TrkA was achieved by tamoxifen injection in 4-week-old fl/fl;NaV1.8CreERT2TrkA  mice. OA was induced by partial medial meniscectomy (PMM) in 12-week-old mice, and OA-pain-related behavior was analyzed for 12 weeks followed by comprehensive histopathological examinations. OA-associated joint pain was markedly improved without cartilage protection in sensory-neuron-specific conditional TrkA knock-out (cKO) mice. Alleviated hyperalgesia was associated with suppression of the NGF/TrkA pathway and reduced angiogenesis in fibroblast-like synovial cells. Elevated pain transmitters in the DRG of OA-induced mice were significantly diminished in sensory-neuron-specific TrkA cKO and global TrkA cKO mice. Spinal glial activity and brain-derived neurotropic factor (BDNF) were significantly increased in OA-induced mice but were substantially eliminated by sensory-neuron-specific deletion. Our results suggest that augmentation of NGF/TrkA signaling in the joint synovium and the peripheral sensory neurons facilitate pro-nociception and centralized pain sensitization.Tropomyosin receptor kinase A (TrkA/NTRK1) is a high-affinity receptor for nerve growth factor (NGF), a potent pain mediator. NGF/TrkA signaling elevates synovial sensory neuronal distributions in the joints and causes osteoarthritis (OA) pain. We investigated the mechanisms of pain transmission as to whether peripheral sensory neurons are linked to the cellular plasticity in the dorsal root ganglia (DRG) and are critical for OA hyperalgesia. Sensory neuron-specific deletion of  Osteoarthritis (OA) is characterized by progressive, degenerative joint damage involving articular cartilage deformation of joint structure and severe pain. OA is a global disease and is a leading cause of disability that affects over 300 million worldwide . DespiteIn the early stages of OA, both meniscal and hyaline articular cartilage are damaged, followed by architectural alterations of the subchondral bone. As OA progresses, synovitis and cartilage erosion can occur, physiological cartilage remodeling and repair follow, and the subchondral bone responds biologically by thickening . As OA aNGF is not only critical for the growth and survival of neurons, but also plays a significant role in pain transmission and sensation . PreclinTrkA mutations can change protein folding, retention in the endoplasmic reticulum (ER), and/or cause aggregation [NGF binds to NGF receptors, promoting multiple pain signaling pathways, leading to nociceptive pain . TrkA isregation . Differeregation ,21, and regation .One line of investigation has explored the concept that alteration of sensory neurons  and funcTrkA in the peripheral sensory neurons , a global deletion , and a cartilage-specific deletion  in mice. Our main results show that sensory-neuron-specific deletion of TrkA significantly reduces OA-associated joint pain, spinal glial activity, and central pain transmitters but does not preserve cartilage integrity. The mitigation of hyperalgesia is attributable to the suppression of the NGF/TrkA pathway in fibroblast-like synovial cells and synovial angiogenesis. Our findings support the hypothesis that NGF/TrkA signaling in the joint synovium towards peripheral sensory neurons facilitates pro-nociception and pain centralization.Here, we investigated the pathological role of the NGF/TrkA pathway in OA-related pain transmission after partial medial meniscectomy (PMM)  at 12 wePKCδ null mice [TrkA, a cognate receptor of NGF, by global tamoxifen-inducible ablation of the gene in fl/fl;RosaCreERT2TrkA mice with five consecutive days of intraperitoneal (i.p) injection of tamoxifen at 4 weeks of age. Pain sensitivity and histopathological changes in the knee joint following OA induction by PMM surgery were analyzed. Pain tests measuring mechanical allodynia using von Frey filaments and other pain-associated behavior including increased frequency of grooming activity (data not shown) indicated that global deletion of TrkA in mice  shows insensitivity to OA pain compared to Cre-negative control mice that develop OA-associated hyperalgesia measured by withdrawal force thresholds  , while fl/fl;RosaCreERT2TrkA-negative mice displayed significant sensitivity in 8 weeks after PMM  sensory neurons, according to our previous studies with ull mice . In orde < 0.01) A. Five a < 0.01) , and the < 0.01)  showed nfter PMM . The aceive mice .TrkA mice (Cre positive) and fl/fl;RosaCreERT2TrkA-negative littermates, these two groups showed no statistically significant difference in the development of OA-like pathological progression, as observed in Safranin-O fast green staining  and their litter mate controls, fl/fl;RosaCreERT2TrkA-negative mice (n = 11). Immunofluorescence staining of TrkA and NGF in the lumbar L3-L5 DRG isolated at 12 weeks after PMM surgery were monitored. The expression levels of TrkA were significantly (p < 0.00001) reduced in fl/fl;RosaCreERT2TrkA-positive mice in the lumbar DRG (TrkA significantly (p < 0.013) reduced the expression of NGF  and their littermate controls, fl/fl;AggrecanCreERT2TrkA-negative mice (n = 6), followed by harvesting of knee joints at 12 weeks after PMM surgery. Development of mechanical allodynia by von Frey filament testing in the ipsilateral hind paw was compared with fl/fl;AggrecanCreERT2TrkA-negative mice and fl/fl;AggrecanCreERT2TrkA-positive mice every week until 12 weeks after PMM surgery. Mice with a cartilage-specific deletion of TrkA did not exhibit significant differences in pain sensation  sensitivity to heat after 8 weeks after PMM, but there was no differences between cKO mice or negative control mice. In the acetone tests, fl/fl;AggrecanCreERT2TrkA-negative mice displayed significant cold sensitivity in 12 weeks after PMM (p < 0.05), while fl/fl;AggrecanCreERT2TrkA-positive mice displayed no significant sensitivity compared with pre-PMM or cKO mice  in C57Bl/6 mice at 13 weeks after PMM surgery, there was no improvement in pain reduction from chronic osteoarthritis A. HoweveTrkA using Cre recombination under control of the promoter for voltage-gated sodium channel (NaV1.8/SCN10A). fl/fl;NaV1.8CreERT2TrkA mice were generated by crossing fl/flTrkA mice with V1.8CreERT2Na, and conditional ablation was induced by systemic administration of 2 mg tamoxifen for 5 consecutive days starting at 4 weeks of age. OA was induced by PMM in 12-week-old fl/fl;NaV1.8CreERT2TrkA-positive mice and their corresponding littermate controls , followed by pain testing every week and harvesting knee joints, DRG, and spinal cords (SCs) at 12 weeks after PMM surgery.To understand the function of NGF/TrkA signaling in peripheral sensory neurons, we selectively and conditionally deleted fl/fl;NaV1.8CreERT2TrkA-negative and fl/fl;NaV1.8CreERT2TrkA-positive mice following PMM or sham surgery. Sensory-neuron-specific deletion of TrkA in mice  abolished OA-associated hypersensitivity , as shown in TrkA. Hence, loss of TrkA did not significantly improve cartilage structure protection compared to mice in the fl/fl;NaV1.8CreERT2TrkA-negative control group , reflecting the efficiency of sensory neuron-specific deletion of TrkA in fl/fl;NaV1.8CreERT2TrkA-positive mice. TrkA intensity measured in ImageJ software from the National Institutes of Health  significantly increased sixfold from a baseline of 2.96 to 17.71 upon OA induction . However, TrkA intensity decreased to 1.82 upon sensory-neuron-specific loss of TrkA   E. These RT2 mice D,E.fl/fl;NaV1.8CreERT2TrkA-positive mice on the basis of immunofluorescence staining . However, levels of NGF expression return to close to the control level 1.92  (NGF expression was significantly reduced in staining F and quastaining G. NGF inive PMM) F,G. Thesfl/fl;NaV1.8CreERT2TrkA-negative and fl/fl;RosaCreERT2TrkA-negative mice compared to sham control mice. PGP9.5-positive nerve fiber density was calculated as the signal intensity per nerve fiber surface area, as reflected by the number of nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) blue. PGP9.5-positive structures (green) in the knee joint synovium were significantly reduced upon loss of TrkA in both fl/fl;NaV1.8CreERT2TrkA-positive and fl/fl;RosaCreERT2TrkA-positive mice compared with both negative control fl/fl;NaV1.8CreERT2TrkA-negative and fl/fl;RosaCreERT2TrkA-negative mice . However, PGP9.5 expression levels in OA induced mice were completely reduced to 0.43 and 0.63 in fl/fl;NaV1.8CreERT2TrkA-positive and fl/fl;RosaCreERT2TrkA-positive OA mice, respectively   antibody A. The reive mice A, and thive mice B. PGP9.5ctively) A,B.TrkA in the peripheral sensory neurons. Hence, inactivation of NGF/TrkA signaling can effectively block OA-associated pain transmission.These results indicate that the degree of OA pain correlated with increased peripheral nerve fibers in knee joints of OA mice and deletion of TrkA in sensory neurons. Immunofluorescence staining for CD31 in the synovial vasculature of the ipsilateral knee joint of 12 weeks after PMM surgery showed no obvious aggressive angiogenesis in fl/fl;NaV1.8CreERT2TrkA-positive mice and fl/fl;RosaCreERT2TrkA-positive mice compared to fl/fl;NaV1.8CreERT2TrkA negative and fl/fl;RosaCreERT2TrkA negative PMM mice. CD31 expression increased strongly (18-fold) in the ipsilateral knee joint of both fl/fl;NaV1.8CreERT2TrkA-negative mice and fl/fl;RosaCreERT2TrkA-negative mice at 12 weeks after PMM surgery, as shown by immunofluorescence staining with anti-CD31 antibody  and to 24.26 in fl/fl;RosaCreERT2TrkA-negative PMM mice (p < 0.0004), respectively. This elevation of CD31 signals was severely reduced to intensity to 2.66 in fl/fl;NaV1.8CreERT2TrkA-positive mice (p < 0.0007) and 4.32 in fl/fl;RosaCreERT2TrkA-positive PMM mice (p < 0.0015) compared to their respective negative control PMM mice  and 18.18 with fl/fl;RosaCreERT2TrkA-negative PMM mice (p < 0.013), respectively. However, this increase in VEGF levels was strongly mitigated (85% and 97%) by loss of TrkA to intensity 1.59 (p < 0.025) in fl/fl;NaV1.8CreERT2TrkA-positive OA mice and 1.36 (p < 0.015) in fl/fl;RosaCreERT2TrkA-positive PMM mice  in the ipsilateral knee joint of antibody E. Howeveive mice F. As expPMM mice E,F.Collectively, our results indicate that NGF/TrkA signaling in sensory nerves is important for angiogenesis and that inhibition of this pathway may alleviate both OA-enhanced angiogenesis and pain sensation.fl/fl;NaV1.8CreERT2TrkA-negative and fl/fl;RosaCreERT2TrkA-negative mice with sham control mice). However, the OA-induced elevation of these cytokines was impeded substantially upon TrkA loss in fl/fl;NaV1.8CreERT2TrkA-positive and fl/fl;RosaCreERT2TrkA-positive mice.We next measured tumor necrosis factor-α (TNF-α/TNF) and interleukin-1β (IL-1β/IL1B) expression, the most common pro-inflammatory cytokines in OA pathology. To identify the tissue source of inflammatory cytokine production, we examined expression levels of TNF-α and IL-1β. Protein levels of TNF-α G,H and Ifl/fl;NaV1.8 CreERT2TrkA-negative and fl/fl;RosaCreERT2TrkA-negative mice at 12 weeks after PMM surgery, as shown in immunofluorescence staining  and the intensity increased from 1.6 to 25.8 in fl/fl;RosaCreERT2TrkA-negative PMM mice (p < 0.0005). However, expression of TNF-α in OA-induced mice was reduced to 9.84 in fl/fl;NaV1.8CreERT2TrkA-positive mice (p < 0.0018) and 3.21 in fl/fl;RosaCreERT2TrkA-positive mice (p < 0.001)  H.fl/fl;NaV1.8CreERT2TrkA-negative mice and increased 34.7-fold in fl/fl;RosaCreERT2TrkA-negative mice at 12 weeks after PMM surgery, as shown by immunofluorescence staining with anti-IL-1β antibody  and increased from 0.53 to 18.5 in fl/fl;RosaCreERT2TrkA-negative mice (p < 0.028). The expression decreased to an intensity of 5.97 in fl/fl;NaV1.8CreERT2TrkA-positive PMM mice (p < 0.029) and 5.29 in fl/fl;RosaCreERT2TrkA-positive OA-induced mice (p = 0.073)  J.TrkA-deleted OA mice showed substantial reductions in the expression of pro-inflammatory cytokines, this inhibition was only partial because pro-inflammatory cytokine activities still remained in the knee joints of PMM-induced TrkA cKO mice and facilitated the destruction of joint integrity. This result may explain why loss of TrkA greatly relieves pain transmission , as confirmed in the intensity quantification  and increased from 1.09 to 23.5 in fl/fl;RosaCreERT2TrkA-negative mice (p < 0.001). However, expression of MMP-13 in OA-induced TrkA cKO mice remained high in both sensory-neuron-specific deletion  and in global deletion  of TrkA , fails to eliminate MMP-13, a cartilage-degrading enzyme in PMM-induced OA mice. This finding indicates that TrkA/NGF signaling impacts pain transmission but not the joint pathology, at least in part, because it has no effect on the MMP-13 activity in mediating cartilage destruction.Immunofluorescence of MMP-13 showed NGF/TrkA is not a critical mediator in joint destruction in OA. Deletion of fl/fl;NaV1.8CreERT2TrkA-positive and fl/fl;NaV1.8CreERT2TrkA-negative mice . SP intensity in sham control mice was 0.43 and was significantly increased to 78 in fl/fl;NaV1.8CreERT2TrkA-negative PMM mice (p < 0.0003) and 37.25 in fl/fl;RosaCreERT2TrkA-negative OA mice (p < 0.00005). However, loss of TrkA resulted in 99% reduction of SP expression to control level 0.80 in fl/fl;NaV1.8CreERT2TrkA-positive OA mice (p < 0.00030) and 77% reduction to 8.65 in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.004) (Immunofluorescence staining for substance P (SP: green) co-stained with the neuronal marker NeuN (red) in DRG A and the< 0.004) A,C.fl/fl;NaV1.8CreERT2TrkA-positive mice (p < 0.006) and 79% reduction in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.003) compared to their Cre negative littermate control OA mice. CGRP intensity in sham mice was 2.09, and it was significantly increased to 54.24 in fl/fl;NaV1.8CreERT2TrkA-negative OA mice (p < 0.003) and 48.75 in fl/fl;RosaCreERT2TrkA-negative OA mice (p < 0.014). Over 90% reduction of CGRP expression in both TrkA cKO mice returned the level down to 6.91 in fl/fl;NaV1.8CreERT2TrkA-positive OA mice (p < 0.01) and 11.66 in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.01)  visualized with NeuN (red) co-staining showed 91% reduction in  < 0.01) B,D.TrkA in sensory neurons significantly reduces pain molecules SP and CGRP in DRG. SP and CGRP are also known as angiogenesis neuropeptides and it is suggested that the NGF/TrkA signaling in sensory nerve has significant effects on angiogenesis for alleviating pain sensation and OA-induced hyperalgesia.This indicates that the deletion of fl/fl;NaV1.8CreERT2TrkA-negative mice was increased sixfold in spinal cords (p < 0.0004) and fourfold in fl/fl;RosaCreERT2TrkA-negative OA mice (p < 0.0031) but was reduced by 83% in OA-induced fl/fl;NaV1.8CreERT2TrkA-positive mice (p < 0.0029) and 100% in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.0009). Quantitative analyses of IBA-1 was calculated as the intensity of IBA-1(green) area  area A,B. Thisfl/fl;NaV1.8CreERT2TrkA-negative mice was increased 16-fold and in fl/fl;RosaCreERT2TrkA-positive OA mice in spinal cords compared to sham control mice, which increased 8-fold . However, loss of TrkA was reduced completely in OA-induced fl/fl;NaV1.8CreERT2TrkA-positive mice (p < 0.00008) and by 93% in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.003)  C,D.fl/fl;NaV1.8CreERT2TrkA-positive mice and drastically reduced in fl/fl;RosaCreERT2TrkA-positive OA mice. This change in astroglial activation was due to deletion of TrkA in sensory neurons, which impedes retrograde transportation of augmented NGF levels upon OA induction. Hence, astroglial activity to produce inflammatory cytokines and pain molecules is significantly abolished. Interestingly, complete reduction of astroglial cell activity is achieved in sensory-neuron-specific deletion in that NGF and pain molecule retrograde transportation is solely accountable to deletion of TrkA in sensory neurons.This finding indicated that astroglial cell activation due to OA induction was abolished in fl/fl;NaV1.8CreERT2TrkA-negative and fl/fl;RosaCreERT2TrkA-negative OA mice at 12 weeks after PMM compared to sham control mice. TNF-α expression was increased fourfold in SC of OA-induced fl/fl;NaV1.8CreERT2TrkA-negative mice and increased sixfold in fl/fl;RosaCreERT2TrkA-negative mice compared to the sham surgery mice . However, TNF-α expression in SC was 93% reduced in fl/fl;NaV1.8CreERT2TrkA-positive mice (p < 0.0016) and 72% reduced in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.001). TNF-α intensity in sham-operated control mice was 4.6 and greatly increased to 20.3 upon OA-induction in fl/fl;NaV1.8CreERT2TrkA-negative mice (p < 0.0041) and 26.8 in fl/fl;RosaCreERT2TrkA-negative mice (p < 0.0005). However, TNF-α expression in OA-induced TrkA cKO mice was significantly reduced to 5.7 in fl/fl;NaV1.8CreERT2TrkA-positive OA mice (p < 0.0016) and 10.8 in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.001)  and interleukin-1β (IL-1β) expression in SCs to see whether OA-induced glial cell activity produced inflammatory cytokines. Expression levels of TNF-α E,F and I< 0.001) E,F.fl/fl;NaV1.8CreERT2TrkA-negative mice and increased 12-fold in fl/fl;RosaCreERT2TrkA-positive OA mice at 12 weeks after PMM surgery, as shown by immunofluorescence staining with anti-IL-1β antibody  and 87% reduced in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.03)  and 11.37 in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.015). A total of 100% reduction occurred at intensity 0.5 in fl/fl;NaV1.8CreERT2TrkA-positive OA mice (p < 0.00002) and a 87% reduction in 2.24 in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.03) (IL-1β expression was increased 5-fold in the SC of antibody G. Howeve < 0.03) G,H. IL-1 < 0.03) G,H.TrkA exhibited partial reduction of pro-inflammatory cytokines in the knee joints of OA-induced fl/fl;NaV1.8CreERT2TrkA-positive mice, expression of TNF-α and IL-1β was reduced by over 90% in spinal cords. This indicated that glial cell activity due to retrograde pain transportation was significantly reduced by the sensory neuron-specific deletion of TrkA.Even though sensory-neuron-specific deletion of TrkA completely inhibited CGRP expression in the SC of OA-induced fl/fl;NaV1.8CreERT2TrkA-positive mice and fl/fl;RosaCreERT2TrkA-positive mice  and a 7-fold increase to 14.1 in OA-induced fl/fl;RosaCreERT2TrkA-negative mice (p < 0.003). However, expression of CGRP in PMM-induced mice was reduced to 0.86 in fl/fl;NaV1.8CreERT2TrkA-positive PMM mice (p < 0.012) and to 2.73 in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.004), indicating complete inhibition of glial activity , a key pain mediator in the central nervous system (CNS), and whether BDNF is diminished upon sensory-neuron-specific deletion of fl/fl;NaV1.8CreERT2TrkA-positive mice and fl/fl;RosaCreERT2TrkA-positive mice following PMM surgery  and increased 14-fold to 11.29 in fl/fl;RosaCreERT2TrkA-negative OA mice (p < 0.00003). However, expression of BDNF was reduced to 0.41 in fl/fl;NaV1.8CreERT2TrkA-positive PMM mice (p < 0.016) and to 2.67 in fl/fl;RosaCreERT2TrkA-positive OA mice (p < 0.0004). This showed a complete inhibition of the central pain mediator BDNF . We previously observed that augmentation of NGF caused increased expression of TrkA, which is the cognate receptor for NGF . In our TrkA (as the cognate NGF receptor) fails to protect from OA-associated hyperalgesia. This finding suggests that NGF signaling in cartilage may not be catabolic or that TrkA deletion in cartilage is not sufficient to block the progression of OA. In contrast, deletion of TrkA in sensory neurons modulated the activity of NGF, angiogenesis, and pro-inflammatory cytokines in the knee joint of mice upon OA induction after PMM surgery. More importantly, mice experienced remarkable pain relief during early to advanced stages of OA in our study. The protection from OA-induced hyperalgesia upon sensory-neuron-specific deletion of TrkA is directly supported by our observations that loss of TrkA decreased peripheral nerve fibers in knee joints and reduced angiogenesis.In knee joints, NGF is produced by fibroblast-like synoviocytes and/or chondrocytes under OA conditions. Yet, cartilage-specific deletion of TrkA in OA-induced mice did not fully deactivate pro-inflammatory cytokine production, in agreement with the idea that the levels of TNF-α and IL-1β correlate with joint pathology but not with the level of pain.Expression of pro-inflammatory cytokines was partially reduced in the synovium but was completely abolished in the spinal cord. This result indicates that these pro-inflammatory cytokines in the synovium play important roles in joint integrity. The complete ablation of cytokine production observed in the spinal cord was the result of the inactivation of glial cells, which are able to produce these inflammatory factors when provoked. NGF may upregulate IL-1β through TrkA and NF-κB/NFKB1-dependent caspase-1/CASP1 activation in human monocytes . Pro-infTrkA deletion or global TrkA deletion. Therefore, we propose that MMP-13 is a critical factor for joint pathology protection, but its expression is not completely dependent on the NGF/TrkA pathway.The matrix metalloprotease MMP-13/MMP13 is a major collagen-degrading enzyme responsible for cartilage destruction in OA, and its inhibition has been considered a strategy to block OA development . Our stuTrkA in sensory neurons significantly reduced expression of pain molecules such as SP and CGRP in DRG and spinal cords that also act as angiogenic neuropeptides. NGF/TrkA signaling in sensory neurons stimulated angiogenesis factors such as VEGF and CD31, and, as a consequence, contributed to angiogenesis. This result suggests that NGF/TrkA signaling in sensory nerves contributes significantly to angiogenesis while alleviating pain sensation and OA-induced hyperalgesia.Deletion of TrkA inactivation. OA induces expression of the brain-derived pain mediator BDNF by peripheral NGF, and this induction of BDNF was completely abolished upon sensory-neuron-specific deletion of TrkA, which inactivates the NGF/TrkA signaling pathway.Activated glial cell activity in our OA mouse model may have involved retrograde centralized pain transportation as a key mechanism that improves hyperalgesia upon peripheral sensory neuronal inhibition of NGF/TrkA signaling. Both microglial and astroglial cell activities were modulated, indicating that hyperalgesia in both early and advanced OA can be treated by TrkA is as efficient as a global deletion and is sufficient for alleviating pain. Therefore, disease intervention would benefit from local inactivation of TrkA to eliminate the risk and safety issues of systemic treatments . Beyond localized inhibition of TrkA, other interventions will be required for cartilage repair and protection in advanced OA.Our findings strongly support an emerging concept for the etiology of OA in that the severity of joint pain is due to pathological changes in the synovium as well as cellular and molecular plasticity in the sensory neurons of the innervating DRG. Our results also support the mechanistic interpretation that NGF/TrkA signaling is one of the key determinants for the painful symptomatic transition of OA. Peripheral inhibition of NGF/TrkA signaling can effectively reduce the pain mechanism that operates via retrograde centralized pain sensitization. Hence, sensory-neuron-specific deletion of Clinical studies with analgesics, including non-steroidal anti-inflammatory drugs (NSAIDs), opioid agonists, and calcitonin, have been evaluated for therapeutic benefits to treat chronic pain . Our finV1.8 is typically selectively expressed at high levels in sensory ganglion neurons but not within the CNS. Moreover, no loss of neuronal cells nor any differences in motor function or other abnormal behavior in tamoxifen-inducible Cre-ERT2 recombinase deletion has been reported [V1.8 expressing neurons) are similar.One study limitation is that our findings on NGF/TrkA signaling in nociceptive responses associated with OA were based on a post-traumatic surgical mouse model for OA that may not strictly correlate with the age-related degenerative human OA. However, our results can be further tested for human applications using human-derived sensory-neuron-like stem cells. Another limitation is that targeting NGF/TrkA signaling is effective only for the alleviation of nociception but not for preserving joint pathology. Therefore, future studies may require the intercalation of OA disease-modifying drugs that inhibit nociception and preserve joint integrity simultaneously. A new experimental model could include chondrocytic three-dimensional cultures with primary cells from human patients and mouse models in which NGF/TrkA signaling is modified using nanoparticles with controlled release. Immunofluorescence microscopy and Western blots are both semi-quantitative because immunofluorescence and chemiluminescence each have a limited dynamic range. We were not able to present additional assessments of NGF/TrkA in Western blotting due to the size limitation of DRG, where the NGF receptor TrkA is selectively expressed in nociceptive DRG neurons . Howeverreported . The behTrkA in mice abolished OA pain. (2) Sensory neuron-specific deletion of TrkA alleviated OA pain as effectively as whole-body deletion of TrkA in mice. (3) Cartilage-specific deletion of TrkA in mice led to development of joint pain and damage in OA, showing no improvement on OA progression. (4) Peripheral sensory neuron-specific deletion of TrkA modulated OA-induced sensory neuron plasticity and abolished OA pain by interfering with the NGF/TrkA signaling pathway of retrograde pain transmission. (5) Sensory neuronal TrkA deletion did not downregulate the cartilage-degrading enzyme, hence leading to cartilage degeneration progresses. (6) Peripheral inhibition of NGF/TrkA signaling can effectively reduce pain, but other interventions are needed for cartilage repair and protection. In conclusion, this study provides compelling evidence for the role of NGF/TrkA in OA-related pain perception and supports the idea that this pathway is a viable target for novel disease-modifying pharmacotherapies.Our findings can be summarized as follows: (1) Conditional whole-body deletion of fl/flTrkA) with CreERT2Rosa mice from Jackson (008463: B6.129-Gt (ROSA)26Sortm1(cre/ERT2) Tyj/J) for tamoxifen-inducible fl/fl;RosaCreERT2TrkA mice. For tamoxifen-inducible fl/fl;AggrecanCreERT2TrkA mice, TrkA floxed mice were crossed with CreERT2Aggrecan mice Crm/J). Tamoxifen-inducible fl/fl;Nav1.8CreERT2TrkA mice were generated by mating TrkA floxed mice with CreERT2Nav1.8 mice, obtained from John Wood’s lab.Experimental mice were generated by crossing TrkA floxed mice  for 5 consecutive days at 4 weeks of age. The dose was optimized and ensured for the safety of growing young mice bone health [Nav1.8-CreERT2 constructs were specific for sensory neurons, and the NaV1.8 driver does not support Cre recombination in the CNS [Tamoxifen-inducible conditional deletion of e health . Our Nav the CNS ,34,35. T the CNS .Mice were housed under standard laboratory conditions (in a temperature-controlled (21 ± 1 °C) room with a normal 12 h light/12 h dark cycle). Animal studies were performed according to guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the Jessie Brown Veterans Affairs Medical Center’s Institutional Animal Care and Use Committee . Surgeries were performed under anesthesia, and all efforts were made to minimize pain and suffering.All the surgical operations were performed under a microscope in an aseptic setting. Mice were positioned supine and given anesthesia with 1.5% isoflurane  in oxygen via a facemask. Osteoarthritis was induced in mice by partial medial meniscectomy (PMM) by cutting the medial menisco-tibial ligament (MMTL) and removing the medial meniscus (MM). Briefly, MMTL was cut to dislodge MM from the tibia, and then a portion of MM of around 1mm was cut with a surgical knife.fl/fl;RosaCreERT2TrkA-negative mice (n = 11), fl/fl;RosaCreERT2TrkA-positive mice (n = 5), fl/fl;AggrecanCreERT2TrkA-negative mice (n = 6), fl/fl;AggrecanCreERT2TrkA-positive (n = 13) mice, sensory-neuron-specific fl/fl;Nav1.8CreERT2TrkA-negative (n = 9), and fl/fl;Nav1.8CreERT2TrkA-positive (n = 12) mice. Sham surgery was performed without PMM on five wild-type mice and on fl/fl;Nav1.8CreERT2TrkA-negative (n = 8) and fl/fl;Nav1.8CreERT2TrkA-positive (n = 7) mice.We performed PMM on Longitudinal pain behavior assessments were performed weekly in PMM or sham control groups from weeks 1 to 12 after surgery.Mechanical allodynia by von Frey testing: Allodynia was evaluated on the basis of hind paw withdrawal from mechanical stimuli ,37. AfteActivity monitor: Animals were tested in clean vivarium plastic cages (42 × 25 × 20 cm) enclosed in a cage rack Photobeam Activity System . One set of photobeams was placed at foot level above the cage floor (with adjacent beams 5 cm apart) to measure ambulation . Another set of photobeams was placed 5 cm above the cage floor to measure rearing . Activity was monitored in a dark room for 30 min.2, and knee joints, DRG, and lumbar spinal cords were harvested for histological analysis. Safranin-O fast green staining was performed using 0.1% Safranin-O solution and 0.05% fast green solution for histological assessment of tissue morphology. The degree of joint damage was graded using the OARSI scoring system [At 12 week after surgery, mice were euthanized via COg system .Knee joints, the lumbar part of the spinal cords, and L3-5 DRG were harvested, fixed, and embedded. All samples were cut at 5 μm thickness. After removing paraffin, antigens were retrieved, permeabilized, and blocked. Sections were then incubated overnight with primary antibodies: anti TrkA , anti-rabbit tumor necrosis factor-alpha (anti-TNF-α) , anti-rabbit interleukin (IL)-1β (anti-IL-1β) , anti-rabbit nerve growth factor (anti-NGF) , anti-CD31 , anti-rabbit protein gene product 9.5 (anti-PGP9.5) , anti-substance P , anti-CGRP , anti-MMP13 , anti-BDNF , and anti-NeuN  primary antibodies, followed by incubation with Alexa 488 or Alexa 555 fluorescent antibody conjugate . Immunofluorescence was examined using a fluorescence microscope .t-test and analysis of variance (ANOVA), as well as a post hoc Tukey test. p  ≤  0.05 was considered significant.GraphPad Prism 8.02  software was used for statistical analyses. All data are presented as mean ± SEM. Pain data at each time point were normalized to those obtained pre-surgery and were analyzed using a general linear model for repeated measures. The differences between PMM and sham groups were assessed with Student’s TrkA in the peripheral sensory neurons, cartilage-specific deletion, and global deletion of TrkA. Conditional deletion of TrkA in peripheral sensory neurons improved OA-associated hyperalgesia by suppressing (i) NGF production in fibroblast-like synoviocytes, (ii) synovial angiogenesis, (iii) pain molecules, and (iv) glial cell activation. However, the collagen-degrading enzyme MMP-13 was not reduced, and thus sensory-neuron-specific deletion of TrkA failed to prevent cartilage damage in the joints of OA mice.We investigated the mechanisms of pain transmission via the ligand NGF and its cognate receptor TrkA by utilizing OA mice with conditional deletion of


Pseudorabies (PR) is a domestic and wild animal infectious disease caused by the pseudorabies virus (PRV) and is one of the major infectious diseases that endanger the global swine industry. Studies have reported that PRV may achieve cross-species transmission from pigs to humans in recent years. Therefore, in-depth exploration of the relationship between PRV and host proteins is of great significance for elucidating the pathogenic mechanism of PRV and anti-PRV infection. Here, we report that heat shock protein 27 (HSP27) ubiquitinates and degrades cyclic GMP-AMP synthase (cGAS) and attenuates cGAS-mediated antiviral responses, thereby promoting PRV infection. Overexpression of HSP27 promoted PRV proliferation in vitro, while knockdown of HSP27 inhibited PRV infection. Importantly, we found that HSP27 inhibited PRV infection or poly(dA:dT)-activated IFN-β expression. Further studies found that HSP27 may inhibit cGAS-STING-mediated IFN-β expression through targeting cGAS. In addition, we found that HSP27 can suppress the expression of endogenous cGAS in different cells at both gene transcription and protein expression levels, and that HSP27 interacts with and ubiquitinates cGAS. In conclusion, we reveal for the first time that HSP27 is a novel negative regulator of the cGAS-STING signaling pathway induced by PRV infection or poly(dA:dT) activation and demonstrate that HSP27 plays a crucial role in PRV infection. Innate immune response is the body’s first line of defense against viral infection. Virus infection could induce host innate immune responses, and this plays an important as well as decisive role in the outcome of the infected host. The crucial point is that the host can establish an antiviral status to antagonize the invasion of the virus via identifying the components of invading virus through pattern recognition receptors (PRRs), which activate signaling pathways to produce type I interferons (IFN-I) . The relFor DNA virus, Toll-like receptor 9 (TLR9), cyclic GMP-AMP (cGAMP) synthase (cGAS), DAI (DLM-1/ZBP1), absent in melanoma 2 (AIM2), and IFN gamma-inducible protein 16 (IFI16) serve as the main PRRs that recognize viral DNA ,6. PRRs Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. PRV is the causative agent of Aujeszky’s disease . Although the natural host of PRV is swine, it also threatens a wide range of other mammals [Pseudorabies virus (PRV) is a typical DNA virus. It belongs to the family  mammals ,11,12,13 mammals . PRV Bar mammals . For exa mammals . PRV UL5 mammals . In addi mammals ,19. HoweBcl2-associated athanogene (BAG) 3, which is a chaperone-mediated selective autophagy protein, plays a pivotal role in modulating the life cycle of a wide variety of viruses. During PRV infection, researchers found that PRV protein UL56 served as a novel BAG3 interactor by co-immunoprecipitation and co-localization analyses. The overexpression of pUL56 induced a significant degradation of BAG3 at protein level via the lysosome pathway. Overexpression of BAG3 significantly suppressed PRV proliferation, while knockdown of BAG3 resulted in increased viral replication in HEK293T cells . PeroxirHeat-shock proteins (HSPs) were a conserved protein family whose major roles seemed to promote the correct folding and assembly of target proteins as well as prevent their aggregation . Heat sh2 incubator in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) new bovine serum (NBS). HEK293T cells were cultured at 37 °C under 5% CO2 incubator in DMEM containing 10% (v/v) fetal bovine serum (FBS). PRV gE/gI-deleted vaccine strain Bartha-K61 was propagated and titrated in BHK-21 cells.PK15, BHK-21, and HEK293 cells were cultured at 37 °C under 5% COA plasmid encoding Myc-tagged HSP27 was constructed by standard molecular cloning techniques. The expression plasmids HA-cGAS, Myc-STING, FLAG-TBK1, FLAG-IRF3/5D (the active mutant of IRF3), and Myc-Ub were all constructed in the lab. All recombinant plasmids were identified by sequencing.The antibodies and chemical reagents used in this study were as follows: anti-Myc tag (60003-2-Ig), anti-Flag tag (20543-1-AP), anti-HA tag (51064-2-AP), anti-IRF3 (11312-1-AP), anti-HSP27 (18284-1-AP), and anti-GAPDH (60004-1-Ig) were purchased from Proteintech ; anti-TBK1 (38066) and anti-STING (D2P2F) were purchased from Cell Signaling Technology ; anti-phospho-IRF3 (AP0623) was purchased from ABclonal Technology ; anti-cGAS (D163570) and anti-Ub (D220023) were purchased from Sangon Biotech ; horseradish peroxidase (HRP) affinity-purified goat anti-mouse IgG (115-035-003) and anti-rabbit IgG (111-035-003) were purchased from Jackson ImmunoResearch Laboratories . Lipofectamine 2000 (11668019) and lipofectamine 3000 (L3000015) were purchased from Invitrogen . Evo M-MLV Mix Kit with gDNA Clean for qPCR (AG11728) was purchased from Accurate Biology . TransStart Top Green qPCR SuperMix (+Dye II) was purchased from Transgen Biotech . NP-40 lysis buffer (P0013F), RIPA lysis buffer (P0013K), protease and phosphatase inhibitor (P1050), Protein G Agarose (P2009), caspase 3 inhibitor Ac-DEVD-CHO (C1206) and proteasome inhibitor MG132 (S1748) were purchased from Beyotime Biotechnology . Endosomal acidification and autophagy inhibitor chloroquine (tlrl-chq) and poly (dA:dT) (tlrl-patn) were obtained from InvivoGen . Western lightning plus chemiluminescence reagent (NEL105001EA) was purchased from PerkinElmer .BHK-21, HEK293, or HEK293T cells in 6-well plates were transiently transfected with indicated plasmids or siRNA oligos using Lipofectamine 2000  according to the manufacturer’s instructions. PK15 cells in 6-well plates were transfected with indicated plasmids, siRNA oligos, or poly (dA:dT)  using Lipofectamine 3000  according to the manufacturer’s instructions. The siRNA targeting HSP27  and siRNA of negative control  were synthesized by Guangzhou Ribo Biotechnology . At 24 h post-transfection, the effects of HSP27 overexpression or interference were identified by Western blotting.Cellular RNA was extracted from RNAiso Plus  and transcribed into cDNA by Evo M-MLV Mix Kit with gDNA Clean for qPCR . A TransStart Top Green qPCR SuperMix (+Dye II)  was used for relative real-time quantitative PCR (RT-qPCR). The mRNA expression levels of IFN-β and cGAS were normalized to that of β-actin using the ΔΔCt method, respectively. The primer sequences used were as follows: Homo sapiens IFN-β-qF, 5′-TTGTTGAGAACCTCCTGGCT-3′; Homo sapiens IFN-β-qR, 5′-TGACTATGGTCCAGGCACAG-3′; Homo sapiens cGAS-qF, 5′-AGGAAGCAACTACGACTAAAGCC-3′; Homo sapiens cGAS-qR, 5′-CGATGTGAGAGAAGGATAGCCG-3′; Homo sapiens β-actin-qF, 5′-TGGCACCCAGCACAATGAA-3′; Homo sapiens β-actin-qR, 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′; Sus scrofa IFN-β-qF, 5′-TCCACCACAGCTCTTTCCAT-3′; Sus scrofa IFN-β-qR, 5′-CTGGAATTGTGGTGGTTGCA-3′; Sus scrofa cGAS-qF, 5′-GCACCGGGAGCTACTATGAG-3′; Sus scrofa cGAS-qR, 5′-CTCTCCACAGTGACACCTTCT-3′; Sus scrofa β-actin-qF, 5′-CAAGGACCTCTACGCCAACAC-3′; Sus scrofa β-actin-qR, 5′-TGGAGGCGCGATGATCTT-3′.Cells were lysed with NP-40 lysis buffer , or RIPA lysis buffer  containing a protease and phosphatase inhibitor  for 30 min on ice. For immunoprecipitation assay, the supernatant after centrifugation was incubated with anti-Myc antibody or anti-HA antibody  for 12 h at 4 °C, and the resuspended Protein G Agrose  was added and incubated for 4 h at 4 °C. The complexes were centrifuged and washed 5 times with pre-chilled lysis buffer. Precipitates or cell extracts were suspended in 1x SDS loading buffer, boiled at 95 °C for 10 min, and used for Western blotting analysis. These samples were isolated in 10% SDS-PAGE gel and transferred to PVDF membranes . PVDF membranes were sequentially incubated with the indicated primary antibodies and HRP-labeled goat anti-mouse IgG or goat anti-rabbit IgG . These bound protein bands were detected using Western Lightning Plus chemiluminescence reagent .2 incubator. After freeze-thawing of the cell suspension 3 times, the supernatant was collected by centrifugation. The DNA was extracted from the viral stock using TIANamp virus DNA/RNA Kit , and the copy number of PRV UL37 gene was detected by absolute RT-qPCR using the Premix Ex Taq Probe qPCR ; the viral stock was serially diluted 10 times, and the PRV titers were detected in BHK-21 cells by the Reed-Muench method. The primers and probe sequences of absolute RT-qPCR used were as follows: PRV-UL37-qF, 5′-GGACTACATGTTCCCCACGG-3′; PRV-UL37-qR, 5′-TAGAACGGCGTCAGGAATCG-3′; PRV probe, 5′-(FAM) CCACGGCCGTCACGA (Eclipse)-3′.For in vitro virus infection, treated or untreated PK15 cells or HEK293 cells were washed 3 times with PBS and infected with PRV Bartha-K61 strain (MOI = 0.001) for 24 h or 48 h in a 37 °C, 5% COHEK293 cells cultured in 6-well plates were transfected with Myc-HSP27 plasmid or pCMV-Myc plasmid  using Lipofectamine 2000. At 24 h post-transfection, the cells were treated with proteasome inhibitor MG132 , endosomal acidification and autophagy inhibitor chloroquine , caspase 3 inhibitor Ac-DEVD-CHO , or DMSO for 12 h, respectively. The supernatant after cell lysis was collected and subjected to Western blotting analysis.The experiment was performed as described previously ,33. HEK2p value of <0.05 was considered statistically significant, and p values of <0.01 or 0.001 were considered highly statistically significant.Data analyses were performed using GraphPad Prism 9.0 software. One-way ANOVA or two-way ANOVA were used to evaluate statistical significance. Among them, a To explore the role of HSP27 in the process of PRV infection, PK15 cells were transfected with EV or Myc-HSP27 plasmid and then infected with PRV (MOI = 0.001) for 24 h or 48 h. We observed that endogenous and exogenous HSP27 proteins could be detected simultaneously only in the Myc-HSP27 transfection group, and the exogenous HSP27 protein expression showed an increasing trend with the prolongation of PRV infection time A. OverexWe next transfected PK15 with siNC or siHSP27 for 24 h and then infected it with PRV (MOI = 0.001) for 24 h or 48 h. In contrast to the above results of overexpressing of HSP27, knockdown of endogenous HSP27 expression in PK15 cells reduced the viral copy number of PRV A, and siTo further explore whether HSP27 assisting PRV infection is related to IFN-β production, PK15 cells were transfected with Myc-HSP27 plasmid for 24 h and then infected with PRV. Results showed that PRV infection could indeed significantly enhance IFN-β mRNA expression level in PK15 cells; however, this activation was significantly reduced when HSP27 was overexpressed A. PolydB. MoreovGiven that PRV is a DNA virus that activates IFN-β production mainly through the cGAS-STING signaling cascade ,35, we nTo further clarify which one or several of adaptor molecules in the cGAS-STING pathway were specifically inhibited by HSP27, we cotransfected PK15 cells with Myc-HSP27 and HA-cGAS, Myc-STING, FLAG-TBK1, or FLAG-IRF3/5D, respectively. The results showed that HSP27 clearly reduced the gene transcriptional expression of IFN-β activated by cGAS A, STING Next, in order to explore whether HSP27 affects the protein expression of endogenous cGAS, STING, TBK1, and IRF3, we transfected EV or Myc-HSP27 plasmid into PK15, HEK293 and BHK-21 cells for 24 h, respectively. The results showed that endogenous cGAS proteins were degraded to varying degrees in different types of HSP27-overexpressing cells, while the protein expression of other adaptor molecules did not change significantly A. SimilaTo verify whether there is a physical interaction between HSP27 and cGAS, we cotransfected HEK293T cells with Myc-HSP27 and HA-cGAS for 24 h. Cells were harvested and lysed for co-immunoprecipitation experiment. The results showed that HA-tagged cGAS protein could be immunoprecipitated after coating Protein G Agarose with Myc-tagged antibody A. LikewiFor the sake of exploring whether HSP27-mediated cGAS degradation is dependent on the endosomal acidification and autophagy pathway, the caspase-3-dependent apoptosis pathway, or the ubiquitin-proteasome pathway, HEK293 cells were first transfected with Myc-HSP27 plasmid for 24 h, followed by treatment with endosomal acidification and autophagy inhibitor chloroquine (CQ), caspase 3 inhibitor Ac-DEVD-CHO (DEVD), ubiquitin-proteasome inhibitor MG132, or DMSO for 12 h, respectively. It was found that HSP27-mediated cGAS degradation was blocked by MG132, but not CQ or DEVD C, suggesAlthough the natural host of PRV is swine, it can also infect a variety of livestock and wildlife. In recent years, some cases of pseudorabies have been reported at home and abroad, but there is no conclusive evidence. In 2019, domestic researchers successfully isolated a human PRV strain hSD-1/2019 from the cerebrospinal fluid of patients for the first time , which pPRV has broad cell tropism and can proliferate in a variety of cell lines, such as PK15, Vero, MDBK and BHK-21 cells. Our research group also found that PRV can replicate effectively in human-derived cell lines, such as HEK293 cells. Therefore, PK15 cells, BHK-21 cells and HEK293 cells were used in this study. The results showed that HSP27 can not only degrade the protein expression of exogenous cGAS, but also weaken the gene transcription and protein expression of endogenous cGAS. Moreover, HSP27 could degrade the expression of porcine-derived, murine-derived and human-derived cGAS, indicating that HSP27 has specificity and universality for the recognition and degradation of cGAS. Because HEK293T cells lack endogenous cGAS , HEK293TMany studies have shown that HSP27 regulates the proliferation of RNA viruses, mainly through the NF-κB signaling pathway, RLR signaling pathway, or autophagy pathway. HSP27 promotes the replication of foot-and-mouth disease virus (FMDV) by interacting with the structural protein VP2 of FMDV and activating the autophagy-related pathway EIF2S1-ATF4-AKT-MTOR . HSP27 iThere are four main modifications of cGAS: ubiquitination, phosphorylation, acetylation, and palmitoylation ,41,42,43In this study, we found that overexpression of HSP27 contributed to PRV infection, and knockdown of HSP27 suppressed PRV infection in vitro. HSP27 inhibited PRV infection or poly(dA:dT)-activated IFN-β expression. Interestingly, we found that HSP27 attenuated cGAS-STING-mediated IFN-β expression through targeting cGAS. Further studies found that HSP27 bound directly to cGAS and promoted cGAS degradation by ubiquitination, and impaired the cGAS-mediated signaling cascade and IFN-β expression, thereby promoting PRV infection . In conc


Primary effusion lymphoma (PEL) is a fatal B-cell lymphoma caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. Inducing KSHV lytic replication that causes the death of host cells is an attractive treatment approach for PE; however, combination therapy inhibiting viral production is frequently needed to improve its outcomes. We have previously shown that the KSHV lytic protein K-bZIP can SUMOylate histone lysine demethylase 4A (KDM4A) at lysine 471 (K471) and this SUMOylation is required for virus production upon KSHV reactivation. Here, we demonstrate that SUMOylation of KDM4A orchestrates PEL cell survival, a major challenge for the success of PEL treatment; and cell movement and angiogenesis, the cell functions contributing to PEL cell extravasation and dissemination. Furthermore, integrated ChIP-seq and RNA-seq analyses identified interleukin-10 (IL-10), an immunosuppressive cytokine, as a novel downstream target of KDM4A. We demonstrate that PEL-induced angiogenesis is dependent on IL-10. More importantly, single-cell RNA sequencing (scRNA-seq) analysis demonstrated that, at the late stage of KSHV reactivation, KDM4A determines the fates of PEL cells, as evidenced by two distinct cell populations; one with less apoptotic signaling expresses high levels of viral genes and the other is exactly opposite, while KDM4A-K417R-expressing cells contain only the apoptotic population with less viral gene expression. Consistently, KDM4A knockout significantly reduced cell viability and virus production in KSHV-reactivated PEL cells. Since inhibiting PEL extravasation and eradicating KSHV-infected PEL cells without increasing viral load provide a strong rationale for treating PEL, this study indicates targeting KDM4A as a promising therapeutic option for treating PEL.IMPORTANCE PEL is an aggressive and untreatable B-cell lymphoma caused by KSHV infection. Therefore, new therapeutic approaches for PEL need to be investigated. Since simultaneous induction of KSHV reactivation and apoptosis can directly kill PEL cells, they have been applied in the treatment of this hematologic malignancy and have made progress. Epigenetic therapy with histone deacetylase (HDAC) inhibitors has been proved to treat PEL. However, the antitumor efficacies of HDAC inhibitors are modest and new approaches are needed. Following our previous report showing that the histone lysine demethylase KDM4A and its SUMOylation are required for lytic reactivation of KSHV in PEL cells, we further investigated its cellular function. Here, we found that SUMOylation of KDM4A is required for the survival, movement, and angiogenesis of lytic KSHV-infected PEL cells. Together with our previous finding showing the importance of KDM4A SUMOylation in viral production, KDM4A can be a potential therapeutic target for PEL. Primary effusion lymphoma (PEL), characterized by a pleural or peritoneal lymphomatous effusion without solid tumor mass, is therefore also known as body cavity-based lymphoma (BCBL) . It is aThe human oncogenic gammaherpesvirus KSHV, also known as human herpesvirus-8 (HHV-8), is an oncogenic DNA virus exhibiting biphasic life cycle: latency and lytic replication . Therape12–In particular, our previous report showed that the deposition of histone lysine demethylase 4A (KDM4A) on the KSHV episome limits the enrichment of heterochromatin mark H3K9me3 on certain parts of the viral genome that contain immediate early (IE) and early (E) genes, allowing rapid switch-on of the viral lytic cycle , 26. KnoKO), we confirmed that loss of KDM4A could induce cell death, with minimal virion production, in KSHV-reactivated PEL cells. These results indicate that KDM4A would be a new promising therapeutic target for treating PEL.To study this, we first performed Gene Ontology (GO) analysis using differentially expressed genes (DEGs) obtained from KDM4A-WT and KDM4A-K471R upon KSHV reactivation to determine the cellular mechanism by which KDM4A SUMOylation regulates during KSHV reactivation. We found that in addition to inhibiting cell apoptosis, KDM4A also promotes cell movement and angiogenesis, the cellular functions contributing to lymphoma dissemination , 31. UsiTo study how KDM4A SUMOylation influences cellular gene expression in PEL cells after KSHV reactivation, we reanalyzed our RNA-seq data from TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -K471R BCBL-1 cells  focusingInterestingly, GO analysis revealed that cell movement, cardiovascular system development, cancer, and cellular assembly and organization are the major activation pathways . The amoKO cell line complemented with KDM4A-WT, but not KDM4A-K471R, induced a significantly higher angiogenesis (~2-fold) in HMEC-1 following KSHV reactivation  from BCBL-1 cells could enhance endothelial cell angiogenesis. As expected, CM from KSHV-reactivated TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT BCBL-1 cells induced a slight (<1.5-fold change) but significantly higher angiogenesis in HMEC-1 when compared with CM from TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-K471R cells . We susptivation . These dTo identify the SUMOylated KDM4A target gene responsible for angiogenesis, genes differentially activated in TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and KDM4A-K471R BCBL-1 cells after KSHV reactivation in the angiogenesis pathway were analyzed by RT-qPCR. Among them, IL-10, an immunosuppressive cytokine contributing to viral persistence in and pathogenesis of KSHV-associated lymphoproliferative disorders –35, show–We have previously shown that SUMOylation stabilizes KDM4A binding on the KSHV genome and enables viral gene transactivation . To deteTo determine whether IL-10 could be one of the active factors secreted by BCBL-1 cells that enhances endothelial cell angiogenesis, we measured IL-10 levels in the CM of KDM4A-WT- and KDM4A-K471R-expressing cells using ELISA. Consistently, KSHV reactivation induced IL-10, and its level was higher in both KSHV latent infected and lytic reactivated BCBL-1 cells expressing KDM4A-WT when compared with KDM4A-K471R . Next, wAs GO analysis showed that SUMOylated KDM4A is negatively associated with cell death, the role of KDM4A in protecting against KSHV reactivation-induced cell death was further evaluated. Since KSHV reactivation by no means guarantees that all virally infected cells undergo a similar cell fate , we therNext, we performed a trajectory analysis using combined scRNA-seq data from TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -K471R BCBL-1 cells. Three states were defined  that repThe split of C2 (L) into states 2 and 3 of the trajectory suggests virally infected cells undergo different fates in response to viral reactivation. Elucidating the differences between these two states may help reveal cell fate decisions of lytic KSHV-infected cells. To this end, the DEGs between these two states  in the combined trajectory . ConsistKO BCBL-1 cells after KSHV reactivation. Consistent with our hypothesis, the results showed that compared with the control, knockout of KDM4A significantly reduced cell number (KO BCBL-1 cells when compared with the control (~70–80%)  . These r~70–80%) . Next, wThe biphasic life cycle of KSHV is tightly epigenetically regulated . Therefo42–Although induced lytic reactivation of KSHV from latently infected B lymphoma cells can be a potential therapy for PEL, KSHV encodes viral genes that inhibit apoptosis in both latent and lytic infection, hampering this therapy. During latent infection, inhibition of apoptosis is important to maintain persistent infections. In lytic reactivation, KSHV inhibits apoptosis to prevent cell death before viral replication and release . TherefoUnexpectedly, bioinformatic analyses of bulk RNA-seq data revealed that KDM4A also participates in the regulation of cell movement and angiogenesis, thus possibly contributing to the dissemination of PEL. PEL is a lymphomatous effusion localized in body cavities, and this tropism depends on lymphocyte migration. Our data here suggest that targeting KDM4A may interrupt PEL homing. In addition, a previous report showed that angiogenic factor vascular endothelial growth factor (VEGF) secreted by PEL cells interacted with endothelial cells, induced angiogenesis, and contributed to the invasive behavior of PEL invasion . Here, oin vivo that is worth further investigation in future research.Importantly, IL-10 is a multifunctional cytokine that regulates cell growth and differentiation, immune response, and tumor development, particularly in hematopoietic neoplasms , 51, 52.PEL is a lymphomatous effusion localized in body cavities without tumor mass that cannot be surgically removed. Since the current CHOP treatment efficiency is restricted, finding new therapeutic approaches for PEL remains a challenge. Targeting dominant viruses with drugs that reactivate KSHV lytic replication is an attractive option for treating KSHV-associated hematologic malignancy, such as PEL. However, the therapeutic effectiveness of current lytic induction therapies in PEL has yet to be improved. Therefore, therapeutic candidates that can selectively target KSHV lytic replicated PEL cells for death without increasing viral load need to be identified. In this study, we demonstrate that KDM4A is involved in protecting PEL cells from death, PEL cell migration, and angiogenesis induced by viral reactivation. Most importantly, we identified IL-10 as a novel target gene of KDM4A. Together with our previous findings, inhibition of KDM4A or its SUMOylation appeared to achieve a desirable clinical goal of concomitantly inducing incomplete viral lytic replication that does not increase viral load, increases cell death, and prevents PEL dissemination.The doxycycline (Dox)-inducible TREx-MH-K-Rta BCBL-1 cell line, with Myc-His-tagged K-Rta, was maintained in RPMI 1640 containing 15% FBS, 20 μg/mL blasticidin, and 200 μg/mL hygromycin . The doxycycline (Dox)-inducible TREx-MH-K-Rta-shKDM4A BCBL-1 cell line was maintained as described for TREx-MH-K-Rta BCBL-1 and supplemented with 1 μg/mL puromycin (Invitrogen). TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-K471R BCBL-1 were maintained as described for TREx-MH-K-Rta-shKDM4A BCBL-1 cells and supplemented with 100 μg/mL G418 (AMRESCO).KO BCBL-1 cells were generated by transfected TREx-MH-K-Rta BCBL-1 cells with the plasmid PX330 that expresses guide RNA  targeting exon 3 of KDM4A and Cas9 using X-tremeGENE HP . Three days after transfection, RFP-positive cells were isolated by fluorescence-activated cell sorting (FACS) and plated in 96-well plates at 1 cell/well. The expression of KDM4A was analyzed by immunoblotting assays. PCR fragments (PCR primer sequence: 5′-CTT GCC AGG TTT CTC ATC TGT C-3′ and 5′-GGT TTC CAC TCA CTT ATC GCT-3′) amplified from genomic DNA extracted from the 6th clone (#6) of TREx-MH-K-Rta-KDM4AKO BCBL-1 cells were subjected to Sanger sequencing.KDM4A knockout TREx-MH-K-Rta-KDM4Al-glutamine, 20 ng/mL EGF , and 5 μg/mL hydrocortisone. All cells used in this study were cultured at 37°C and supplied with 5% CO2.293T was cultured in Dulbecco’s modified Eagle’s medium  containing 10% FBS . Human dermal microvascular endothelial (HMEC1) cell line was cultured in MCDB131 medium  supplemented with 10% FBS, 1% KO BCBL-1 cells.IL-10 knockdown plasmids  were purchased from RNA Technology and Gene Manipulation Core in Academia Sinica. pLenti4-CMV/TO-KDM4A-WT and -KDM4A-K471R were used to ectopically express KDM4A in TREx-MH-K-Rta-KDM4ANC_009333.1). Cells with low-quality cell barcodes or with a high percentage of reads mapping to the mitochondrial genome were excluded. A total of 7,172 and 7,427 cells from TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -K471R BCBL-1, respectively, were included for clustering analysis using k-means model and visualized by t-distributed stochastic neighbor embedding (t-SNE). All genes in TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -K471R BCBL-1 cells were used to construct the pseudotime trajectory using Partek Flow . DEGs were identified using Partek Flow (Partek), filtered by average read >1 (|log2 fold change| ≥ 1) and P-value < 0.05, and then subjected to GO analysis using ingenuity pathway analysis (IPA) software .The single-cell suspension with viability >95% was loaded onto the 10x Genomics Single-Cell-A Chip for target capture of ~8,000 cells/chip. The cDNA library was prepared using Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 , according to the manufacturer’s protocol. The library was sequenced by NovaSeq 6000  at a depth of ~150 M reads per sample. The raw sequencing data were processed by Cell Ranger  analysis pipeline and aligned with the human reference genome (GRCh38) and the HHV-8 reference genome . Cells were observed for 24 h in a humidified CO2-equilibrated chamber with a Nikon Eclipse Ti microscope  equipped with a motorized stage . The images were captured every 5 min. For the reactivation experiments, 0.2 μg/mL Dox was added the day before tracking. The movement of cells in each group was tracked for 279 frames. All movement cells in the captured fields were selected by trajectory tracking with ImageJ software. Measurements of a single cell trajectory were produced by 279 displacements, and all displacements from the same cell line were used to plot the histograms. Results were displayed with box and whisker plots.1 × 104 HMEC-1 in MCBD131 medium (100 μL/well) were placed onto the BME and incubated for 6 h at 37°C. The tube structures were captured by an inverted light microscope (100×). The average tube length in each field was measured by ImageJ. For each treatment, the tube length was calculated using the average of 10 microscopic fields.Basement membrane extract (BME)  was coated in 96-well plates (50 μL/well) for 1 h at 37°C. Then, 2 × 104/100 μL) cells were seeded into 96-well plates. After treatments, the cell viability was examined by MTT assay . A final concentration of 0.5 mg/mL MTT was added, and the formazan crystals were solubilized in lysis buffer (10% SDS-0.01N HCl). The optical density (OD) was determined by a microplate spectrophotometer at a wavelength of 570 and 660 nm.BCBL-1 (1 × 105/300 μL) were seeded into 48-well plates. Three days after treatment, cells were resuspended by gentle pipetting and then taken out (20 μL) for cell counting. Cell suspensions were mixed with equal volume of trypan blue and counted three times by Countess 3 FL cell counter .BCBL-1 cells (4 × 106 cells/mL) were cultured for 24 h and then treated with Dox for 48 h. To analyze apoptotic cells, 1 × 107 BCBL-1 cells were washed three times with ice-cold PBS and stained with Zombie Aqua dye  for 15 min at room temperature. The cells were then washed three times with ice-cold PBS and fixed in 4% paraformaldehyde/PBS for 20 min at room temperature. After fixation, cells were washed once with ice-cold permeabilization buffer  and incubated with permeabilization buffer for 40 min on ice. The cells were then stained with anti-KSHV ORF 45  followed by Alex Fluor 488-conjugated AffiniPure goat antimouse IgG and with anti-KSHV ORF 8.1  followed by Cy3-conjugated AffiniPure goat antimouse IgG. The cells were then washed three times with ice-cold permeabilization buffer and analyzed by flow cytometer  and CytExpert software (Beckman Coulter).TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -K471R BCBL-1 cells (2 × 10http://perlprimer.sourceforge.net/) . The expression level of mRNA was normalized against GAPDH. Real-time qPCR analysis was carried out in the CFX96 Connect real-time PCR detection system .Total RNA was isolated by TRIzol . cDNA was generated using the SuperScript III first-strand synthesis kit  using Oligo-(dT). qPCR primer pairs were designed by PerlPrimer (https://bohdan-khomtchouk.github.io/docs/Chromatin-Immunoprecipitation-ChIPs-Protocol-from-Farnham-Lab.pdf). Chromatin DNA prepared from 1 × 107 TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R cells was used for ChIP assays using anti-KDM4A rabbit polyclonal antibody (NC_009333.1) by Partek Flow (Partek).ChIP assay was performed using protocol from Dr. Farnham’s laboratory (antibody  and rabbGAGGATTCAACAGTGATGGGAC-3′) was designed to amplify the potential KDM4A binding site on the IL-10 promoter.ChIP DNA was confirmed for successful IP using SYBR Green-based real-time qPCR analysis in the CFX Connect real-time PCR detection system (Bio-Rad). The specific primer set  following the manufacturer's instructions. The sensitivity of the ELISA was 1.5 pg/mL.


ESM1 expression was significantly elevated in human cervical cancer tissues, correlating with patients’ poor prognosis. Moreover, ESM1 mRNA and protein upregulation was detected in local cervical cancer tissues and various cervical cancer cells. In established and primary cervical cancer cells, ESM1 shRNA or CRISPR/Cas9-induced ESM1 KO hindered cell proliferation, cell cycle progression, in vitro cell migration and invasion, and induced significant apoptosis. Whereas ESM1 overexpression by a lentiviral construct accelerated proliferation and migration of cervical cancer cells. Further bioinformatics studies and RNA sequencing data discovered that ESM1-assocaited differentially expressed genes (DEGs) were enriched in PI3K-Akt and epithelial-mesenchymal transition (EMT) cascades. Indeed, PI3K-Akt cascade and expression of EMT-promoting proteins were decreased after ESM1 silencing in cervical cancer cells, but increased following ESM1 overexpression. Further studies demonstrated that SYT13 (synaptotagmin 13) could be a primary target gene of ESM1. SYT13 silencing potently inhibited ESM1-overexpression-induced PI3K-Akt cascade activation and cervical cancer cell migration/invasion. In vivo, ESM1 knockout hindered SiHa cervical cancer xenograft growth in mice. In ESM1-knockout xenografts tissues, PI3K-Akt inhibition, EMT-promoting proteins downregulation and apoptosis activation were detected. In conclusion, overexpressed ESM1 is important for cervical cancer growth in vitro and in vivo, possibly by promoting PI3K-Akt activation and EMT progression. ESM1 represents as a promising diagnostic marker and potential therapeutic target of cervical cancer.The expression, biological functions and underlying molecular mechanisms of endothelial cell-specific molecule 1 (ESM1) in human cervical cancer remain unclear. Bioinformatics analysis revealed that  Cervical cancer is a common gynecological malignancy, the fourth-common cause of incidence and death of cancer among women –3. The mESM1 gene is located on chromosome 5 q11.2 [Endothelial cell-specific molecule 1 (ESM1), also known asendocan, is an acysteine-rich proteoglycan. The  5 q11.2 . ESM1 wa 5 q11.2 . Recent  5 q11.2 –16. More 5 q11.2 , vascula 5 q11.2 , and can 5 q11.2 .ESM1 is  5 q11.2 –22.et al. reported that ESM1 can suppress the progression and metastasis of prostate cancer cells by regulating TIMP-1/MMP-9 expression [More recently, studies have found that ESM1 could participate in various physiological processes in cancer cells, including cell proliferation, angiogenesis, migration, and invasion, as well as therapy resistance. It therefore plays a crucial role in carcinogenesis and tumor progression , 23, 24.pression .Epithelial-to-mesenchymal transition (EMT) plays a crucial role in cancer progression , 32, as ESM1 transcripts in human cervical cancer tissues. A total of 319 tissues were collected, including 13 normal cervical tissue samples and 306 cervical cancer tissue samples. As shown, the number of ESM1 mRNA transcripts in the cervical cancer tissues was significantly higher than that in the normal cervical tissues  database and the Genotype-Tissue Expression (GTEx) project were consulted to analyze ues Fig. . We furtset Fig. . In addiP < 0.01) and disease classification in cervical cancer patients  and lymphatic vascular invasion (P <0.05)  Fig. . Therefon = 12). ESM1 mRNA expression in cervical cancer tumor tissues was about four-fold higher than that in adjacent normal tissues  and paired adjacent normal tissues (“N”) were from a total of 12 different primary cervical cancer patients Fig. . Furtherpic)Fig. . CollectESM1 mRNA expression was decreased significantly  was studied. shESM1-a and shESM1-b led to robust ein Fig.  downreguein Fig.  and in vein Fig. .ESM1 KO screening. Compared to the control cells with the CRISPR/Cas9 empty vector (“Cas9-C”) ESM1 mRNA expression was significantly decreased in the koESM1-cervical cancer cells  lls Fig. . ESM1 ovlls Fig.  and promlls Fig. . Moreovells Fig. , in vitrlls Fig. . In addills Fig. . The ESMion Fig. . Ectopicion Fig.  and in vion Fig. . TogetheTo explore the possible molecular mechanisms underlying ESM1-driven cervical cancer cell progression. Gene Set Enrichment Analysis (GSEA) was employed to analyze the differentially expressed genes (DEGs), by comparing ESM1 high-expression cervical cancer tissues with the ESM1 low-expression cervical cancer tissues retrieved from TCGA database. As shown, DEGs were enriched in PI3K-Akt cascade Fig.  and cellAs shown, Akt, S6, and 4EBP1 phosphorylation as well as mTOR and PI3K expression were significantly downregulated in shESM1-expressing cervical cancer cells Fig. . While eNext, the bioinformatics analysis was performed in OE-ESM1 and control SiHa cells see Fig. . DEGs beSYT13 (synaptotagmin 13) expression was significantly higher in cervical cancer tissues (n = 306) than that in normal cervical tissues (n = 13)  Fig.. Moreove 13) Fig.. Next, tlls Fig. . Moreoveues Fig. .SYT13 mRNA expression, and strongly inhibited PI3K-Akt cascade, leaving ESM1 expression unaffected  or Cas9-C control vector (“Cas9-C”) were subcutaneously (s.c.) injected to the flanks of the nude mice to establish xenograft tumors. The ko-ESM1 SiHa xenografts grew significantly slower than the Cas9-C xenografts  dramatically reduced gastric cancer metastasis in a mouse model, and significantly increased animal survival [Located on human chromosome 11p11.2,  protein . Studies protein , 46. SYT protein . Nakanissurvival . Kanda esurvival .et al., found SYT13 silencing inhibited activation of multiple pro-cancerous signalings, including cyclin-dependent kinase 2 (CDK2), focal adhesion kinase (FAK) and more importantly PI3K-Akt cascade, without affecting JAK-STAT and NF-κB signalings [SYT13 could play an important role in regulating multiple signal cascades in human cancer. Ichikawa et al., reported that SYT13 overexpression in estrogen receptor (ER)-positive breast cancer correlated with several key oncogenes, including estrogen receptor 1 (ER1), Akt, and cyclin-dependent kinases 4 (CDK4) . Kanda egnalings .Here, the bioinformatics studies and RNA sequencing data revealed that SYT13 could be a primary target of ESM1 in cervical cancer. SYT13 is upregulated in cervical cancer patients, and its overexpression correlated with poor overall survival. Ectopic ESM1 overexpression in cervical cancer cells increased SYT13 expression. Whereas siRNA-induced silencing of SYT13 decreased PI3K-Akt cascade in cervical cancer cells, without affecting ESM1 expression. Importantly, SYT13 silencing potently inhibited ESM1-overexpression-induced PI3K-Akt activation and cervical cancer cell migration/invasion. Therefore, ESM1-driven cervical cancer progression could be due to promoting SYT13 expression. The underlying mechanisms may warrant further characterizations.Cancer screening, HPV vaccine application combining with the current treatment strategies, including neoadjuvant chemotherapy and immunotherapy, have made cervical cancer a potential preventable and treatable disease (mainly in developed countries) , 50. YetCell Counting Kit-8 (CCK-8) was purchased from Dojindo Co. . Puromycin was provided by Sigma-Aldrich Chemicals . Antibodies for anti-cleaved caspase-3 (#9664), anti-cleaved-poly (ADP-ribose) polymerase (PARP) (#5625), anti-cleaved-caspase-9 (#20750), anti-E-Cadherin (#3195), anti-p-Akt S473 (#4060) and antiphospho-S6 (#4858) were provided by Cell Signaling Tech . Anti-ESM1(#103590), anti-N-Cadherin (#245117), anti-Vimentin (#92547), anti-slug (#302780) and all other antibodies were obtained from Abcam . RNA reagents and other transfection reagents were obtained from Thermo-Fisher Invitrogen . Annexin V and propidium iodide (PI) were purchased from BD Biosciences .https://gdc.xenahubs.net) was downloaded to analyze ESM1 transcript expression in cervical cancer tissues and normal cervical tissues, and GSE9750 dataset was used to further verify the results. Additionally, Kaplan-Meier survival analysis was used to determine the prognosis of cervical cancer patients. Subgroup analysis by different clinical characterizations was performed based on TCGA database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted by Gene Set Enrichment Analysis (GSEA).The Cancer Genome Atlas (TCGA) database  were purchased from the Cell Bank of Shanghai Institute of Biological Science . The primary human cervical cancer cells (priCC-1), derived from one written-informed consent patient at age 55 in authors’ institutions with gynecology and obstetrics (FIGO) stages IIB cervical cancers (squamous cell carcinoma), were cultured as described . Cells wThe cervical cancer tissues and surrounding normal cervical tissues were obtained from 12 primary cervical cancer patients with written-informed consent . Tissues were homogenized by the tissue lysis buffer , followed by further biochemical analyses. The protocols of this study comply with the Declaration of Helsinki and were approved by the Ethics committee of Affiliated Kunshan Hospital of Jiangsu University.Three lentiviral shRNAs targeting non-overlapping sequences of human ESM1, as well as the scramble control (“shC”) were designed and validated by Genechem Co. Cells were inoculated into six-well plates in complete medium, and transfected with ESM1 shRNAs and scramble control respectively. Stable cells were screened for two weeks in complete medium with puromycin (5.0 µg/mL). Silencing of ESM1 in stable cells was verified by qPCR and Western blotting.Lentiviral CRISPR/Cas-9 ESM1 KO construct and CRISPR/Cas-9 empty vector, provided by Genechem, were transfected into SiHa and HeLa cells . ESM1 KOThe full-length ESM1 cDNA-expressing lentiviral construct and the empty vector, provided by Genechem , were transfected into SiHa and HeLa cells. After being selected by puromycin, ESM1 overexpression in stable cells was verified by qPCR and Western blotting.ESM1 overexpressed cervical cancer cells were transfected with a small interference RNA (siRNA) targeting SYT13 and negative control siRNA by Lipofectamine 2000 . 48 h after transfection, cells were used for further experiments.Severe combined immunodeficiency (SCID) mice  were purchased from the Animal Center of Jiangsu University . These mice were assigned into two random groups. Genetically modified SiHa cells were resuspended in Matrigel-containing serum-free medium, then injected subcutaneously into the right flanks of nude mice. Body weights and tumor volumes were recorded every seven days. Immunohistochemistry (IHC) staining was conducted by the procedure described previously . Animal Cell counting Kit-8 (CCK-8) cell viability assay, colony formation assay, nuclear 5-ethynyl-2’-deoxyuridine (EdU) staining assay, wound healing assay, “Transwell” and “Matrigel Transwell” analyses were performed following the protocols described previously –59. PropThe IHC staining protocol has been reported in Liu et al. . TUNEL sP value <0.05.All statistical analyses were performed using SPSS 22.0 (IBM) and R . All data were with normal distribution and were presented as means ± standard deviation (SD) from at least three separate experiments. The differences among the groups of each treatment were analyzed by one-way ANOVA (three or more groups) or Student’st-test (two groups). RNA sequencing data was analyzed using R. software (version 4.2.1). “limma” package, “clusterprofiler” package, “ggplot2” package, “survival” package, and “survminer” package were installed and used. The difference was considered statistically significant as In conclusion, overexpressed ESM1 is important for cervical cancer growth in vitro and in vivo, possibly by promoting PI3K-Akt activation and EMT progression.Supplementary Figuresaj-checklist formAuthor contribution form


Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase,  Simil in MKs b,c. ThesBeCyclop, we tightly controlled cGMP levels in MKs by light. We show that PDE5 is the major PDE, counteracting cGMP levels in BM-derived MKs, and could verify our optogenetic results by cGMP imaging in BM MKs ex vivo using cGMP sensor mice. Our reversible approaches allowing to trigger the intracellular process and directly analyse the effect in a high spatio-temporal resolution revealed that PDE5 is highly active in BM-derived MKs as cGMP levels rapidly declined after removing the stimulus. Our findings are corroborated by research on mouse FLC-derived MKs showing that PDE5 expression is highest in mature FLC-derived MKs [BeCyclop due to the untransduced cell fraction. Further studies are required using MK/platelet-specific optogenetic mouse lines to spatio-temporally light-manipulate molecules or proteins in MKs and platelets in vivo. The finding that BM MK express PDE5 also informs future therapeutic strategies to increase cGMP concentrations and potentially modulate platelet biogenesis with clinically used PDE5 inhibitors.Optogenetics has become successful as an easily applicable tool for precise spatio-temporal manipulation of molecular events in living cells or organisms. By now, optogenetic applications have expanded from neuroscience to other fields such as the cardiovascular system. We established to the best of our knowledge the first-time optogenetics in primary MKs. By expressing the photo-activated guanylyl cyclase, ived MKs . Our stu. 3. 3.1fl/fl; Pf4-Cretg/+cGi500-L2 [MK/platelet-specific cGMP sensor mice (-Cretg/+ ) were us-Cretg/+ . Mouse B. 3.2Riociguat , tadalafil (Sigma-Aldrich/Supelco), DMSO, forskolin (Sigma-Aldrich), vinpocetine, EHNA (Enzo Life Sciences), BAY60-7550, TAK-063 , milrinone, vardenafil, zaprinast (Santa Cruz Biotechnology), sildenafil (Merck), IBMX (Thermo Fisher Scientific) and DEA/NO (Axxora).. 3.3−1 penicillin, 50 mg ml−1 streptomycin) containing 1% of recombinant TPO (homemade) and 100 U ml−1 recombinant Hirudin (Hyphen Biomed) at 37°C under 5% CO2.BM cells were obtained from femur and tibia of C57BL/6 mice by flushing, and lineage depletion was performed using an antibody cocktail of anti-mouse CD3, clone 17A2; anti-mouse Ly-6G/Ly-6C, clone RB6-8C5; anti-mouse CD11b, clone M1/70; anti-mouse CD45R/B220, clone RA3-6B2; anti-mouse TER-119/Erythroid cells, clone Ter-119  and magnetic beads . Lineage-negative (Lin-) cells were cultured in DMEM medium  for 5 min at room temperature and lysed either immediately or after 3 min in the dark with 0.1 M hydrogen chloride. Concentrations of cGMP or cAMP were measured by DetectX Direct cyclic GMP or AMP Enzyme Immunoassay Kit, respectively, and analysed by the optical density at 450 nm with a Thermo Scientific Multiskan microplate spectrophotometer.Cell density was adjusted to 5 × 10. 3.6MK and platelet lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Membranes were incubated with an anti-PDE3A antibody (GeneTex) or an anti-PDE5 antibody (Abcam) overnight. Horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence solution (MoBiTec) were used for visualization. Immunoblots were recorded directly using an Amersham Imager 600 .. 3.7fl/fl; Pf4-Cretg/+cGi500-L2 [2, 2 mM CaCl2, 5 mM HEPES and 10 mM D-glucose; pH = 7.4) with or without added drugs. Real-time FRET/cGMP imaging was performed by recording CFP and YFP fluorescence as described in detail elsewhere [Femurs and tibias were dissected from MK/platelet-specific cGMP sensor mice (-Cretg/+ ). Bone tlsewhere . The rel. 3.8t-test with GraphPad Prism software. p-values < 0.05 were considered significant . ns: non-significant. Statistical analysis of cGMP sensor imaging data was performed with Origin 2019 . Since datasets were not normally distributed, statistical differences were analysed non-parametrically by Mann–Whitney U-test.Results are shown as mean ± s.e.m. Statistical significance was assessed by an unpaired


Moringa oleifera Lam. in lipopolysaccharide (LPS)-induced inflammation models. MIC-1 decreased nitric oxide production and reduced the expression of pro-inflammatory markers  in C2C12 myoblasts. The daily oral treatment of MIC-1 (80 mg/kg) for three days significantly reduced the expression of pro-inflammatory markers in gastrocnemius muscle tissue of LPS-treated C57BL/6 male mice. Transcriptomic analysis provided further insights into the inhibitory effects of MIC-1 on the LPS-induced inflammation, which suggested that MIC-1 affects inflammation and immunity-related genes in myoblasts and skeletal muscle tissue. MIC-1 inhibited the nuclear accumulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the LPS-treated myoblasts. Our data support the hypothesis that the MIC-1’s effects in the muscle cells are mediated through the inhibition of the NF-κB translocation in the nucleus, which, in turn, results in immunomodulating and anti-inflammatory responses at the gene expression levels.This study aims to investigate the anti-inflammatory effects of moringa isothiocyanate-1 (MIC-1) extracted from seeds of  Moringa oleifera Lam., a plant used as food and medicine, with concentrations up to 40% in the hydro-alcoholic moringa seed extract [Moringa isothiocyanate-1  is an isothiocyanate that has similar biological activities as other natural isothiocyanates, such as sulforaphane from cruciferous vegetables. Isothiocyanates are the class of plant secondary metabolites originating from the sulfur-containing compounds glucosinolates. MIC-1 is found in  extract . MIC-1’s extract , 3. SixtPrevious reports showed that MIC-1 has anti-inflammatory, antioxidant, and antimicrobial activities , 5–8. ThThe anti-inflammatory mechanisms of MIC-1 have also been investigated by using in vivo models. We previously showed that the daily oral supplementation with MIC-1 (80 mg/kg bw) for 3 days protects from systemic inflammation induced by LPS in mice , and theSince MIC-1 and other isothiocyanates have anti-inflammatory properties, MIC-1 may be used to promote skeletal muscle health. Pre-clinical and clinical studies have suggested that sulforaphane ameliorates muscle damage caused by inflammation –20. SulfMoringa oleifera seed extract as previously reported [MIC-1 purity >98%) was isolated from % was isoC2C12 myoblasts were cultured in a growth medium  at 37°C in a humidified atmosphere with 5% carbon dioxide. MIC-1 solutions were made in ethanol. Myoblasts were treated with 0.475% ethanol (vehicle), or 1–10 μM MIC-1, with or without 1 μg/mL LPS for 24 h before the in vitro assays.Cell viability was measured by using MTT, which was added to the growth medium to create purple formazan crystals . AbsorbaNitric oxide (NO) production was evaluated by using Griess method . The conImmunofluorescence staining was used for NF-κB nuclear translocation assessment as previously described . Fixed cEighteen C57BL/6 male mice were randomly separated into vehicle control, LPS-treated, or LPS-MIC-1-treated group as previously described . MIC-1  [P-values). Reproducibility of RNA-seq data is shown in principal component analysis (PCA) plots (RNA-sequencing data (Gene Expression Omnibus accession #GSE209706) were analyzed by using the transcript abundances from pseudoalignments in Kallisto (v0.46.11), an RNA-seq quantification program , 29. ThePKM > 1) , 31. MorA) plots ; DESeq2 Reverse transcription quantitative real-time PCR was performed to measure the expression of pro-inflammatory genes as previously described . Power SP < 0.05) by using GraphPad .Data are expressed as mean ± standard deviation (SD) of three experiments in triplicate. One-way analysis variance or Student’s t-test were used to determine statistical differences . MIC-1 did not significantly affect the cellular viability at the tested concentrations . Next, wP-value < 0.001 and FPKM > 1), identified by DESeq2 analysis using the pairwise comparisons , were further clustered by using k-means to identify clusters of genes showing common responses to these treatments. This list of 3,567 genes were clustered in five clusters , identified by DESeq2 analysis using the pairwise comparisons , were further clustered by using k-means to identify clusters of genes showing common responses to these treatments. Five k-means clusters were obtained from this list of 1,049 genes (MIC-1 (80 mg/kg bw) was orally administered to C57BL/6 male mice for three days before LPS treatment . Thereafter, the gastrocnemius muscle tissues of animals were collected 16 h after the LPS injection for transcriptomic analyses by RNA-seq . We comp49 genes . GO anal49 genes . GO anal49 genes ; S2 FileTo validate the transcriptomic analyses, we evaluated the effects of MIC-1 treatment on the pro-inflammatory cytokines in murine muscle tissues by RT-qPCR. As expected, our results showed that MIC-1 decreased LPS-induced gene expressions of TNF-α, Ifn-α, IL-1β, and IL-6 in muscle tissue . These rNF-κB is transcription factor that regulates pro-inflammatory cytokines, and our previous study showed that MIC-1 inhibited the nuclear translocation of NF-κB in macrophages . To testM. oleifera seeds. Our previous studies documented that MIC-1 activates anti-inflammatory processes in macrophages by inhibiting the NF-κB pathway [MIC-1 is a chemically stable anti-inflammatory isothiocyanate from  pathway , 7, 8. HLPS, a membrane component of Gram-negative bacteria, is used to induce inflammation and sepsis in pre-clinical studies –12, 25. NF-κB is a transcription factor that regulates innate and adaptive immune responses mediated by inflammation. As a result, NF-κB activation contributes to the etiology of various inflammatory diseases. Our results showed that MIC-1 reduced the LPS-induced inflammation, at least partially, via NF-κB pathway in skeletal muscle. MIC-1 decreased the NF-κB’s nuclear translocation, as visualized by immunofluorescence staining. This observation may be validated further with cell fractionation experiments . The obsMIC-1 may also regulate inflammation via other pathways, such as interferons (Ifn) signaling pathway. MIC-1 downregulated the expression of a type I interferon (Ifn-α), which attracts immune response cells to the infected areas. Interferons can induce immune response independently of NF-κB pathway, for example via activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway , 37. AltThe effective and safe dosages of MIC-1, given orally, used in rodent models can be translated to 6.5–16 mg/kg bw (390–960 mg/60 kg bw) in human equivalent doses , 8, 40. S1 FigDESeq2 analysis of the 3 replicates for C2C12 samples (A) and muscle tissue samples (B) was conducted to create PCA plots. The data were first turned into a DESeq2 object, then ’regularized log’ transformed and plotted in a PCA plot.(TIF)Click here for additional data file.S1 FileGene counts, gene list, and GO terms of comparisons between control (Ctrl) vs lipopolysaccharide (LPS)-treated samples, and LPS-treated samples vs LPS-moringa isothiocyanate 1 (MIC-1)-treated samples.(XLSX)Click here for additional data file.S2 FileGene counts, gene list, and GO terms of comparisons between control (Ctrl) vs lipopolysaccharide (LPS)-treated samples, and LPS-treated samples vs LPS-moringa isothiocyanate 1 (MIC-1)-treated samples.(XLSX)Click here for additional data file.


Our results showed that network prediction of NRICM101 by a high throughput target screening platform showed that NRICM101 has multiple functions that may affect cytokine regulation to prevent human lung cell injury. In addition, NRICM101 revealed protective effects against TNF-α/IL-1β-induced normal human lung HEL 299 cell injury through JNK and p38MAPK kinase signaling. Next-generation sequencing (NGS) analysis of NRICM101 on TNF-α/IL-1β-injured HEL 299 cells indicated that inflammatory pathway, cell movement of macrophages, cellular infiltration by macrophages, and Th1/Th2 immuno-regulation pathways were included. Thus, NRICM101 is a therapeutic agent, and it can improve COVID-19 syndrome to confer beneficial effects through multiple targeting and multiple mechanisms.COVID-19 pandemic has been a global outbreak of coronavirus (SARS-CoV-2 virus) since 2019. Taiwan Chingguan Yihau (NRICM101) is the first traditional Chinese medicine (TCM) classic herbal formula and is widely used for COVID-19 patients in Taiwan and more than 50 nations. This study is to investigate  Scutellaria baicalensis, HA.), Mongolian Snakegourd Fruit , Indigowoad Root , baked Liquorice Root , Saposhnikovia Root , Peppermint Herb , Mulberry Leaf , Fineleaf Nepeta , Heartleaf Houttuynia , and Magnolia Bark   were selected using three criteria: P < 0.005, adj P < 0.05 and absolute fold change of ≥1.5). The differentially expressed genes were selected by StringTie (StringTir v2.1.4) and DEseq (DEseq v1.39.0) with genome bias detection/correction using Welgene Biotech’s in-house pipeline. The p-value was calculated using the hypergeometric p-value calculated as the probability of random drawing [HEL 299 cells were treated with TNF-α, IL-1β , and NRICM101 (100 μg/mL) for 12 h. Total RNA was extracted using TRIzol ® reagent . Purified RNA was using a Bioanalyzer 2100  with an RNA 6000 LabChip Kit  and measured at OD drawing ,29.t-test (p < 0.05) [For network and signaling pathways analysis of the whole transcriptome sequencing database, a total of 331 human target genes were prepared and subsequently analyzed using a core analysis tool in IPA software  and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The network analysis rankings were calculated based on statistical significance using Fisher’s exact  < 0.05) ,29.in vitro study, three independent experiments were conducted. Data are presented as the mean ± standard (SD) deviation. One-way analysis of variance followed by Dunnett’s test and Tukey’s post hoc test was conducted to analyze the differences between two groups and among multiple groups by SPSS software version 25.0 . ***P < 0.001 and ###P < 0.001 were considered to indicate a statistically significant difference.For each To predict target proteins of NRICM101, we used high throughput target screening platform analysis. The canonical pathway of NRICM101 was showed in in silico prediction, we further investigated the potential protective effects of NRICM101 on TNF-α/IL-1β-induced cell injury in HEL 299 cells. The results demonstrated that cell viability significantly decreased in TNF-α/IL-1β  treatment  showed the raw sequencing data of NRICM101-treated TNF-α/IL-1β-injured HEL 299 cells. To further determine the mechanism of action (MOA) of the genes and associated functions, the Ingenuity pathway analysis (IPA) database and KEGG database were used. The most significantly enriched pathways were selected and shown in https://www.biomedicinej.com/cgi/editor.cgi?article=1378&window=additional_files&context=biomedicine)). In addition, those results suggest NRICM101 regulated TNF-α/IL-1β medicated inflammatory pathway , Heartleaf Houttuynia  and Peppermint Herb  potentially blocked spike (S) protein of SARS-CoV-2 and host’s angiotensin-converting enzyme 2 (ACE2) interaction [S. baicalensis, HA.), Peppermint Herb , Fineleaf Nepeta , Magnolia Bark  and Mulberry Leaf  inhibited 3C-like protease (3CLpro) or main protease (Mpro) activity of SARS-CoV-2 [S. baicalensis, HA.), has a high binding affinity and inhibitory effect on Papain-like Protease (PLpro) [In silico study showed quercetin and kaempferol of Heartleaf Houttuynia  had high binding affinities for 3C-like protease (3CLpro) and RNA-dependent RNA polymerase (RDRP) of SARS-CoV-2 [M. officinalis, NK.), exerted a high binding affinity on angiotensin-converting enzyme 2 (ACE2) [I. indigotica, NE.), had high binding affinities for 3C-like protease (3CLpro) of SARS-CoV-2 [https://www.biomedicinej.com/cgi/editor.cgi?article=1378&window=additional_files&context=biomedicine)). The experimental studies showed that all active components of NRICM101 may be a potent anti-SARS-CoV-2 agent for COVID-19.Traditional Chinese medicine (TCM) formula, Taiwan Chingguan Yihau NRICM101), has been administered orally to COVID-19 patients in Taiwan [01, has beraction –34. In aRS-CoV-2 . In our 2 (ACE2) . SinigriRS-CoV-2 ,43. The It has been demonstrated that an association between poor outcome and cytokine-release syndrome (CRS) of COVID-19 ,45. In tFigs. 4https://www.biomedicinej.com/cgi/editor.cgi?article=1378&window=additional_files&context=biomedicine) showed a total of 213 genes were upregulated, and 118 genes were down-regulated. A network of the associations of different genes was generated following IPA analysis, as shown in https://www.biomedicinej.com/cgi/editor.cgi?article=1378&window=additional_files&context=biomedicine)). In the present study, in vitro study results are not only support our in silico results but also suggest a protective role and immuneregulation function of the NRICM101 against normal human lung cell injury.The first purpose of this study was to investigate the effects of NRICM101 on TNF-α/IL-1β-induced normal human lung HEL 299 cell injury. Our results demonstrated the protective effects of NRICM101 against TNF-α/IL-1-induced injury , cytotox8+ T cells, and then decreased IL-4, IL-5, and IL-13. IL-15 inhibits viral replication, reduces viral loads and reduces SARS-CoV-2-induced inflammation and fibrosis [in silico and in vitro studies showed that IL-15 production signaling is involved in TNF-α/IL-1β-induced normal human lung cell injury after NRICM101 treatment. Our results suggest that NRICM101 may affect T cell proliferation, activation, and NK cells activation. In future experiments, we will focus on IL-15 production signaling and design a series in vivo experiments of functional assays to include T cell proliferation, T cell activation, and NK cell activation and Th1, Th2 regulation.IL-15 is an immune-regulatory cytokine and plays an important role in anti-viral properties . IL-15 ifibrosis –50. Our S. baicalensis, HA.) can attenuate cytokine-induced and chemokine-induced inflammation [H. cordata, HC.) and Liquorice  extract, glycyrrhizin suppressed lipopolysaccharide (LPS)-stimulated expression of PGE2, iNOS, IL-1β, TNF-α and IL-6 levels in LPS-induced RAW264.7 inflammatory models and LPS-induced inflammatory in endometrial epithelial cells [In vitro study showed that IL-1β-induced activation of inflammatory factors  was suppressed by Mulberry Leaf  [G. glabra, NG.) inhibits IL-1β-induced inflammatory response in mouse chondrocytes [G. glabra, NG.) attenuates LPS-induced acute kidney injury [Previous studies demonstrated that baicalin and baicalein in Scutellaria Root  . 18β-Glydrocytes . In addiy injury . Our finin silico and in vitro approaches require further in vivo and clinical experimental verification. Collectively, our results revealed the potential of NRICM101 as a therapeutic agent based on TCM that may confer beneficial effects on COVID-19 patients through multiple targeting and multiple mechanisms. Furthermore, the major bioactive compounds of NRICM101 are worthy of attention as striking candidates for anti-viral agent and protective agent discovery studies on COVID-19. It may be useful for further studies on the therapeutic properties of NRICM101 and its constituents on a molecular level, with the aim of findings that might contribute to improving care for COVID-19 patients.Although most of the bioactive components of NRICM101 were selected for this study, the list of compounds in