonhuman mammals, such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV). Pegivirus includes several viruses infecting different groups of mammals comprising humans.Flaviviridae are widespread all over the world. DENV infection is endemic in more than 100 countries, and accounts for approximately 400 million infections per year with 96 million exhibiting a severe disease condition, and thus poses a major public problem Flavivirifor DENV , and TBEnfection . Furthernfection , no specFlaviviridae infections very often include significant systemic inflammatory reactions. Generally, the common mechanism of inflammation implies four steps: sensing of damage-/pathogen-associated molecular patterns (DAMPs/PAMPs) by PRRs (pattern recognition receptors); triggering of molecular signaling cascades, such as NF-κB, cGAS-STING, and IFN pathways; the release of proinflammatory cytokines and chemokines; and the recruitment of inflammatory cells [Flaviviridae infections.The symptoms of human ry cells . Severe ry cells ,10,11,12ry cells ,14, and ry cells ,17,18,19ry cells ,21,22. Iry cells ,25,26,27ry cells ,29, and Flaviviridae. Flaviviridae RNA genome is approximately 10–11 kb and is represented by a long ORF flanked by 3′ and 5′-UTR with a 5′-end cap [It is well studied that inflammatory reactions are triggered by the propagation of -end cap ,31. The -end cap ,33 into -end cap .Structural flaviviral proteins build up viral particles and play the role in antiviral immune response. E protein is a glycoprotein of 53 kDa, forming the shell of flaviviruses, responsible for binding of the virus to the receptors ,36, and Flaviviridae species, and may be essential in the viral life cycle processes, including RNA synthesis, the formation of the replication complex, and virion assembly/maturation/secretion [cis and trans cleavage of the viral polyprotein, generating nonstructural proteins, and the C-terminal NTPase/RNA helicase. The NS2B cofactor is necessary for NS3 protease activity [Nonstructural proteins of flaviviruses, though not being a part of viral particles, play different roles in the viral life cycle, and have multiple functions beyond this. NS1 is a 40–50 kDa glycoprotein, depending on the glycosylation status, which is cleaved from NS2A , and traecretion . It alsoecretion ,55. NS2Becretion . NS2B inecretion . It alsoecretion ,59. NS2Aecretion ,59,60. Nactivity . NS4A haactivity , has a tactivity . It alsoactivity . It playactivity ,66. NS4Bactivity ,68, whicactivity ,69. NS5 activity . Its N-tactivity , and itsactivity . Viral ractivity ,74. In aFlaviviridae interact with PRRs acting as PAMPs and may interfere with downstream signaling pathways. Nonstructural proteins of flaviviruses, besides their roles in the viral life cycle, interact with host cells and interfere with cellular functions. They can activate or inhibit immune signaling pathways leading to the activation or abrogation of inflammatory and antiviral responses. In this review, Flaviviridae nonstructural protein interactions with TLRs, RLRs, cytoplasmic DNA sensors, and NLRs, as well as their interference with downstream cascades, are discussed. A discussion of the JAK-STAT signaling pathway in view of Flaviviridae nonstructural proteins is omitted, as this aspect is covered by several recent reviews [The key factors involved in triggering inflammatory and antiviral responses are PRRs. They include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), AIM2-like receptors, C-type lectin receptors, and cytoplasmic DNA and RNA sensors ,76,77. T reviews ,80,81,82TLR is a type I transmembrane glycoprotein consisting of three domains: N-terminal extracellular domain with a variable number of leucine-rich repeats (LRRs) motifs, which is responsible for the recognition of a pathogen; a transmembrane domain; and a cytoplasmic Toll-interleukin 1 receptor (TIR) homology domain, for the recruitment of adaptor proteins, such as MyD88 (myeloid differentiation primary response 88) and TRIF (TIR domain-containing adapter inducing IFN-β) ,84. CurrThe sensing of the ligands by TLRs stimulates two signaling cascades: the MyD88-dependent pathway, which mainly leads to the production of proinflammatory cytokines and chemokines; and TRIF-dependent pathway, which leads to the production of IFNs and proinflammatory cytokines . The TRIIn the MyD88-dependent pathway, after PAMPs sensing, TLRs homo- or heterodimerize and recruit MyD88, which forms a complex with IRAK1 and IRAK4 (IL-1 receptor associated kinase family members) . IRAK4 aIn specific immune cells subsets, such as plasmacytoid dendritic cells, TLR7- and TLR9-MyD88-dependent signaling can trigger the production of type I IFNs. This requires the recruitment of TRAF3 (TNF receptor associated factor 3) and IKKα to the MyD88-IRAK-TRAF6 complex. Then, IRAK1 and IKKα phosphorylate transcription factor IRF7 (interferon regulatory factor 7) , which tThe activation of TLR3/TLR4 results in the interaction of TRIF with a multiprotein signaling complex consisting of TRAF6, TRADD, Pellino-1 (a member of the Pellino family E3 ubiquitin ligases), and RIP1 (receptor-interacting protein 1), which activates the TAK1 and IKK complex, and, finally MAPK and NF-κB ,115. In Thus, both MyD88-dependent and TRIF-dependent TLR signaling result in the activation of NF-κB and IFN responses, by different mechanisms.Flaviviridae. TLR3 is a classic receptor for viral dsRNA and can recognize the dsRNA of ZIKV, WNV, DENV, ATMUV (Avian Tembusu Virus), and JEV [TLRs sense the dsRNA of many viruses including  and JEV ,121,122. and JEV . Interes and JEV . TLR7 is and JEV ,129,130.DENV interference with TLR signaling is extensively studied. DENV, as described above, causes severe hemorrhagic fever, with vascular leakage, and the establishment of the reasons for this is the focus of many studies. DENV NS1 protein behaves as a virulence factor in many of these studies. NS1 exists in different cellular locations in several oligomeric forms, having different functions. During DENV infection, monomers of NS1 arrange in dimers in the ER, and these dimers remain membrane-associated, either in vesicular compartments within the cell or on the cell surface membrane . Three dE. coli derived NS1 was used, which exhibited wrong protein folding and was monomeric under all the conditions [However, in the case of DENV, there are also controversial data. For example, DENV-infected patients suffering from a more severe form of dengue with hemorrhagic fever in comparison to the patients with milder dengue, displayed higher NS1 serum levels correlated with low TLR4 expression and impaired TLR4 signaling during the acute febrile phase of the disease  Table 1Table 1. nditions . Additionditions . Modhiranditions .Anti-DENV antibodies to NS1 are also capable of exhibiting an interesting double-faced effect. From one side, a bunch of studies indicated that both vaccine/infection-derived and monoclonal anti-NS1 antibodies have protective activity in vivo ,154,155.Data on the WNV evasion of TLR signaling are concerned with the interaction of the WNV NS1 protein with TLR3 . SeveralFlaviviridae-Pestivirus, had a similar effect on MyD88, by interacting with it and thus decreasing MyD88 levels and MyD88-mediated TLR4 signaling in macrophages of BVDV-infected calves [Pestivirus genus), which increased the activation of MyD88 and subsequently, elevated IFN-α levels in porcine monocyte-derived macrophages [Several HCV nonstructural proteins are also involved in TLR signaling . This isd calves   like receptors) are cytosolic PRRs  expresseRIG-I/MDA5 signaling is dependent on the adapter protein MAVS , which is also variously known as the IFN-β promoter stimulator-1 (IPS-1), VISA , or CARDIF (CARD adaptor inducing IFN-β). MAVS is located on the outer membranes of mitochondria, peroxisomes, and in the mitochondrial-associated endoplasmic reticulum membrane (MAM) , and conSensing and signaling activities of RIG-I and MDA5 should be under tight control in order to maintain immune homeostasis in a normal physiological state and to provide an effective antiviral defense during infections. The short descriptions of only some of these control mechanisms, for which flavivirus nonstructural proteins’ interference has been demonstrated, are mentioned below.The relocation of RIG-I and MDA5 from the sites of RNA recognition to mitochondria and MAMs is an essential event in RLR signaling and is mediated by the members of the 14-3-3 protein family. The 14-3-3ε protein forms a complex with RIG-I and TRIM25 (Tripartite motif-containing protein 25), stabilizes their interaction, and mediates their translocation to MAVS on MAMs . SimilarFlaviviridae species, including YFV, TBEV, WNV, JEV, DENV, CSFV, and ZIKV [Flaviviridae infections, including WNV, DENV, HCV, CSFV, and ZIKV [RIG-I can sense plenty of and ZIKV ,220,221.and ZIKV ,223,224.The suppression of the initial sensors of the RLR pathway, MDA5 and RIG-I, is demonstrated for several flaviviral nonstructural proteins . Direct Flaviviral nonstructural proteins, predominantly proteases, can inhibit RLR signaling also through its adaptor MAVS, by different mechanisms . ZIKV NSRLR signaling may also be inhibited by the effects on scaffold 14-3-3 proteins, which normally help to relocate RIG-I and MDA5 to mitochondria . ZIKV NSZIKV strategies of RLR inhibiting are also redundant, as they include suppression of MDA5, RIG-I, MAVS, 14-3-3ε, and 14-3-3η by its NS2A, NS3, NS4A, and NS5 proteins ,244,246 Activation of the NF-κB pathway and of the initial stages of IFN response are the processes downstream of TLR and RLR signaling. The detailed mechanisms of NF-κB pathway activation and of the initial stages of IFN response have been discussed above, in TLR signaling and RLR signaling sections, and the ways of interference with these pathways by flavivirus nonstructural proteins are discussed below .Flaviviridae have evolved strategies to target the NF-κB pathway, which may have several outcomes. The most widespread strategy exploited by a large number of viruses is the inhibition of NF-κB which dampens proinflammatory and immune responses of the host [Viruses including the host ,250,251.the host . Activatthe host . The actthe host ,254 and the host . InteresHCV NS2 and NS5B proteins are shown to upregulate NF-κB activation ,256 Tab. The mecDENV NS2B3, contrary to HCV NS3, promotes activation of NF-κB , which rIn addition, two different mechanisms of interaction with TRAF6 are found for flaviviral nonstructural proteins but both promote viral replication . CSFV NSThe unique mechanism of NF-κB inhibition is described for JEV NS5. NS5 interacts with the proteins of nuclear transport which help to translocate IRF3 and p65 (NF-κB subunit) to the nucleus, thus inhibiting downstream NF-κB and IFN signaling , DENV NBeyond cytoplasmic RNA sensors, cytoplasmic DNA sensors are known, such as cGAS . cGAS is activated by binding to cytoplasmic dsDNA, and catalyzes the cyclization of AMP and GMP in the cytoplasm producing the noncanonical cyclic dinucleotide 2′3′-cGAMP, which serves as the second signal, binding to STING (stimulator of IFN genes) dimers . This reFlaviviridae and may limit their replication [Interestingly, cGAS, initially known as a sensor of microbial DNA, was found playing an important role in limiting infections by RNA viruses. cGAS detects members of lication ,279. Onelication ,280. Howlication . ZIKV allication . JEV canlication .DENV NS2B3 has been demonstrated to cleave human STING and thus to inhibit IRF3 activation and IFN response in several studies ,286,287 YFV and HCV NS4B protein have also been shown to interact with STING and thus to disrupt its interactions with downstream proteins ,293,294 The family of NOD-like receptors (NLRs) consists of more than 22 members, which are characterized by the presence of a central NOD or nucleotide binding (NBD) domain, also known as NACHT, flanked with a C-terminal LRR (leucine-rich repeat) domain, and a variable N-terminal interaction domain . NLRs arThe first NLRs found to detect bacterial components were NOD1 and NOD2, also called NLRC1 and NLRC2. They sense bacterial peptidoglycan, and this triggers the release of auto-inhibitory conformation, oligomerization of NOD1/2, and recruitment of the receptor-interacting protein kinases 2 (RIP2) via CARD–CARD interactions, and activation of NF-κB and MAPKs, which drive the expression of proinflammatory genes and antimicrobial responses ,299. OveThe most widely studied NLRs, NLRP3, NLRP1, and NLRC4, represent a part of a multi-protein complex called inflammasome. Microbial PAMPs and DAMPS are sensed by these NLRs and trigger inflammasome activation . InflammThe NLRP3 inflammasome complex also includes the adaptor protein ASC (an apoptosis associated speck-like protein containing a CARD) composed of two domains (PYD and CARD) and an effector protein pro-caspase-1 . ActivatThe basal expression levels of NLRP3, pro-IL-1β, and pro-IL-18 are usually low, and a two-step process is required for priming and activation of the inflammasome ,316. TheFlaviviridae, which cause inflammatory reactions, are capable of activating inflammasomes. This was demonstrated for several viruses, including JEV, DENV, WNV, HCV, and ZIKV, and in some cases, viral proteins are involved in these processes  aimed at bove 90% . Experimlymerase . These dlymerase . One of lymerase . In addilymerase . In thislymerase . DENV NSlymerase . Furtherlymerase , and thuparanase . Currentparanase  and hepaparanase ,372 may Flaviviridae may pose a larger threat than before, and cause significant outbreaks or even a pandemic. With this scenario, the continuation of flavivirus studies, the development of antivirals, and their translation into clinical practice are extremely essential.In view of global warming, growing freedom of movement all over the world, and the SARS-CoV-2 pandemic, which brings long-term negative effects on human health, in particular on the immune, nervous, and cardiovascular systems,


Plasma TSLP in COVID-19 patients significantly correlated with duration of hospitalization, while SARS-CoV-2 induced TSLP secretion from bronchial epithelial cells in vitro. Based on our findings, TSLP may be considered an important therapeutic target for COVID-19 treatment.Thymic stromal lymphopoietin (TSLP) is an epithelium-derived pro-inflammatory cytokine involved in lung inflammatory responses. Previous studies show conflicting observations in blood TSLP in COVID-19, while none report SARS-CoV-2 inducing TSLP expression in bronchial epithelial cells. Our objective in this study was to determine whether TSLP levels increase in COVID-19 patients and if SARS-CoV-2 induces TSLP expression in bronchial epithelial cells. Plasma cytokine levels were measured in patients hospitalized with confirmed COVID-19 and age- and sex-matched healthy controls. Demographic and clinical information from COVID-19 patients was collected. We determined associations between plasma TSLP and clinical parameters using Poisson regression. Cultured human nasal (HNEpC) and bronchial epithelial cells (NHBEs), Caco-2 cells, and patient-derived bronchial epithelial cells (HBECs) obtained from elective bronchoscopy were infected in vitro with SARS-CoV-2, and secretion as well as intracellular expression of TSLP was detected by immunofluorescence. Increased TSLP levels were detected in the plasma of hospitalized COVID-19 patients , the levels of which correlated with duration of stay in hospital : 0.01–0.21). In cultured NHBE and HBECs but not HNEpCs or Caco-2 cells, TSLP levels were significantly elevated after 24 h post-infection with SARS-CoV-2 ( Thymic stromal lymphopoietin (TSLP), an epithelium-derived pleiotropic cytokine, is considered a master regulator of the early innate immune response to a range of airborne environmental factors and pathogens. The release of TSLP from the airway epithelium stimulates innate lymphoid cells, T cells, dendritic cells, and mast cells to augment their effector functions and promote the amplification of innate and adaptive immune responses in infected tissues ,2,3. In A major cell type in the lung innate immune system is the airway epithelial cell, which has the capacity to produce a wide range of alarmins and pro-inflammatory cytokines in response to many triggers including respiratory viruses that can rapidly overwhelm immune responses. TSLP, interleukin (IL)-25, and IL-33 are three alarmin-type cytokines that are initially generated by epithelial cells in response to infection by RNA viruses including coronaviruses ,10,11,12In this study, we investigated the production of TSLP during SARS-CoV-2 infection in hospitalized COVID-19 patients and its association with other factors including duration of hospital stay. The effects of SARS-CoV-2 on TSLP production was also determined in cultured human nasal, intestinal, and bronchial epithelial cells.n = 11, confirmed COVID-19-positive by polymerase chain reaction testing for spike protein (SP), provided by Alberta Health Services) and experiencing symptoms requiring hospitalization were admitted to the non-intensive care unit (non-ICU) of the University of Alberta Hospital in Edmonton, Alberta, Canada in the period 18 October–25 November 2020. Clinical parameters upon hospitalization such as heart rate, respiratory rate, body temperature, oxygen saturation in blood (SpO2), comorbidities, and the interval between the date of the COVID-19-positive test result and the date of hospitalization were assessed from electronic medical records on the first day of hospitalization. Patients testing positive for SARS-CoV-2 (Inclusion criteria were that (1) participants provided informed consent prior to any study specific procedures, (2) they were 18–80 years old, and (3) that they were admitted to hospital for COVID-19 symptoms. Exclusion criteria were any participants (1) receiving an investigational drug product within five half-lives of the drug, (2) receiving inhaled corticosteroids prior to hospital admission, (3) receiving biological agents within the preceding six months, or (4) unable to provide informed consent. n = 11) on the first date of hospitalization into BD Vacutainer® EDTA tubes  and centrifuged at 1000× g for 15 min at 4 °C. For comparison, we recruited 10 age- and sex-matched healthy control participants without a history of COVID-19 infection for plasma collection. Plasma supernatants were collected, aliquoted, and snap-frozen on dry ice and ethanol for storage at −80 °C before sample analysis. Plasma samples from COVID-positive patients were also collected from the Canadian BioSample Repository, which is a biobank facility located at the University of Alberta campus . Venous blood samples were collected from nonvaccinated COVID-19-positive patients (2 in a humidified incubator. Cells were cultured on ethanol-washed glass coverslips until they reached ~80% confluency. Attached cells were then subjected to mock infection (vehicle-treated with BEGM media) or SARS-CoV-2 virus infection  for 24 h in a biosafety level 3 containment facility in a humidified incubator at 37 °C and 5% CO2. Infection was terminated by the addition of 4% buffered paraformaldehyde, and coverslips were then processed for immunolabeling. Human bronchial epithelial cells ) and the intestinal cell line Caco-2 from the American Tissue Culture Collection (ATCC) as well as nasal epithelial cells (HNEpC) from PromoCell  were cultured under sterile conditions in bronchial epithelial growth media (BEGM) plus supplements  at 37 °C with 5% COg for 5 min at 4 °C, and the pellet was resuspended in BEGM before being transferred to coverslips or flasks and grown to ~80% confluency prior to infection as described above for NHBE cells.To test TSLP release from freshly isolated airway epithelial cells, we obtained human bronchial epithelial cells (HBECs) from bronchial brushings from six noninfected individuals undergoing elective bronchoscopies for unidentified lung masses at the University of Alberta Hospital Endoscopy unit. Patients with suspected or confirmed SARS-CoV-2 infection were excluded from sample collection. Bronchial brushings were collected using cytology brushes from the surface of apparently healthy right or left main stem bronchus that was distant from any apparent lung mass and were placed in BEGM transport medium to be subsequently analyzed. Samples were then vortexed, and brushes were removed from media. HBECs were centrifuged at 300× Frozen plasma samples were thawed and subjected to analysis using Meso Scale Discovery Multiplex Assay kits  following manufacturer’s protocols. Specific kits used in this report were an ultra-sensitive S-Plex human TSLP kit with a lower limit of detection (LLOD) of 9.1 fg/mL (lower limits of quantification (LLOQ) 34 fg/mL), with an approximately 1000-fold higher sensitivity than other commercially available assay kits for human TSLP. V-Plex human Cytokine Panel 1 biomarker multiplex kits were also used for IL-1α, IL-5, IL-7, IL-12/IL-23p40, IL-15, IL-16, IL-17, vascular endothelial growth factor A (VEGF-A), tumor necrosis factor b (TNF-β)/lymphotoxin α (LTα), CCL2/monocyte chemoattractant protein 1 (MCP-1), CCL3/macrophage inhibitory protein-1α (MIP-1α), CCL4/MIP-1β, CCL11/eotaxin, CCL13/MCP-4, CCL17/thymus and activation-regulated chemokine (TARC), CCL22/monocyte-derived chemokine (MDC), CCL26/eotaxin-3, and CXCL10/interferon-γ-induced protein 10 (IP-10), with an LLOD of 0.04–9.33 pg/mL.Coverslips with attached NHBEs or HBECs were fixed in 4% buffered paraformaldehyde (pH 7.4) at room temperature for 20 min, washed with phosphate-buffered saline (PBS), and permeabilized with 0.1% Triton X-100 in PBS, pH 7.4 for 10 min. After washing and blocking in PBS with 2% bovine serum albumin and 5% goat serum, cells were labeled with primary rabbit anti-TSLP antibody  and in some cases double-labeled with diluted human sera obtained from volunteers who had been vaccinated against SARS-CoV-2 spike protein (SP) (Hobman lab). Primary antibodies were detected using anti-rabbit IgG conjugated to Alexa 647 for anti-TSLP  and anti-human IgG conjugated to Alexa 488 for anti-SP  respectively. Counterstaining was carried out with rhodamine- or Alexa Fluor 647-conjugated phalloidin stain for actin cytoskeleton and Hoechst stain for nuclei . The specificity of immunostaining was verified via isotype controls. All images were collected on an Olympus IX81 epifluorescence microscope and analyzed using cellSens software , and Volocity image analysis software  was used to determine fluorescence intensity. Cell outlines or regions of interest (ROI) were automatically defined using phalloidin (cytoskeleton stain) and the mean fluorescence intensity was calculated using the sum of all pixel values within each ROI . 2 on admission, number of comorbidities, and the interval between the date of the COVID-19-positive test result and the date of hospitalization were considered as potential confounders. The goodness of fit of the models was tested by Akaike’s information criteria (AIC) [Demographic, clinical, and cytokine data were described as mean (standard error of mean (SEM)), median (interquartile range (IQR)), or frequency (%) for continuous, ordinal, and categorical variables, respectively. To compare the levels of cytokines between the infected (COVID-19 patients) group and uninfected controls, we used a one-way analysis of covariance (ANCOVA) for each cytokine adjusting for sex and age. Spearman’s correlations were computed for the association between TSLP and all other cytokines with Šidák’s correction. As data regarding clinical factors were not available for the control group, we restricted the additional analyses to the infected group only. To find the association between TSLP levels and period of hospitalization (days), we used a generalized linear (Poisson) regression model. Based on prior evidence, age, sex, heart rate, respiratory rate, body temperature, and SpOia (AIC) , and potp-value < 0.05 was considered as statistically significant.For cellular data, we presented intracellular TSLP expression as median (IQR) and analyzed the difference in TSLP release between mock- and SARS-CoV-2-infected NHBEs and HBECs by Mann–Whitney test. For the subgroup analysis including isotype controls, mock- and SARS-CoV-2-infected cells, we used the Kruskal–Wallis test with Dunn’s post hoc test for multiple comparisons. All analyses were performed in STATA v17.0  and violin plots were constructed in GraphPad Prism Version 9.0 . A The majority of healthy controls and hospitalized COVID-19 patients recruited for this study were males  and were of a similar age  . COVID-1p = 0.25). We also observed a > 2-fold increase in IL-15 in COVID-19 patients (2.3 ± 0.1 for controls vs. 5.7 ± 0.8 pg/mL for COVID-19 patients) and a 10-fold increase in CXCL10/IP-10 over controls . In contrast, TNF-β/LTα levels were lower in COVID-19 patients (0.16 ± 0.04 pg/mL) than controls . A similar decrease in COVID-19 patients was observed for CCL13/MCP-4  and CCL22/MDC . All other cytokines measured in this study did not differ significantly between the two groups. We observed a significant positive correlation between TSLP and IL-17 , CCL2/MCP-1 , and CCL11/eotaxin  but not with other measured cytokines. However, these associations did not remain significant after corrections for multiple testing   to 997.6 ± 241.4 fg/mL in hospitalized COVID-19 patients . Althoug > 0.05) .p = 0.03) : 0.11; 95% confidence interval (CI): 0.01 to 0.21 per 100 fg/mL increase in plasma TSLP level,  = 0.03) . We did p < 0.0001) (+) indicating infection by SARS-CoV-2 virus showed a significant increase in intracellular TSLP compared to mock-infected and SP- SARS-CoV-2-infected cells   A,B. This 0.0001) . Furtherp < 0.0001)  A,B. HBECpulation C. Howeveed cells C.Additionally, we determined if other epithelial cell types that are SARS-CoV-2-compliant ,18,19,20Our findings indicate that plasma levels of TSLP, IL-15, and CXCL10/IP-10 were elevated in COVID-19 patients over that of normal uninfected controls. Although the difference in TSLP levels did not achieve a statistically significant level, it has to be noted that a clinically significant observation may not always satisfy statistical criteria for significance. Our analysis also suggests, for the first time, that plasma levels of TSLP at the time of hospitalization could plausibly indicate the period of hospitalization in COVID-19 patients. In parallel with this, we confirmed that SARS-CoV-2 infection induces TSLP production in cultured bronchial epithelial cells as a likely source of plasma TSLP in infected patients.The function of TSLP appears to be that of a master cytokine that has the potential to initiate an inflammatory cascade during a virus infection, leading to the cytokine storm observed during SARS-CoV-2 infection. However, whether SARS-CoV-2 infection initiates an increase in TSLP production has been contradictory ,13,14,15SARS-CoV-2 infections trigger systemic autoimmune and inflammatory responses which may be regulated through the release of TSLP. Several clinical manifestations of SARS-CoV-2-induced autoimmunity ,22 and rElevated plasma TSLP levels in this report are supported by our observations using an immunofluorescence approach in cultured epithelial cells to detect in vitro TSLP expression during SARS-CoV-2 infection. A significant increase in TSLP immunofluorescence was observed upon infection by SARS-CoV-2 in NHBEs, suggesting the de novo synthesis of this cytokine in response to the virus. NHBE cells have previously been used as a model for SARS-CoV-2 studies to examine host immune responses and complement complement pathway activation ,27. SARSInterestingly, no significant differences in supernatant TSLP was observed when comparing mock to SARS-CoV-2 infection after 24 h in two other epithelial cell cultures from different tissue sources . This suggests that the production and release of TSLP from bronchial epithelial cells is not a universal response to SARS-CoV-2 infection amongst epithelial cells in the body and appears to be specific to the lower airway epithelium. It would be interesting to explore TSLP expression in other epithelial cells from other tissue sources beyond this study.+ cytotoxic T cells, as well as epithelial cells [In addition, we found significantly elevated IL-15 in plasma from hospitalized COVID-19 patients at equivalent levels to those previously reported . IL-15 ial cells . Numeroual cells ,30.A more substantial 10-fold increase in CXCL-10/IP-10 was observed in hospitalized COVID-19 patients compared with healthy controls. CXCL10/IP-10 is a pro-inflammatory chemokine that attracts a broad range of leukocytes including monocytes/macrophages, B cells, T cells, and other cell types . It has A major strength of this study is that our findings demonstrate for the first time that plasma TSLP may be a determinant of the period of hospitalization in COVID-19 patients. The clinical significance of this finding is that SARS-CoV-2-induced TSLP could be a useful indicator of disease severity, which is measured as length of stay in hospital, as well as an important target for therapeutic intervention at early stages of COVID-19 disease. The SARS-CoV-2-induced cytokine storm is a major clinical risk factor for disease severity leading to ARDS-like illness in COVID-19 . The tarDespite these important findings, our study had some limitations that should also be addressed. The sample size was necessarily small, as this was designed as a pilot proof of concept study to evaluate the levels of TSLP following SARS-CoV-2 infection. However, a larger sample size would be required to achieve adequate power to confirm our initial findings. Secondly, we were unable to measure TSLP levels in patients immediately after testing positive. Therefore, the temporality of plasma TSLP levels between the initial virus infection and the day of hospitalization could not be determined. Prospective clinical and experimental studies are required to investigate temporal changes in TSLP over the time. Finally, we were unable to distinguish between long-form and short-form TSLP proteins, which are shown to have opposite immune functions , due to In conclusion, our study has generated novel findings regarding the involvement of TSLP in SARS-CoV-2 as a determinant of the period of hospitalization. Our findings also suggest that this is an important inflammatory mechanism and speculate that this mechanism may be amenable to therapeutic intervention in patients with COVID-19.