Chronic inflammation of the gut is a multifactorial, incurable condition that involves interactions between our immune cells and the cells that line the surface layer of gut, known as the epithelium. Interleukin-10, a protein well known to be released by our immune cells, has a protective role in countering inflammation, partly due to the fact that it can block the NFκB pathway, a signalling pathway within cells that promotes genes involved in inflammatory responses. However, we recently showed that laboratory cultures of gut epithelial cells can also synthesise interleukin-10, which acts as a positive regulator of the NFκB pathway to support gut health. In this study here, we investigated further the impact of the role of interleukin-10 on the NFκB pathway and its targets within the gut using a whole animal approach, and confirmed that NFκB activation is indeed positively regulated by interleukin-10, affecting the expression of downstream target genes and their encoded proteins. This strengthens the importance of the interleukin-10/NFκB signalling pathway axis in maintenance of gut health and response to damage, inflammation, and infection. Understanding cell-specific biological roles of interleukin-10 and its interactions with NFκB could prove useful for future therapeutic intervention for interleukin-10 regulated inflammatory conditions.−/−Il10 mice, compared to wild-type C57BL6/J counterparts. Upon TNF injection, tissue from the small intestine showed upregulation of NFκB p65[RelA] activity, which was totally diminished in −/−Il10 mice and correlated with reduced levels of TNF, IκBα, and A20 expression. In serum, whilst IgA levels were noted to be markedly downregulated in IL-10-deficient- mice, normal levels of mucosal IgA were seen in intestine mucosa. Importantly, dysregulated cytokine/chemokine levels were observed in both serum and intestinal tissue lysates from naïve, as well as TNF-injected −/−Il10 mice. These data further support the importance of the IL-10-canonical NFκB signaling pathway axis in regulating intestinal mucosa homeostasis and response to inflammatory triggers in vivo.Interleukin-10 (IL-10) is an anti-inflammatory cytokine that has a major protective role against intestinal inflammation. We recently revealed that intestinal epithelial cells in vitro regulate NFκB-driven transcriptional responses to TNF via an autocrine mechanism dependent on IL-10 secretion. Here in this study, we investigated the impact of IL-10 deficiency on the NFκB pathway and its downstream targets in the small intestinal mucosa in vivo. We observed dysregulation of TNF, IκBα, and A20 gene and protein expression in the small intestine of steady-state or TNF-injected  IL10 and IL10R genes, both associated with an early onset of IBD, a greater severity of disease, and a lack of response to conventional treatments, including anti-tumour necrosis factor (TNF) biologics therapy  activity within the small intestine, which leads to a dysregulation of key NFκB target genes −/−Il10) transgenic mice, developed by Kuhn and colleagues  . Data were normalized against total cellular protein as measured in RIPA lysates by Pierce bicinchoninic acid (BCA) assay .Small intestine tissue samples were homogenized in a 300 μL ice-cold radio-immunoprecipitation assay (RIPA) lysis buffer  for 20 min on ice followed by centrifugation at 10,000× Murine serum and RIPA-extracted small intestinal tissue lysates were analysed, as per manufacturer instructions, using the following ELISA kits: Cxcl1/KC ; Cxcl10/IP-10 ; IL-1β ; IL-12/IL-23p40 allele-specific , and TNF . Immunoglobulin changes in murine serum (dilution 1:1000) were screened using the Ig Isotyping mouse uncoated ELISA kit , as were intestinal tissue levels of IgA (dilution 1:10). All ELISAs were measured at optical density (OD) 450 nm using a Tecan Sunrise plate reader .w/v BSA for 2 h, followed by overnight incubation with primary antibodies in sterile PBS pH7.4 containing 0.5% BSA, 0.05% Tween 20. Primary antibodies used were anti-TNFAIP3 mouse monoclonal antibody, clone 59A426 at 1:200 dilution , and an anti-IκBα polyclonal antibody at 1:1000 dilution . Secondary HRP-linked antibodies used were anti-mouse IgG1 (#7076) and anti-rabbit IgG (#7074), each at 1:2000 . Chemiluminescence detection was performed using SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific), with imaging and densitometry analysis on a ChemiDoc XRS+ system .RIPA-extracted intestinal tissue lysates (N = 3 mice per group) were loaded to nitrocellulose (100 ug protein) using a slot blot vacuum system for immunodetection of A20 (TNFAIP3) and IκBα protein. Membranes were first blocked with 5% p < 0.05.Results are expressed as mean ± standard error of mean (SEM). Independent sample groups were first assessed for normality and equality of variances, and then as appropriate using either a one-way ANOVA or non-parametric Kruskal–Wallis test followed by pairwise comparisons of treatments. Statistical analyses of datasets utilized Prism GraphPad v9.1.0  and StatsDirect v3.0.171 . Differences were considered statistically significant when p < 0.05 ANOVA (−/−Il10 mice (N = 8). However, a significantly attenuated TNF-induced response was observed for both Cxcl1/KC (9.3-fold change) and Cxcl10/IP-10 (2.2-fold) in serum of IL-10 deficient mice (N = 3), as compared to TNF-treated C57BL/6J mice . IL-12p40 levels in serum in response to TNF were not seen to be significantly attenuated in IL-10-deficient mice  with TNF  resulted in markedly elevated serum levels of NFκB-dependent inflammatory mediators Cxcl1/KC (68.3-fold), Cxcl10/IP-10 (27.1-fold), and IL-12p40 (16.5-fold) above those resting levels detected in naïve C57BL/6J mice (N = 5); all 05 ANOVA . No signent mice .p < 0.01 ANOVA); p < 0.01). Serum from TNF-induced IL-10-deficient mice was found to have significantly elevated levels of IgG1, and likewise, levels of both κ and λ light chains were both raised compared to untreated genotype controls ; We also examined for any differences in serum levels of immunoglobulins, as well as for specific changes in κ and λ light chains. At rest, there was a significant impairment noted for IgA and the κ light chain in IL-10-deficient mice ; see Il10−/− mice, likely lymphoid and myeloid cells , but this increase in p65 activation was not observed in the TNF-treated, IL-10-deficient small intestine ; see Tnf mRNA was observed in the C57BL/6J small intestine . In contrast, there was an attenuated TNF-induced Tnf response observed in Il10−/− mice ; see p < 0.001 ANOVA), with significantly attenuated levels seen in intestinal tissue lysates of TNF-induced, IL-10-deficient mice ; Following i.p. injection of mice with TNF, we examined the activation profile of the NFκB canonical pathway. Using small intestinal tissue lysates from naïve and TNF-injected mice, an ELISA was performed to assess p65[RelA] transcription factor activation. No significant difference in DNA-binding activity of p65 was observed under resting conditions in the e tissue A. Upon ip < 0.01 ANOVA, N = 3) when compared to non-injected controls (N = 5); see −/−Il10 mouse intestine (N = 9 mice) were significantly lower compared to those seen in wild-type intestine at rest (p < 0.05), and the response to TNF was markedly attenuated ; We also observed that TNF induced significant proinflammatory IL-1β and Cxcl1/KC responses in wild-type C57BL6/J intestinal tissue (both Given observed changes in serum (monomeric) IgA in IL-10-deficient mice, and in response to i.p. injection of TNF, we measured mucosal (polymeric) IgA in intestinal tissue lysates but observed that levels were not significantly different in treatment groups C.Tnfaip3 and Nfkbia, encoding key inhibitory regulators of NFκB, i.e., A20 and IκBα, respectively. The basal transcription of Tnfaip3 and Nfkbia genes from total RNA isolated from small intestinal mucosa was similar in both genotypes  relative to wild-type tissue ; IL10-deficient intestinal mucosa compared to wild-type tissue, it was notable that staining was markedly reduced in Il10−/− intestinal epithelial cells. Similarly, IκBα staining was almost undetectable in lamina propria of both strains, but it was severely reduced in Il10−/− intestinal epithelial cells under resting conditions; see The defect we observed in RelA activation in response to TNF in IL-10-deficient mice prompted us to check further downstream targets of the NFκB pathway, including enotypes A. We alsignaling . At restTnfaip3 and Nfkbia expression in the small intestine . In contrast, TNF induction of Tnfaip3 and Nfkbia in the intestinal tissue of Il10−/− mice was defective ; Tnip1 and Tnip2 expression in the small intestine were observed in response to TNF injection of wild-type mice, with no differences observed in Il10 knockout mice ; p < 0.001 ANOVA, N = 3); see Post i.p. injection of TNF in wild-type C57BL/6J mice, we observed a significant upregulation in out mice C. ImmunoIl10ra in the small intestine in both genotypes under resting conditions and post i.p. injection of TNF . Post-TNF injection, both Il10 and Il10ra were significantly upregulated in C57BL/6J mouse intestinal tissue, with significantly lower levels of TNF-induced Il10ra transcript detected in Il10−/− mice; p < 0.05 .Given that IL-10 receptor levels are important for IL-10 signaling, we also measured the expression of n of TNF . Basal eκB in immune cells such as macrophages and dendritic cells [κB in vitro, supporting its role as a major regulator of intestinal homeostasis [Interleukin-10 is considered a classical anti-inflammatory cytokine that downregulates pro-inflammatory pathways, including activation of NFic cells . We receeostasis . Here, ieostasis ,31.Tnf mRNA was higher in small intestine from resting −/−Il10 mice, but immunohistochemistry on fixed tissue revealed that this was mainly due to a small increase in TNF-expressing cells localized within the lamina propria, possibly activated T cells or myeloid cells [Il10–/– mice reared in germ-free conditions remained disease-free [κB pathway, as well as in the endothelium, again via activation of the same pathway [Il10-deficient mice showed a defective increase of serum Cxcl10 in response to TNF injection, implying that the source of the chemokine is probably from epithelial cells, and not from immune cells where IL-10 is observed to be a negative regulator of Cxcl10 [Naïve IL-10-deficient mice looked healthy and did not show any signs of inflammation, although they had lower TNF serum levels than the wild-type C57BL/6J controls. id cells . This isase-free , and onlase-free ,35. Upon pathway . The Il1f Cxcl10 . Similarf Cxcl10 .Il10−/− mice [IL-10 is also known to regulate growth and differentiation of B lymphocytes ,40 and i−/− mice . Normal −/− mice ,47.Tnf in the intestinal epithelium of wild-type mice, a result similar to that which others, and our group, have reported previously [Tnf mRNA induction in −/−Il10 small intestinal tissue, supporting our earlier in vitro studies where we clearly showed defective TNF-induced transcriptional activation of the Tnf gene in IL-10-deficient intestinal epithelium [−/−Il10 mice, its further activation in this transgenic model was completely abolished upon TNF injection. These results indicate that IL-10 deficiency has a clear in vivo effect on the canonical NFκB pathway activation in intestinal tissue, supporting earlier data obtained under in vitro culture conditions, as we reported previously [Tnfaip3 and Nfkbia mRNA levels, but within the −/−Il10 small intestinal epithelial cells at rest, A20 and IκBα protein levels, encoded by Tnfaip3 and Nfkbia, respectively, were observed to be extremely low in this transgenic strain compared to C57BL/6J mice. When challenged via i.p. with TNF, lower levels of Tnfaip3 and Nfkbia mRNA transcripts were observed within the small intestine of −/−Il10 mice compared to intestinal tissue taken from TNF- injected C57BL/6J mice. This defective response was further confirmed by examining the protein levels of the related proteins, A20 and IκBα, respectively. These results agree with our previous observations in which both of these genes were similarly affected by endogenous IL-10 deficiency in resting and TNF-induced enteroid cultures [−/−Il10 mice towards intestinal inflammation. Expression of Tnip1 and Tnip2, encoding for A20-binding-1 (Abin1) and A20-binding-2 regulators of NFκB (Abin2), were not substantially affected in this experimental model. TNF-induced upregulation of Il10ra was also impaired in the small intestine of −/−Il10 mice and this is important since the IL-10Rα subunit is responsible for binding and assembly of the receptor, and therefore can affect downstream signal transduction [TNF injection induced the transcription of eviously ,31,48. Tithelium . Real-tiithelium ,50 and tithelium . In smaleviously . We speccultures . A20 hascultures . Therefosduction .In summary, our study has shown that RelA/NFκB activation within the intestinal mucosa is positively regulated by IL-10 in vivo, affecting the expression of downstream target genes and their encoded proteins important for tissue homeostasis. Spatial information on NFκB activation would be a useful future approach, i.e., to establish in which epithelial cell and/or immune cell types within intestinal tissue these events occur. Two of the downstream targets observed to be affected were known inhibitors of the classical NFκB signaling pathway, IκBα and A20 (TNFAIP3). This was a key finding we had previously demonstrated in vitro, using small intestine crypt stem-cell-derived enteroids deficient in IL-10 , highlig


Orthodontic force was reported to induce tumor necrosis factor-α (TNF-α) expression, which activates osteoclast differentiation during orthodontic tooth movement (OTM). The aim of this study was to investigate the influence of DHA on TNF-α-induced osteoclast formation and OTM in vivo. We examined osteoclast formation and bone resorption within the calvaria of both wild-type (WT) and GPR120-deficient (GPR120-KO) mice injected with phosphate-buffered saline (PBS), TNF-α, TNF-α and DHA, or DHA. DHA inhibited TNF-α-induced osteoclast formation and bone resorption in WT mice but had no effect in GPR120-KO mice. OTM experiments were performed in mouse strains with or without regular injection of DHA, and the effects of DHA on osteoclast formation in the alveolar bones during OTM were examined. DHA also suppressed OTM in WT but not GPR120-KO mice. Our data showed that DHA suppresses TNF-α-induced osteoclastogenesis and bone resorption via GPR120. TNF-α has considerable significance in OTM, and therefore, DHA may also inhibit TNF-α-induced osteoclast formation and bone resorption in OTM.Docosahexaenoic acid (DHA) is an omega-3 fatty acid that has a range of positive impacts on human health, including anti-inflammatory effects and inhibition of osteoclast formation  Docosahexaenoic acid (DHA), a long-chain n-3 polyunsaturated fatty acid with 22 carbon atoms and 6 double bonds, is one of the polyunsaturated fatty acids. It has been shown to have benefits on several disorders, including cardiovascular disease , diabetein vitro  are seven transmembrane domain receptors that play crucial roles in many cellular functions and in the regulation of several physiological and pathological processes –20. Longin vivo . Thus, tin vivo , 22–27.in vitro  activation and nuclear factor of activated T-cell cytoplasmic (NFATc1) induction . MoreoveOrthodontic tooth movement (OTM) with the application of orthodontic force involves the remodeling of alveolar bone and reconstruction of the periodontal ligament. Multiple factors are involved in the mechanism underlying OTM, including cytokines, growth factors, components of the bone matrix, and neurotransmitters, which mediate the differentiation and function of osteoblasts and osteoclasts, causing bone remodeling –38.Several studies have demonstrated that mechanical force loading induced the production of TNF-α , 40. Morin vitro experiments. Furthermore, we examined the influence of DHA on OTM and the levels of osteoclast activity mediated via GPR120.The present research was performed with the aim of evaluating the effects of DHA injection on TNF-α-induced osteoclastogenesis and bone destruction in mice through GPR120, and elucidating the underlying mechanisms by (dE1/dE1) mice)  to be used as WT mice. GPR120-KO mice were generated by crossing mice expressing Cre-recombinant activity under the influence of chicken actin promoter and C57BL6/J mice bearing Ffar4-flowed gene (Ffar41) mice) . All ani1) mice) , and rec1) mice) .n = 4), which received daily subcutaneous supracalvarial injection of the following for 5 consecutive days: negative control, phosphate-buffered saline (PBS); TNF-α (3 μg/day); DHA (100 µg/day) and TNF-α (3 μg/day); and DHA (100 µg/day). On day 6, all mice were sacrificed, and the calvariae were resected and then fixed immediately in 4% PBS-buffered formaldehyde. After fixation for 24 h at 4°C, 14% ethylenediaminetetraacetic acid (EDTA) was used to demineralize all calvariae for 3 days at room temperature. After dehydration in a tissue processer , all calvariae were embedded using paraffin and then cut into 5-µm sections perpendicular to the sagittal suture using a microtome. Paraffin sections were stained with TRAP solution mixed with acetate buffer (pH 5.0), naphthol AS-MX phosphate (Sigma-Aldrich), Fast Red Violet LB Salt (Sigma-Aldrich), and 50 mM sodium tartrate. After staining, all sections were counterstained with hematoxylin. Cells were considered as osteoclasts if they were TRAP-positive and contain three or more nuclei , before being embedded in paraffin. The upper left first molars of these mice were cut into horizontal sections at 100, 140, 180, 220, and 260 μm from the apical of the bifurcation area. All the sections were stained with TRAP solution and counterstained with hematoxylin. The mesial side of the labiolingual long axis of the distobuccal root was regarded as the compression side. Finally, we measured the number of osteoclasts on the alveolar surface of the compression side on the sections of different levels to analyze osteoclast formation .Mice treated with daily subcutaneous supracalvarial injection were sacrificed on day 6. Their calvariae were fixed immediately in 4% PBS-buffered formaldehyde and then scanned using microfocus computed tomography (micro-CT) . Software TRI/3DBON64  was used to create 3D images of the calvariae. The resorption area was regarded as the black area between the sutures, in contrast to the bone surface, which appears white in the 3D CT images. To quantify the bone resorption area, a 50 × 70 pixel rectangle was selected at the intersection between the sagittal and coronal sutures, and the amount of bone resorption area to the total area was measured using ImageJ software  .2HPO4, 10 mM NaHCO3, 60 mM sorbitol, 70 mM NaCl, 1 mM CaCl2, 0.5% [w/v] glucose, 0.1% (w/v) bovine serum albumin [BSA], and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). EDTA was prepared at a concentration of 5 mM with 0.1% BSA in PBS and filtered through a 0.2-µm filter. Calvariae were subjected to digestion via collagenase and EDTA, for 20 and 15 min, respectively, at 37°C on a shaker. Fraction 1 (collagenase), fraction 2 (EDTA), fraction 3 (collagenase), fraction 4 (collagenase), fraction 5 (EDTA), and the digests were collected. Fractions 3–5 were considered as the osteoblast high fractions. Cells were cultured in alpha minimal essential medium (α-MEM) (Wako), containing 10% fetal bovine serum (FBS) and 100 IU/ml penicillin G and 100 μg/ml streptomycin, overnight. The adherent cells were collected using trypsin-EDTA . The cells were cultured in α-MEM containing 10% FBS for 3 days with the culture media changed every 2 days. Adherent cells were used as osteoblasts  was prepared in isolation buffer  in 800 μl of TRIzol reagent . RNA was also prepared from osteoblasts of the mouse strains from the in vitro experiments. Cells were incubated in α-MEM with either PBS, TNF-α (100 ng/ml), TNF-α (100 ng/ml) and DHA (100 ng/ml), or DHA (100 ng/ml) for 3 days. Total RNA was used to synthesize cDNAs of each group of samples with oligo-dT primers (Invitrogen) and reverse transcriptase in a total volume of 20 μl. Each reaction contained 2 μl of cDNA and 23 μl of a mixture consisting of SYBR Premix Ex Taq  and 50 pmol/μl of primers. TRAP, RANKL, and OPG mRNA expression levels were evaluated using real-time RT-PCR using a Thermal Cycler Dice Real-Time System (Takara). The PCR cycling conditions were started with an initial denaturation step at 95°C for 10 s, followed by 45–60 cycles of amplification, with each cycle consisting of a denaturation step at 95°C for 5 s and an annealing step at 60°C for 30 s. To determine the relative expression levels of RANKL mRNAs, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The primer sequences used for cDNA amplification were as follows: GAPDH, 5′-GGTGGAGCCAAAAGGGTCA-3′ and 5′-GGGGGCTAAGCAGTTGGT-3′; TRAP, 5′-AACTTGCGACCATTGTTA-3′ and 5′-GGGGACCTTTCGTTGATGT-3′; RANKL, 5′-CCTGAGGCCAGCCATTT-3′ and 5′-CTTGGCCCAGCCTCGAT-3′; OPG, 5′-CTTAGGTCC AACTACAGAGGAAC-3′ and 5′-ATCAGAGCCTCATCACCTT-3′; and Cathepsin K 5′-GCAGAGGTTGTACTATGA-3′ and 5′-GCAGGCGTTGTTCTTATT-3′ 2D3 (Sigma-Aldrich) with or without TNF-α (100 ng/ml) and with or without DHA in 96-well plates. Co-culturing was continued for 4 days after which it was ended by fixation with 4% formaldehyde and stained with TRAP as described previously . These cells were collected as osteoclast precursors. GPR120-KO osteoclast precursors and WT or GPR120-KO osteoblasts were co-cultured in the presence of 10eviously . Cells wOsteoblasts isolated from WT or GPR120KO mice were seeded in 96-well plates in α-MEM at a density of 3 × 10³ per well overnight at 37°C. Cells were then stimulated with PBS, TNF-α (100 ng/ml), TNF-α (100 ng/ml) and DHA (100 ng/ml), or DHA (100 ng/ml) for 1 h. After this, we removed the culture medium and washed it three times using PBS. We then fixed it in 4% (w/v) formaldehyde solution in PBS for 15 min at room temperature. The fixative solution in the 96-well plates was removed and washed by PBS before adding 0.5% Triton X-100 (v/v) in PBS for 15 min. All the wells were washed with PBS and blocked with 3% BSA in PBS. Wells were incubated with p65 (D14E12) XP Rabbit mAb  diluted in 3% BSA in PBS to 1:400 overnight at 4°C. Cells were washed and incubated with Alexa Fluor 555 conjugate  in 3% BSA in PBS for 1 h in the dark at room temperature. Finally, cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min before visualizing fluorescent cells under a fluorescence microscope .g tractive force. After 12 days of force loading, the distance between the upper first molar and the second molar was measured with silicone impressions of the maxilla, and the distance was compared between groups. These alveolar bones were also used for histological analysis.OTM was performed as described previously . Twelve-p < 0.05 represented statistical significance.All data were expressed as the mean ± standard deviation (SD). Statistical analyses were performed using Scheffé’s test and t-test. In all analyses, in vivo. TNF-α and DHA were injected into the calvariae of both WT and GPR120-deficient (GPR120-KO) mice. Following 5 consecutive days of TNF-α injection, large numbers of TRAP-positive cells with three or more nuclei were recognized along the suture mesenchyme on histological sections. In contrast, WT mice co-administered TNF-α and DHA showed a significant reduction in the mean number of TRAP-positive cells compared to those treated with TNF-α alone  or the DHA-treated group. In contrast, the group treated with both TNF-α and DHA showed a lower RANKL mRNA expression than the group treated with TNF-α. On the contrary, the expression level of RANKL mRNA was similar in GPR120-KO osteoblasts treated with TNF-α and with TNF-α plus DHA 2D3 and 10−6 M prostaglandin E2. The numbers of TRAP-positive cells were measured in co-cultures, and these results indicated a significant decrease in the TRAP-positive cell number associated with DHA treatment in co-culture using WT osteoblasts but not GPR120-KO osteoblasts , TNF-α (100 ng/ml) and DHA (100 ng/ml), or DHA (100 ng/ml) in the presence of 10To investigate the role of DHA in the transcription factor NF-κB pathway, we divided the WT and KO cells into four groups and applied stimulation of PBS as a control, TNF-α (100 ng/ml), TNF-α (100 ng/ml) and DHA (100 ng/ml), or DHA (100 ng/ml). We found that NF-κB nuclear localization significantly increased in WT osteoblasts when osteoblasts received TNF-α stimulation, while it was reduced when DHA and TNF-α were co-administered (A 12-day OTM was examined in WT and GPR120-KO mice treated with PBS or DHA. The OTM distance on day 12 was significantly different between control (PBS) and DHA-treated WT mice  , JM. Writing (review and editing), HK. Supervision, AI and IM. Project administration, HK. Funding acquisition, AK, HK, and IM. All authors contributed to the article and approved the submitted version.


Viral infectious diseases threaten human health and global stability. Several vaccine platforms, such as DNA, mRNA, recombinant viral vectors, and virus-like particle-based vaccines have been developed to counter these viral infectious diseases. Virus-like particles (VLP) are considered real, present, licensed and successful vaccines against prevalent and emergent diseases due to their non-infectious nature, structural similarity with viruses, and high immunogenicity. However, only a few VLP-based vaccines have been commercialized, and the others are either in the clinical or preclinical phases. Notably, despite success in the preclinical phase, many vaccines are still struggling with small-scale fundamental research owing to technical difficulties. Successful production of VLP-based vaccines on a commercial scale requires a suitable platform and culture mode for large-scale production, optimization of transduction-related parameters, upstream and downstream processing, and monitoring of product quality at each step. In this review article, we focus on the advantages and disadvantages of various VLP-producing platforms, recent advances and technical challenges in VLP production, and the current status of VLP-based vaccine candidates at commercial, preclinical, and clinical levels. However, they sometimes have safety concerns and deviations in the protective immune response. Recent advances in nucleic acid-based, subunit, and recombinant viral vector-based vaccines may overcome the limitations of traditional vaccines against infectious pathogens by enhancing immunity without compromising safety, efficacy, and tolerability and can also control infectious diseases during outbreaks or pandemics. The significance of such vaccine approaches in rapidly developing vaccine candidates for pandemic threats has been previously reported and is considered an approach for pandemic preparedness and response . Virus-lVLPs are non-infectious multiprotein structures that mimic native viruses but lack the disease-causing viral genome, making them safer vaccine candidates. Because of their optimal size (20–200 nm), they can practically display any epitope in a multivalent format that guarantees a high humoral or cell-mediated immune response in the host.in vitro assembly of VLPs, removal or replacement of the transmembrane region, introduction of a suitable heterologous signal peptide, new bioprocessing modalities, and new macro-scale purification materials can address these problems robustly, at the expense of process complexity , which are sensed by pathogen recognition receptors (PRRs) present either on the cell surface or endosomes of dendritic cells (DC). Presentation of VLP-peptides along with major histocompatibility complex (MHC) class I or class II molecules by DC induces CD8+ or CD4+ T cell responses, respectively . SimultaSeveral platforms have been used for VLP production. The choice of the preferred platform provides flexibility in the manufacturing conditions required for the scaled-up production of VLP vaccines . RemarkaThis article briefly presents the merits and demerits associated with various platforms, recent advances, and technical challenges in VLP production. The current scenario of VLP-based vaccine candidates at commercial, clinical, and preclinical levels are also summarized.2VLP-based vaccines have been developed as the next-generation vaccines. However, to ensure optimal protein folding, post-translational modifications (PTMs), cost-effectiveness, and scale-up production of these vaccines in a timely manner, an appropriate platform must be selected. VLPs can be experimentally generated in the laboratory using recombinant viral proteins that are expressed in a range of expression systems including prokaryotic cells, yeast, insect cell lines, plants and mammalian cell lines. In addition, cell-free expression techniques have been used successfully. In these cases, VLP proteins are expressed first in a cell-based expression system, then assembled in a cell-free environment for proper folding . All sysvia trial-and-error by comparing the translated products of multiple expression systems. Expression in E. coli is often preferred for producing small proteins with limited PTMs; however, PTMs of larger proteins require a more complex expression system, such as yeast, baculovirus, and mammalian cells. Compared to higher eukaryotes, yeasts are considered a cost-effective production system. In addition, yeasts avoid endotoxin and viral contamination problems associated with bacterial and mammalian expression systems  are used to produce approximately 28% of all VLPs , which transfers oligosaccharides to N-linked asparagine, thereby facilitating glycosylation  VLPs (Hansenula polymorpha (methylotrophic yeast) is particularly striking  VLPs . EV71 castriking . H. poly sources . This pl sources .® and Sci‐B‐Vac™. These vaccines contain the HBsAg small and middle proteins (GenHevac B) or middle and large proteins (Sci‐B‐Vac) , HEK293, amniotic fluid CAP-T, and Vero cells, are used to produce VLPs . These ci‐B‐Vac) , 59. Simi‐B‐Vac) . Howeverapeutics . Therefoapeutics . Additioapeutics . Similar55.1Cell concentration at infection (CCI), multiplicity of infection (MOI), and time of harvest (TOH) in the baculovirus system strongly influence protein expression and, consequently, assembly of VLP. 72–120 h post-infection is considered the optimal time of harvest. Outside this period, various parameters can hinder productivity, such as cell lysis, protein degradation, and difficulties in downstream processing owing to the presence of contaminant proteins, hosts, or viral DNA. Incomplete VLP may also be released into the medium along with target mature VLP and can hinder the downstream processing of VLP –66. Diff5.2in-vivo assembly) or in cell-free conditions (in-vitro assembly). The nature of the stabilizing contacts can influence the VLP assembly route  are genetically fused to the truncated circumsporozoite protein (CSP) and serve as protein carriers. The region of the CSP included in the RTS,S vaccine includes immunodominant B-cell epitopes (last 18 four amino acids (NANP) repeats) and three known T-cell epitopes: a highly variable CD4+ T-cell epitope before the Thrombospondin (TSP)-like domain, a highly variable CD8+ T-cell epitope within the TSP-like domain, and a conserved “universal” CD4+ T cell epitope at the C-terminus. These epitopes of Mosquirix enhanced antigen presentation to the immune system and likely facilitated strong anti-CSP antibody and T-cell responses measured in vaccinated individuals. Mosquirix has received approval from the regulatory authorities , 72.in vitro assembly of VLPs requires control of variables such as temperature, pH, and specific assembly solution  and storage. Various studies have demonstrated the effects of several physicochemical parameters  on the formation of VLPs  or nonionic copolymers may provide a solution depending on the degree of sensitivity of the cells , 79–81. 5.4The separation of a complex mixture of proteins from VLP using transduction is challenging because of their similar size and molecular weight. The same problem does not exist in the case of transfection, which thus, significantly decreases the complexity of downstream processing. Serum-free medium further improves or facilitates downstream processing because it does not contain any animal-derived supplements. Controlling various process conditions, such as optimal salt, pH, and ion concentrations, can reduce or avoid protein aggregation, which is a major challenge in downstream processing. However, aggregation during large-scale production of VLPs, owing to their larger size, leads to the loss of a considerable portion of the target protein or reduced immunogenicity . In a re5.5in situ monitoring of the kinetics of VLPs is possible using this technology - polyacrylamide gel electrophoresis, western blotting (WB), enzyme-linked immunoassay (ELISA), and bicinchoninic acid (BCA) protein quantification assays. Flow cytometry is now being exploited as a tool for the study and validation of VLPs . Howeverchnology –93.5.6Critical Quality Attributes (CQAs), such as the biophysical, biochemical, structural, and immunochemical properties of VLPs, are critical parameters in developing vaccine candidates with good stability and delivering consistent products for the preclinical and clinical stages. In addition, these parameters also help in post-licensure life-cycle management to support market demand by upgrading the process or scale-up.Various methods are used to characterize the physical and structural attributes of VLPs, such as transmission electron microscopy (TEM), Atomic force microscopy (AFM) for morphology, size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) for monodispersity , dynamicIn vitro relative potency or mouse potency assays are used to test the release and stability of VLPs. These assays are required to define the quality and quantity of epitopes on recombinant antigens. Therefore, they play a major role in the implementation of process improvements or scale-up.Activity-related attributes are also important for checking the structure–function relationship in VLP-based vaccines or the integrity of key epitopes. Tools such as bilayer interferometry (BLI) and SPR are used to check epitope-specific antigenicity and epitope mapping . In vitrE. coli are heterogeneous in nature because of the many ill-formed or incomplete particles. This heterogeneity can be reduced by post-purification disassembly followed by reassembly, as reported previously  batch analysis. Lot-to-lot comparability relies on the physical and functional properties of the vaccine, which are responsible for predicting the consistency of batches across different lots. This analysis ensures that vaccine lots are equivalent in potency, purity, and physicochemical integrity. Any process modification and formulation change between these phases must also be verified by CQA-based comparability, which is required for Good Manufacturing Practice (GMP) .5.7Nicotiana benthamiana plants  by a Swiss biotech company, Saiba-AG  7/8 and serves as a potent adjuvant by engaging these innate receptors in specific B cells  or mouse potency under proper dissolution conditions to fully recover the antigen is used to assess the stability of vaccines over many months , accelerated, and stress (high-temperature) conditions . Long-teSimilarly, to avoid the freezing–de-freezing cycle, critical attention should be paid to choosing appropriate and compatible cryoprotectants to preserve the native structure of VLPs.66.1E. coli-based VLPs against FMDV infections of different serotypes, making this approach cost-effective. Carvalho et al. showed that the use of sulfated cellulose membrane adsorbers (SCMA) enhances the scalability of vaccines compared to conventional ion exchanger membrane adsorbers by reducing the number of purification steps. This study suggested that SCMA could qualify as a generic platform for purifying VLP-based influenza vaccines  VLPs. This protocol does not include non-preferred industrial size-exclusion chromatographic purification or sucrose gradient ultracentrifugation steps . This apvaccines . HoweverNicotiana species are commonly used to generate plant-made pharmaceutical proteins (PMPs). However, these plants have high levels of phenolics and toxic alkaloids; therefore, the downstream processing of target PMPs is hindered by technical and regulatory difficulties. Hence, lettuce-based production systems are used instead of tobacco-based platforms because of their low levels of secondary metabolites  variants of SARS-CoV-2, respectively. These vaccines contain a stabilized form of the spike protein of SARS-CoV-2 inserted into the eVLP lipid bilayer , 118.The immunogenicity of VLPs can be increased by engineering VLPs, allowing for a more versatile display of antigens . To maxi7Several VLP-based vaccine candidates tested in clinical and preclinical trials are summarized in 8Currently, only a few VLP-based vaccines are commercially available against various pathogens such as zika virus, HCV, HBV, and HPV due to various hurdles in their development processes. Therefore, these hurdles must be addressed to enhance commercialization. Researchers are trying to refine VLP-based vaccines at the formulation level by selecting appropriate adjuvants and stabilizers/protectants, administration routes, and delivery vehicles, such as liposomes, poly(lactide–co-glycolide) microparticles, or alginates to reduce the number of vaccine shots and dosage. Concomitantly, methods that can enhance the shelf life of vaccines and avoid cold chains are under investigation.From an economic point of view, Gardasil is the most successful VLP-based vaccine, yielding over $48.7 billion in 2021. Gardasil has been replaced by Gardasil9 that is an adjuvanted non-infectious recombinant 9-valent vaccine. Gardasil9 has increased concentrations of L1 VLPs for HPV 16 and 18 in order to induce antibody responses in comparison to Gardasil . L1, is PK, NA, KA, and RG were responsible for the conceptualization of the manuscript. SR, AJ, RR and RG: Contributed to latest literature review and manuscript drafting. All authors contributed to the article and approved the submitted version.


Nanotechnology is a fast-evolving field focused on fabricating nanoscale objects for industrial, cosmetic, and therapeutic applications. Virus-like particles (VLPs) are self-assembled nanoparticles whose intrinsic properties, such as heterogeneity, and highly ordered structural organization are exploited to prepare vaccines; imaging agents; construct nanobioreactors; cancer treatment approaches; or deliver drugs, genes, and enzymes. However, depending upon the intrinsic features of the native virus from which they are produced, the therapeutic performance of VLPs can vary. This review compiles the recent scientific literature about the fundamentals of VLPs with biomedical applications. We consulted different databases to present a general scenario about viruses and how VLPs are produced in eukaryotic and prokaryotic cell lines to entrap therapeutic cargo. Moreover, the structural classification, morphology, and methods to functionalize the surface of VLPs are discussed. Finally, different characterization techniques required to examine the size, charge, aggregation, and composition of VLPs are described.  Nanotechnology is an interdisciplinary field devoted to engineering and developing structures ranging from 1 to 500 nm. Structures that correspond to this scale are defined as nanoparticles (NPs). NPs possess promising optical, chemical, and physical properties attractive for biomedical purposes, such as diagnostic, chemical sensing, cellular imaging, drug delivery, therapeutics, and tissue engineering [Given the unique physical, optical, chemical, and therapeutic properties of NPs, there has been an increasing interest in designing methods to develop and characterize them. NPs are prepared through top-down and bottom-up approaches, including laser ablation, sputtering, etching, and mechanical milling techniques. The latter encompasses spinning, chemical reduction, molecular condensation, and green synthesis processes . InsteadAmong protein-based nanomaterials, virus-like particles (VLPs) are self-assembled platforms commercially approved by the US Food and Drug Administration (FDA) since the 1980s . Since VIn contrast to other organic NPs, VLPs are convenient, because they exhibit higher biocompatibility, the capacity for cell internalization, and ease of functionalization for cell targeting. In fact, for the latter, they can be tailored by chemical or genetic methods with various biomolecules  to enhance their bioavailability and, herein, evoke both humoral and cellular immune responses ,12. HoweNowadays, there is a constant effort to develop analytical methods that, alone or in combination with others, enable scientists to assess the influence of physicochemical parameters in the biological activities of nanomaterials. For instance, the therapeutic performance, stability, and morphology ,14 of VLSince the novel coronavirus SARS-CoV-2, there has been an increasing interest in reviewing VLPs as a powerful approach to producing vaccines and nanocarriers . HoweverTherefore, the literature regarding the structure classification, production, morphology, and functionalization of VLPs for biomedical applications was consulted in this review. The research engines PubMed, Google Scholar, Web of Science, and Wiley Online Library databases were used to compile the literature. In addition, the same databases were used to integrate the main aspects of viruses and how the geometry is indispensable to constructing VLPs-based platforms.Viruses are entities characterized by the lack of machinery for self-replication and energy production. Their replication relies on hijacking the cellular systems of the host cells to produce the molecules necessary for their assembly and subsequent escape from the cells. Infectious viruses that have not been internalized in the host cells (not in a replication phase) and residing outside the cell host are called virions. Viruses are ubiquitous entities containing a DNA- or RNA-based genome protected or not by a protein shell known as a capsid and other accessory biomolecules, such as proteins and membranes . The capThe general structures of viral capsids show a diversity of conformations, including icosahedral conformation. Icosahedral capsids comprise 20 triangular subunits, whereas helical capsids are proteins assembled to form helical cylinders . DespiteBesides the capsids, viruses are surrounded or not by a viral envelope that facilitates their fusion and infection of the host cell. In addition to the presence of proteins and glycoproteins, this envelope can contain lipidic membranes acquired from the host. Viral envelopes are necessary to protect the viral genome, increase the packaging capacity, confer structural flexibility, and enable the new viruses to exit from their host cell and avert immune responses . Viruses with envelopes are known as enveloped viruses, such as the varicella-zoster virus , lymphocThe assembly of VLPs uses exclusively viral proteins and excludes the genetic material. Thus, VLPs can be produced from a myriad of wild-type viruses, such as hepatitis B and E, tobacco, and papaya mosaic viruses , and froThe production of VLPs using bacteria, mammalian cells, plants, yeast, and insect cell lines is well-documented ,38,39. HThe capsid self-assembly of VLPs is a spontaneous natural process by which highly ordered structures arise from the interactions between protein monomers, also known as building blocks. The self-association between building blocks is facilitated by a thermodynamic equilibrium based on van der Waals, hydrogen bonding, hydrophobic, and electrostatic interactions during the nucleation and growth phases ,41. As aThe self-assembly of VLPs is assisted by small molecules such as scaffolding proteins and nucleic acids . In this®-B hepatitis B vaccine [® human papillomavirus vaccine [Since the self-assembly of VLPs can be perturbed by changes in the salt concentration, denaturant agents, pH, and temperature , excipie vaccine . Compara vaccine .VLP-based nanocarriers could be produced by producing viruses in their natural host, followed by virion purification, disassembling, and nucleic acid removal see . Then, tThe coat proteins of the VLPs can be heterologously produced and purified from an industrial microorganism and reconstituted under suitable conditions in the presence of their cargo. In this regard, the cargo loading could be obtained by simple encapsulation when the charges of the inner portion of the VLP and the cargo surface are complementary. However, as described in detail below, the loading could also be reached by chemical or genetic modification of the coat protein on both the inner and outer surfaces.Although most VLPs are deficient in viral genetic material, they retain properties from the native virus from which they are produced. These features include their affinity for cellular receptors, host cell entry mechanisms, and immunogenicity . As natiFunctionalized VLPs can trigger an immune response by activating PAMPs. These patterns are conserved molecular motifs associated with pathogen infections . PAMPs aLike other NPs, the surfaces of VLPs can be modified with various ligands to improve their therapeutic efficacy, bioavailability, and cellular interactions. The introduction of multiple molecules on the VLPs surface is known as multivalence. Then, VLPs are multivalent engineered nanoplatforms that form independent ligand–receptor bonds, elicit therapeutic responses, and mitigate endemic diseases. For example, Garg and coworkers developed a VLP-based multivalent vaccine (CJaYZ) against four arboviruses, including the zika, chikungunya, yellow fever, and Japanese encephalitis viruses. Even though the CjaYZ vaccine promoted a neutralizing antibody response against the four viruses in Balb/c mice models, further studies are required to assess the precise antigen amount of individual VLPs and their efficacy in other animal models . The genGiven the structural diversity of VLPs, they have been categorized into three main groups: enveloped, nonenveloped, and chimeric. For example, enveloped VLPs (eVLPs) are expressed using eukaryotic systems and as wild-type viruses. eVLPs are complex structures that own a host–cell-derived membrane and one or more glycoproteins. The viral envelope in eVLPs can be engineered to display heterologous adjuvants and antigens; however, this process might alter their downstream processing due to the possible presence of host cellular contaminants . 49) have been expressed on silkworm larvae to deliver doxorubicin into human colon carcinoma cells [As vaccines, eVLPs stimulate immune responses and are manipulated with chemical or genetic methods. In this regard, a handful of eVLPs have been produced from pathogenic viruses for vaccine development. Some examples include eVLPs derived from the West Nile virus (WNV), dengue virus (DENV), JEV, Rift Valley fever virus (RVFV), and Ross River virus (RRV), among others ,62. For ma cells . In addiNicotiana benthamiana plants. The immunogenicity and tolerance of Ro-VLPs were evaluated in adults, toddlers, and infants [Non-eVLPs are single or multiple capsid protein nanoconstructs that lack cell membranes. Members of this category are produced on eukaryotic or prokaryotic expression systems. The surfaces of non-eVLPs can also be manipulated with chemical and genetic approaches to display epitopes or peptides on their surfaces and, herein, elicit wider immunological responses . For ins infants . Chimeric VLPs (cVLPs) are nanoplatforms from structural components originating from at least two different viral serotypes . In thes+ and CD4+ T cells and antibody responses [For biomedical purposes, cVLPs have been prepared from major structural components of influenza viruses  and human immunodeficiency virus type-1 (HIV-1)  to target colon carcinoma cell lines and for vaccination purposes . In anotesponses . The immesponses . Recombinant proteins are derived from the expression of recombinant DNA within living cells. Once a DNA fragment is inserted into cell lines, the cells are induced to express the protein of interest.An expression vector is used to introduce genetic materials into cells. This vector contains sequences upstream of the cloned gene that controls its transcription and translation . Non-virEscherichia coli, Bacillus subtilis, Pichia pastoris, baculovirus/insect cells, plant cells, and mammalian cells [E. coli, Chinese Hamster Ovary cells, Saccharomyces cerevisiae, murine myeloma cells, etc. [On the other hand, viral vectors are optimized platforms able to transfer genetic material into host cells . Variousan cells . For clils, etc. . Some ofls, etc. . For VLP production, heterologous gene expression is used to clone the gene(s) of interest . For example, the heterologous expression has been used to produce VLPs derived from the coat proteins of (1) single- and double-stranded DNA viruses , canine parvovirus, and JC polyomavirus; (2) single-stranded RNA positive-sense viruses ; (3) single-stranded RNA negative-sense viruses ; and (4) double-stranded RNA viruses .T). This triangulation number is important, because the higher this value is, the more the diameter of the capsid and its loading capacity and number of interactions will increase [T number defines their symmetry and location. The T number is defined as the subdivision of the 20 triangular shapes that conform to an icosahedron. The Caspar-Klug theory (CK theory) dictates the symmetry of the viruses. This theory assumes that most viruses adopt icosahedral arrangements that vary in size from 20 nm to 800 nm, dictated by the number of protein–protein and protein–nucleic acid interactions ,79. Thesincrease ,80 , as well as weak interactions  between the amino acid residues and the capsomers located in the structure. However, interactions may occur between the capsomers [Likewise, it has been explained how the self-assembly of capsomers considers the formation of covalent bonds ; ricin toxin A-chain (RTA) (100 fM); and chemotherapeutic drugs such as doxorubicin, cisplatin, and 5-fluorouracil (<1 nM) into human hepatocellular carcinoma and human epidermoid carcinoma cell lines [For more than a decade, the design of drug delivery systems has been considered successful, especially during the encapsulation of chemotherapeutic agents from materials designed by nanotechnology ,92. For The surface modification of VLPs can be achieved with covalent, noncovalent, and genetic approaches. In genetic procedures, small or entire proteins are accommodated on the VLP surface via loop insertion, N-terminus/C-terminus (N/C-ter) modification, or the mutation of amino acid residues. In loop insertion approaches, epitopes are introduced into the surface-exposed loops of capsid proteins from VLPs to induce strong neutralizing responses . During Post-translational modifications (PTMs) are biochemical events that change the properties of a protein after translation . In suchβ bacteriophage VLP-based vaccine produced in E. coli has been phosphorylated at Thr181 to reduce the aggregation of hyperphosphorylated pathological Tau (pTau) in non-Tg and rTg4510 mice [Nicotiana benthamiana displayed influenza hemagglutinin (HA) glycoproteins and elicited immunoglobulin G (IgG) and immunoglobulin E (IgE) responses in 34% of subjects without hypersensitivity or allergic reactions [For VLPs, PTMs depend upon the expression system used and are convenient for enhancing their immunogenicity, antigen stability, and therapeutic properties. For example, a Q510 mice . In anoteactions . N- and O-glycosylation patterns. These PTMs are entailed during cell adhesion, protein targeting, and immune responses [In contrast, baculovirus expression systems (BES) have permitted other PTMs on VLPs, such as acylation, mannosylation, and disulphide bond formation. Such modifications have enabled the expression of multiple VLPs sharing functional similarity, structural arrangements, and antitumor response enhancement ,105. In esponses . Mw), and elemental composition of NPs [Considering that VLPs are NPs, we can characterize them following the standard techniques used to reveal the morphology, size distribution, zeta potential , molecular weight (n of NPs . Examinin of NPs . The particle morphology of VLPs can be examined with transmission electron microscopy (TEM) and scanning electron microscopy (SEM). However, other types of electron microscopy (EM), such as cryo-EM, have been used to report the morphology of influenza HA VLPs and visualize their interactions with murine DCs .Bombyx mori cytoplasmic polyhedrosis virus VLPs (50–70 nm) [For VLP-based vaccines, TEM has been used to confirm the size and morphology of many VLP-based vaccines, for example, spherical porcine encephalomyocarditis virus (EMCV) VLPs (30–40 nm) , HPV VLP0–70 nm) .Macrobrachium rosenbergii nodavirus VLPs (27-30 nm) targeting epidermal growth factor receptor (EGFR)-positive colorectal cancer cells [In the case of SEM, the size, shape, and surface composition can be analyzed. For VLPs, this tool has been applied in combination with TEM to observe the size and corroborate the morphology of SARS-CoV-2 VLPs , influener cells . In addiDLS, also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QLS), is a noninvasive tool used to measure the Brownian motion of macromolecules in a solution . A DLS aDLS is also used to determine the ζ-potential of NPs. The ζ-potential, also known as the electrokinetic potential, is the potential at a colloid particle’s slipping/shear plane moving under an electric field. The ζ-potential reflects the potential difference between the electric double layer of electrophoretically mobile particles and the dispersant layer around them at the slipping plane. The magnitude of the ζ-potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles in a dispersion. Thus, the ζ-potential is an important indicator of the stability of colloidal dispersions. A high ζ-potential value (+ or −30 mV) will confer stability, and the solution or dispersion will resist aggregation. This value is affected and could be modulated by the VLP functionalization.To assess the magnitude of the ζ-potential, DLS instruments require minimal sample preparation and low-cost laboratory materials. DLS instruments are provided with a laser, light detector, and sample holder. For ζ-potential measurements, particles suspended in the medium scatter the incident laser light in all directions, and hence, the scattering intensity is recorded by the detector . Since tIn terms of the sample analysis, DLS measurement conditions must be defined to avoid variabilities on the resultant ζ-potential; the factors that influence this value are temperature, solvent ratio , pH, presence or not of surfactants, the existence of ions in a solution, and ionic strength ,124,125.For VLPs, DLS instruments have been used to record the ζ-potential of various preparations, such as VLPs derived from GI.1 and GII.4 noroviruses and the feline calicivirus at different pHs, temperatures, and ionic strengths . In anotCD remains one of the gold standards for evaluating the secondary structure, intermolecular interactions, folding, ligand-binding properties, and stability of proteins . The strα and β-sheet contents) and thermal stability at 52 °C of prawn nodavirus capsid VLPs displaying the HBV ‘a’ determinant [Despite the differences between such regions, both have been useful in understanding the stability of protein structures against variable conditions. For VLPs, the CD has been performed to evaluate the influence of temperature, pH, and ionic strength on the stability of VLPs derived from the noroviruses GI.1 and GII.4 . Anothererminant . HoweverUV–Vis spectroscopy is another absorption spectroscopy technique used to characterize organic and inorganic samples. In this method, the absorption of UV–visible radiation causes the excitation of electrons from lower to higher energy levels and is based on the Beer–Lambert law. In proteinaceous samples, the UV absorption occurs from 180 to 230 nm due to adiation . For theadiation . For VLPadiation . In addiadiation . 1H, 13C, 5N, and 31P) to an applied magnetic field [β-VLPs labeled with 19F in cell lysates and combined with microscopy techniques  to observe their cellular internalization [Mw of such nanoplatforms.As aforementioned, NMR spectroscopy is used during the characterization of VLPs. NMR is a reproducible and nondestructive technique that describes the response of nuclei , capillary electrophoresis (CPE), isoelectric focusing, etc. [The  process . There ang, etc. . Electrong, etc. . In thisMw and purity of NPs directed at the tumor microenvironment [β-VLP mutants , who manifested varying degrees of self-assembly and interactions with plasma membrane components [Mw of VLPs is inspected with other analytical techniques such as charge detection mass spectrometry (CDMS).For protein-based NPs, some of these methods have been useful in assessing the ironment . For VLPironment . Moreovemponents . The Mw Mw of DNA fragments, natural products, peptides, and proteins [m/z) [Mw of VLPs derived from Norovirus West Chester GI.1., CPMV, and bacteriophages P22 and T5 [CDMS is a quantitative method that provides information about the proteins . This tens [m/z) . For thens [m/z) . For NP ns [m/z) ,153. For2 and T5 . In anot2 and T5 .In nature, proteins participate as catalysts, cellular and molecular processes mediators, and building blocks in eukaryotic and prokaryotic living forms. In biotechnological research, proteins are used to manufacture novel molecules  able to modulate immune responses, diagnose diseases, and deliver therapeutic compounds . Given tT number; the larger this number is, the larger their size and cargo capacity  are preferred because of their growth rate, high expression yield, and ease of scaling-up. Similarly, the use of other expression systems such as yeast cells  can be scalable, cost-effective, and intrinsically, they functionalize  VLPs by PTMs [Another interesting feature of VLPs is that they are expressed in eukaryotic and prokaryotic cells.  by PTMs ,160. In  by PTMs .TM) can contribute significantly to VLP production because of their high protein expression levels and capacity to express numerous proteins simultaneously, which is convenient for constructing eVLPs, non-eVLPs, and cVLPs for vaccine development [Regarding the use of plants to produce these platforms, they have been reviewed as a low-cost scalable system to express well-assembled VLPs in high quantities . Howeverelopment . HoweverIn addition to the processes from which eukaryotic or prokaryotic cells express the coat proteins of VLPs, the heterologous expression of the coat proteins allows genetic manipulation to improve the VLP properties, such as drug delivery ,169, enzFunctionalization procedures are the cornerstone to enhancing the drug release, interaction with cells, and efficacy of NPs. According to our review, the surfaces of VLPs are decorated with genetic and chemical methods. The former is applied to construct VLPs as nanoreactors or drug delivery systems, whereas the latter is implemented to fabricate VLPs as nanocarriers of genetic material, drugs, magnetic resonance imaging (MRI) agents, photosensitizers, vaccines, and NPs ,175. HowFor instance, genetic methods must consider the size of the biomolecule to be inserted, as it can induce detrimental effects on the assembly and stability. In addition, the resemblance of the VLP-based preparation to the native virus from which it was produced can be disrupted . On the In nanomedicine, the characterization of nanomaterials is a major concern required to understand their interaction and fate in biological systems and their safety and therapeutic efficacy . FeatureVLP-based preparations are rigorously characterized by several analytical techniques commonly used in inorganic nanotechnology and protein chemistry. For vaccine development, the size of VLPs is a crucial first step to decide, since this parameter influences their interaction with immune cells and, hence, the immune response that VLPs provoke. Comparably, controlling their geometry and molecular patterns by modifying the temperature, pH, and salt concentration can result in nanoplatforms with different capacities to induce humoral immune responses and side effects .Mw, and assembly. In view of the importance of the size, shape, and arrangement of VLPs, we reviewed the fundamentals and applications of DLS, TEM, UV–Vis, CD, MS, and NMR techniques to study such parameters, as well as their composition, For NP characterization, spectroscopy techniques such as DLS allow the detection of agglomerates, size analysis, and yield information about the possible shape of the produced nanoplatform. This method is extremely advantageous, since it is easy to perform for homogeneous samples, cost-effective, and does not require elaborate sample preparation procedures. However, its use can represent significant limitations; for instance, samples must undergo constant Brownian motion during the analysis to avoid variabilities throughout data acquisition. Moreover, since numerous calculations are performed in the interface of DLS instruments, various assumptions are made regarding the shape of the NPs, which usually is assumed to be spherical . TherefoSince the development of TEM in the 1930s, it has been a powerful technique widely used to examine, individually, the qualitative features of many biological samples and NPs at a high-resolution capacity . HoweverRegarding the use of other visualization techniques, new protocols for cryogenic electron microscopy (Cryo-EM) can be advantageous in determining the actual structure of VLPs in aqueous systems . FacilitOn the other hand, when developing specific protocols for analyzing VLP-based preparations by liquid chromatography-tandem mass spectrometry (LC-MS/MS), this technique is a versatile, robust, and sensitive methodology used to characterize many protein molecules . HoweverAccording to the experience of our group in NMR analyses ,188, we T number, limitations of characterization techniques, and large-scale application) to use VLPs as vaccines or nanocarriers for therapeutic applications. Moreover, for the interested reader, this review provides insight into the basic principles of the common characterization techniques used in nanomedicine. Therefore, this work can be followed not only by specialists but also by students involved in the biomedical field.Nowadays, there are excellent reports regarding the production of VLPs. However, we consider that this work complements the importance of considering additional factors (i.e., This review discusses the VLPs as self-assembled sophisticated nanometric systems resembling wild-type virus structural and biological properties, showing great potential in the biomedical field. VLPs are constituted by different structural components that make them suitable to present antigens or deliver drugs, genes, or imaging agents into experimental models.This work compiled the recent scientific literature about expressing VLPs in different organisms and cell lines to prepare vaccines or nanocarriers for biomedical purposes. In addition, an insight into other analytical techniques to assess the chemical and physical composition of VLPs derived from various viruses is provided. However, in contrast to conventional protocols for nanosized materials and protein chemistry, new and specific protocols for VLP characterization should be developed. Finally, new analytical techniques could be established to improve the VLP-based preparations to understand better and modulate the encapsidation processes.Further studies must assess their safety, stability, integrity, potential toxicity, and medical potency against variable temperatures and pHs.


The apparent dK values for the complexes of multimeric sTNF with S100A11/A12 estimated from fluorimetric titrations are 0.1–0.3 µM. S100A12/A13 suppress the cytotoxic activity of sTNF against Huh-7 cells, as evidenced by the MTT assay. Structural modeling indicates that the sTNF-S100 interactions may interfere with the sTNF recognition by the therapeutic anti-TNFs. Bioinformatics analysis reveals dysregulation of TNF and S100A11/A12/A13 in numerous disorders. Overall, we have shown a novel potential regulatory role of the extracellular forms of specific S100 proteins that may affect the efficacy of anti-TNF treatment in various diseases.Tumor necrosis factor (TNF) inhibitors (anti-TNFs) represent a cornerstone of the treatment of various immune-mediated inflammatory diseases and are among the most commercially successful therapeutic agents. Knowledge of TNF binding partners is critical for identification of the factors able to affect clinical efficacy of the anti-TNFs. Here, we report that among eighteen representatives of the multifunctional S100 protein family, only S100A11, S100A12 and S100A13 interact with the soluble form of TNF (sTNF) in vitro. The lowest equilibrium dissociation constants ( Tumor necrosis factor (TNF), also known as TNF-α, TNF ligand superfamily member 2 or Cachectin, is a potent pleiotropic (pro/anti)-inflammatory cytokine, existing in a soluble form (sTNF) that acts as a ligand, or a membrane-bound form  that acts as either a ligand or a receptor . The complicated regulation of TNF signaling and the wide range of the cellular events evoked by TNF seem to explain its association with progression of numerous disorders, including inflammatory diseases , pulmonary diseases , metabolic disorders , cancer, cardiovascular disease, infectious and neurodegenerative diseases, fibrosis and Dupuytren’s disease, renal disease and hypernociception ,18,19,20The clinical success of TNF inhibitors  drives 2+-binding proteins of the EF-hand superfamily, some of which also bind Zn2+/Cu2+/Fe2+ , and a significant overlap in the diseases associated with disturbance of S100 proteins or TNF, indicate the possibility of direct or indirect relationships between them. In the present work, we show that Ca2+-loaded forms of the bacterially expressed human S100A1/A2/A3/A4/A5/A6/A7/A8/A9/A10/A11/A12/A13/A14/A15/A16/B/P proteins (up to 2 µM) were passed over the chip. Most of the studied S100 proteins showed no signs of specificity for sTNF, which suggests that the corresponding dissociation constants (dK) exceed 10−4 M. Meanwhile, the SPR sensograms for S100A11/A12/A13 proteins reveal the analyte concentration-dependent effects  to 28 nM (S100A11)  was immobilized on the surface of the SPR sensor chip by amine coupling, flushed with the running buffer for two days and several times with 10 mM glycine pH 3.3 for 150 s to ensure dissociation of sTNF multimer(s) ,53, and  effects . The SPR effects   . Importach 84 nM . The avech 84 nM ,56,57,58ch 84 nM . Finallych 84 nM . Furtherch 84 nM ,62 and 0ch 84 nM , respectch 84 nM ) and 3/4ch 84 nM /bacteriach 84 nM ), respecch 84 nM . Thus, tch 84 nM ,43,49.2+-bound forms of S100A11/A12/A13 proteins of 160 µM for S100A12 [dK values , the conformational changes triggered in the S100 proteins by CaExamination of the IntAct  and BioG2+-bound S100A11/A12 (note that these proteins lack Trp residues) were followed by tryptophan fluorescence of sTNF   with the half-life estimates in the range from 600 s  to 30 h /298 nm) . The tit/298 nm)  with an /298 nm) .2+-free S100A11 results in the lane resembling superposition of the bands corresponding to the pure proteins. Meantime, the equimolar mixing of sTNF and Ca2+-bound S100A11 induces slight bleaching of the bands corresponding to monomeric and dimeric S100A11. Besides, the further 2-fold increase in S100A11 concentration does not cause accumulation of its monomer. These effects indicate formation of the sTNF-S100A11 complex in the presence of Ca2+. The similar, but more pronounced, effects are observed for the S100A12 protein , and is absent for S100A11. Meanwhile, the minor effects, comparable with the accuracy of the experiments, raise concerns about the reliability of these conclusions.To probe the ability of S100A11/A12/A13 proteins to affect physiological activity of sTNF, we have studied the changes in the viability of human hepatoma Huh-7 cells induced by the addition of S100/sTNF alone or their mixture. This cell line has been reported to be sensitive to sTNF ,73,74. IMany of the immortalized cell lines are poorly susceptible to sTNF-induced apoptosis, which is largely ascribed to the activation of NF-κB ,77,78,79Taken together, these results suggest that S100A12/A13-bound forms of sTNF exhibit suppressed cytotoxic activity against Huh-7 cells. The lack of a noticeable effect in the case of S100A11 may be related to its insufficiently high affinity to sTNF . The inh2+-binding loop, and the N-terminal residues in the case of S100A13.Quaternary structures of the complexes between monomeric sTNF and S100A11/A12/A13 monomer have been predicted using the ClusPro docking server  Figure . The preThe modeling indicates that the structural details of sTNF recognition by the S100 proteins demonstrate both common and unique features compared to the predictions for the complexes of other S100 proteins with some of the four-helical cytokines. For instance, residues of the ‘hinge’, EF-loop 2 and helices III and IV of S100A1/A4/A6/P proteins were predicted to bind IFN-β . SimilarThe predicted S100-binding sites of sTNF intersect with the residues shown to interact with therapeutic anti-TNF monoclonal antibodies C. In facThe residue E146 of sTNF, predicted to bind S100A13 C, has prTo reveal the diseases associated with the simultaneous involvement of TNF and either the S100A11, S100A12 or S100A13 protein, we analyzed the human disease databases v7.0  and OpenDisGeNET contains 92 entries related to both TNF and S100A11, which mostly correspond to the various neoplasms . The OTPDisGeNET contains 109 diseases related to the dysregulation of both TNF and S100A12, including numerous neoplasms, arthritis, diabetes and many other maladies . The OTPAlthough the analysis of the DisGeNET database does not reveal the entries related to both TNF and S100A13, the OTP database includes 42 such entries , but nonAnalysis of the OTP database shows that 13 entries are simultaneously related to the TNF and S100A11/A12/A13 , includiE. coli was bought from Prospec-Tany Technogene Ltd.  . Alternatively, recombinant human sTNF with an N-terminal hexahistidine tag from ThermoFisher Sci.   was used for MTT assay. Recombinant human TNF-β (UniProt entry P01374) produced in E. coli was from BioVision Inc.  . Recombinant human S100A1/A2/A3/A4/A5/A6/A7/A8/A9/A10/A11/A12/A13/A14/A15/A16/B/P proteins were expressed in E. coli and purified as previously described [Recombinant human sTNF  produced in escribed ,41,43,51escribed .2, EDTA, TWEEN 20, CHX and DMSO were from Sigma-Aldrich Co.  ProteOn™ GLH sensor chip, amine coupling kit, EDAC and sulfo-NHS were from Bio-Rad Laboratories, Inc.  NAP-5 column was from Cytiva .HEPES, Tris, glycine, sodium chloride, sodium hydroxide, DTT and SDS were from PanReac AppliChem. CaClHuman hepatoma Huh-7 cell line was kindly provided by Eliseeva I.A. . DMEM, penicillin, streptomycin and L-glutamine were bought from PanEco Ltd. . Fetal bovine serum was from Biosera. MTT was purchased from Dia-M .2, 0.05% TWEEN 20, pH 7.4) for two days and several times with 10 mM glycine pH 3.3 for 150 s to ensure dissociation of sTNF multimer(s) [SPR studies of sTNF or TNF-β interaction with S100 proteins at 25 °C were performed using the ProteOn™ XPR36 protein interaction array system . Ligand (50 μg/mL sTNF or TNF-β) in 10 mM sodium acetate pH 4.5 buffer was immobilized on the surface of a ProteOn™ GLH sensor chip  by amine coupling, followed by blocking of the remaining activated amine groups on the chip surface by 1 M ethanolamine solution. The chip surface was flushed with the running buffer  ,53. The 1 and L2) that bind a single analyte molecule (A):dK and dk correspond to equilibrium and kinetic dissociation constants, respectively. The ligand may be exposed to the analyte in various preferential conformations, corresponding to different analyte-binding sites or different conformations of the same site. Thus, L1 and L2 correspond to “open” conformations of the ligand, which differ in their specificity to the analyte. dK and dk values were calculated for each analyte concentration using ProteOn Manager™ v.3.1 software ; the averaged values and standard deviations (n = 3–4) are indicated.The double-referenced SPR sensograms were described within a heterogeneous ligand Model (1), which suggests the existence of two populations of the ligand  ensured selective detection of fluorescence emission spectra of Trp residues of sTNF. The fluorescence spectra were corrected for spectral sensitivity of the fluorimeter and fitted by a log-normal function [Fluorescence measurements were performed using a Cary Eclipse spectrofluorometer . sTNF solution in 10 mM HEPES-NaOH, 150 mM NaCl, 1 mM CaClfunction , using Lfunction  and takefunction .2). The reaction was quenched after 1.5 h by addition of SDS-PAGE sample loading buffer, followed by SDS-PAGE (15%) with silver staining and the gel scanning using a Molecular Imager PharosFX Plus System .Crosslinking of sTNF with S100A11/A12/A13 proteins was performed mainly as described in ref. . The S10l-glutamine. One hundred µL of cell suspension (5 × 104 cells) were seeded in quadruplicates into 96-well plates, and 10 ng of sTNF/S100A11/A12/A13 were added to the cells either alone or in combination of sTNF with one of the S100 proteins . Where indicated, CHX was added to the cells to a final concentration of 1 μg per well. The cells were grown at 37 °C in a humidified 5% CO2 atmosphere for 16 h. 10 μL of 5 mg/mL MTT stock solution were added to the cells, and the plates were incubated under the same conditions for 3 h. The medium was carefully removed and 100 µL of DMSO per well were added to dissolve the formazan crystals. The absorbance at 590 nm was measured using a FilterMax F5 microplate reader (ThermoFisher Sci.). The experiments were repeated at least four times. The quantitative results are presented as mean +/− standard deviation. The differences analyzed using Student’s t-test were considered significant at p-values below 0.05.Huh-7 cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM https://cluspro.bu.edu/login.php, accessed on 1 November 2022) [https://alphafold.ebi.ac.uk/, accessed on 1 November 2022) [https://pymol.org/2/, accessed on 1 November 2022).The models of quaternary structures of sTNF-S100A11/A12/A13 complexes were built using ClusPro docking server (er 2022) . The terer 2022)  entry 3Wer 2022) . Distribhttps://www.disgenet.org/, accessed on 1 November 2022) [https://platform.opentargets.org/, accessed on 1 November 2022) [The data on diseases associated with human TNF (UniProt ID P01375) and either S100A11 (UniProt ID P31949), S100A12 (UniProt ID P80511) or S100A13 (UniProt ID Q99584) were collected from the human disease databases DisGeNET v7.0 (er 2022)  and Opener 2022)  as descrer 2022) . The Dis2+-bound forms of S100A11/A12/A13 proteins in vitro are demonstrated here using SPR spectroscopy, intrinsic fluorescence, and chemical crosslinking techniques. These interactions are not redundant and are selective, as confirmed by their disappearance for TNF-β and 15 other S100 proteins. The lowest values of the respective dissociation constants reach the nanomolar levels, which are comparable to the concentrations of extracellular forms of S100A11/A12/A13 under (patho)physiological conditions. This fact indicates a possible regulatory role for the sTNF-S100 interactions, which is further supported by the cell viability analysis using Huh-7 human hepatoma cells as a model system.The interactions between monomeric/multimeric sTNF and CaWe have shown that exogenous S100A12/A13 rescue Huh-7 cells from the cytotoxic effect of sTNF. The relevance of this phenomenon for treatment of liver cancer remains unclear due to the controversial role of TNF in cancer, exerting both pro- and anticarcinogenic effects . TNF proThe structural modelling of the sTNF-S100 complexes predicts an overlap of the S100-binding sites of sTNF with the epitopes recognized by the therapeutic anti-TNF monoclonal antibodies. Therefore, the S100 binding to sTNF could affect the therapeutic efficacy of anti-TNF antibodies, which may partly explain the fact that not all patients respond to the TNF inhibitors ,98.While S100A12 lacks reported non-receptor extracellular binding partners, S100A13 has previously been shown to bind IL-1α, FGF1 and C-C motif chemokine 5 ,47,48, a


Heart failure with preserved ejection fraction (HFpEF) has represented a therapeutic challenge in recent decades , which h−Epidemiological: Cardiovascular diseases are the leading cause of death in obesity [ obesity ; there i obesity , which—i−Pathophysiological: Over the last 10 years, a highly accredited theory has developed that interprets HFpEF as the ultimate consequence of a series of inflammatory mechanisms present in various chronic diseases, in this sense distinguishing it from HFrEF, in which neurohumoral activation mechanisms prevail as the pathogenetic cause of ventricular dysfunction [function ,7,8. In function  recentlyfunction ; it has function .−Clinical: We have stated that the definition of HFpEF actually includes heterogeneous clinical conditions, which are united by an ejection fraction above a certain cut-off (>50% according to the latest European and American guidelines), but with substantial differences in terms of etiopathogenesis, pathophysiology and clinical presentation. This is one of the reasons, probably the most important one, behind the substantial failure of RCTs on the pharmacological treatment of HFpEF (with the exception of recent studies with SGLT2i), since it is unlikely that a single treatment can provide benefit in such different clinical conditions. Thus, the need arose to identify specific phenotypes, on which to perform RCTs with targeted therapies . There is still no univocally accepted standardisation in the definition of these phenotypes: an attempt—conceptually interesting, but difficult to apply in practice—has been made with the use of ‘big data’, analysing a large number of clinical variables with computer systems, and extrapolating three archetypal models of HFpEF, namely, that of obese, diabetic, and obstructive sleep apnoea patients [patients . More repatients . A specipatients  and in 2patients ; the lat−Therapeutic perspectives: On the basis of these observations, Packer hypothesised a possible favourable role of sacubitril/valsartan, MRA and SGLT2i in the obese phenotype with HFpEF [th HFpEF ; we do nFrom this point of view, an HFpEF phenotype of particular interest is that of obese patients, for several reasons, which are briefly reviewed in the following:A class of drugs that could have a potentially favourable role in the specific phenotype of obese patients with HFpEF is that of the GLP1 RA, which has been demonstrated to effectively reduce body weight, have an effect on epicardial fat , a have These hypotheses are currently being tested in clinical trials, such as the SELECT  RCT enroFrom the outcomes of these studies, we are likely to obtain the answer to the question of the actual role of GLP1 RA in reducing cardiovascular events in obese patients and, in particular, its efficacy in the specific obese phenotype with HFpEF.


Background and Objective: Triptolide (TP), one of the fat-soluble components extracted from the Chinese medicinal herb Tripterygium wilfordii Hook F. (TWHF), possesses strong antitumor bioactivities, but its dose-dependent side effects restrict its wide application. This study was designed to investigate whether inflammatory factors increased the antitumor effects of the nontoxic dose of TP on gastric cancer cells and tried to explore the possible molecular mechanisms.Method: AGS and MKN45 cells were treated with different doses of TP and TNF-α. Cell viability and apoptosis were detected in vitro. In addition, NF-κB mediated prosurvival signals and cytoprotective proteins, especially FLICE-inhibitory protein (FLIP), were detected to determine their effects on TP/TNF-α–induced apoptosis. Moreover, the function of lncRNA H19/miR-204-5p/NF-κB/FLIP axis was investigated in vitro, and the antigastric cancer effect of TP plus TNF-α was proved in the mice xenograft model.Result:In vitro experimental results showed that TP pretreatment promoted apoptosis in AGS and MKN45 cells upon TNF-α exposure. TP/TNF-α–mediated apoptosis was partly mediated by the inhibitory effect of NF-κB–mediated FLIP expression. Oncogene H19 lying in the upstream pathway of NF-κB played a vital role upon TNF-α exposure, and bioinformatics analysis proved that H19 participated in TP/TNF-α–induced apoptosis via binding of miR-204-5p. Lastly, a low dose of TP and TNF-α inhibited the tumor weight and tumor volume of AGS and MKN45 cells in vivo.Conclusion: TP pretreatment increased apoptosis in TNF-α–stimulated gastric cancer cells, which are dependent on the disruption of the H19/miR-204-5p/NF-κB/FLIP axis. Cotreatment of TP and TNF-α is a better option for enhancing the anticancer effect and lowering the side effect of TP. Tripterygium wilfordii Hook F (TWHF), has antimicrobial, immunomodulatory, antitumor, antirheumatic, and antiinflammatory activities associated with serious systemic toxicity (α/LPS stimulation (α (TP/LPS) induced apoptosis in hepatocytes via downregulating NF-κB–mediated cellular FLICE-inhibitory protein (FLIP) expression , and TNF receptor-associated factor 2 (TRAF2) through their death domains, coupled with Fas-associated death domain (FADD) and Caspase-8, create a complex named complex-IIa  induced apoptosis in hepatocytes via downregulating NF-κB–mediated FLIP expression  is supposed to be involved in various biomolecular processes, such as transcriptional modulation, translational regulation, and epigenetic mechanism. Research studies have discovered that the expression of lncRNAs, including MALAT1, H19, MEG3, and TUSC7, markedly control gastric cancer cell migration, proliferation, invasion, metastasis, cell cycle, tumorigenicity, and apoptosis . In addipoptosis .α enhanced the antitumor effect of TP against gastric carcinoma. Moreover, we screened lncRNAs related to the pathogenesis of gastric cancer and tried to investigate the effect of the H19/miR-204-5p/NF-κB axis in TP/TNF-α–induced apoptosis in gastric carcinoma.In this study, we explored whether TNF-α (300-01A) and Cell Counting Kit-8  were obtained from Peprotech Inc.  and MedChemExpress , respectively. Trizol reagent (R401-01), SYBR Green Master Mix (Q111-03), and Reverse Transcription Kit (R312-02) were obtained from Vazyme Biotech Co., Ltd. .TP  was supplied by SelleckChem . Human recombinant TNF-β-Actin (HRP-60008) were purchased from Proteintech .Antibodies against FLIP (56343), X-linked inhibitor of apoptosis protein , cleaved caspase-8 (9746), and cleaved caspase-3 (9661) were purchased from Cell Signaling Technology . Antibody against NF-B p65 (ab16502) was purchased from Abcam . Antibodies against cellular inhibitor of apoptosis protein 1 (CIAP1) and cellular inhibitor of apoptosis protein 2 (CIAP2) (10022-1-AP), Lamin B1 (12987-1-AP), and 6 AGS and MKN45 cells (6 mice per group). When tumor volume reached 30–50 mm3, the mice were administered with TP intragastrically (125 μg/kg or 250 μg/kg) or 0.5% CMC–Na at the volume of 10 ml/kg once a day. Tumor volumes were measured every 3 days, and all the mice were sacrificed 3 weeks after TP treatment. Tumors were collected for further detection.All experimental procedures involving mice complied with the ARRIVE guidelines and were permitted by the Animal Ethics Committee, Zhengzhou University. To explore the antitumor effects of TP, six-week-old female athymic BALB/c nude mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. and maintained under pathogen-free conditions. Nude mice were subcutaneously injected into the inguinal region with 5 × 10α, nude mice were subcutaneously injected into the inguinal region with 1 × 107 AGS and MKN45 cells (6 mice per group). The mice were administrated with TP (125 μg/kg) and TNF-α (5 μg/kg). TP was administrated once a day, and an intratumoral injection of TNF-α was administered twice a week. Mice used in this experiment were sacrificed 3 weeks after TP administration.To investigate the antitumor effects of TP and TNF-2. TP was added 2 h before the addition of human recombinant TNF-α in all in vitro experiments.Cell lines used in this experiment, including AGS, MKN45, and HEK293T cells, were purchased from China Cell Culture Center . Cells were cultured in Dulbecco Modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) provided by Gibco  and incubated at 37°C in a humidified atmosphere with 5% CO3 in each well. Fresh DMEM without FBS containing different concentrations of TP as well as human recombinant TNF-α was added to replace the old medium 24 h after seeding. CCK-8 reagents were added at the indicated time, and cell viability was detected with Varioskan LUX .Cell viability was detected with the CCK-8 assay in the 96-well plates. In brief, exponentially growing cells were plated at the density of 5 × 10α-IRES-puro vector, or pEX-1 vector carrying H19 sequence. Fresh DMEM medium was added to the plates to replace the transfection medium after a 24-h transfection. Next, TP (25 nmol/L) and TNF-α (5 ng/ml) were added to the medium 48 h after transfection. Then, cell supernatant was collected at the indicated time after TNF-α application. The sequences of miR-204-5p, H19 siRNA, and their negative controls are presented in Cell transfection was carried out according to our published research . BrieflyDual-luciferase reporter assay was conducted according to the published research . In brieβ-actin for the detection of H19. The relative mRNA expression was calculated using the ΔΔCT method, and the primers used for qPCR are listed in Cellular RNA was isolated using Trizol reagent, and complementary DNA was synthesized from 1-μg RNA for each sample with Reverse Transcription Kit after the quantification of RNA concentration with Nanodrop 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. The experiment was conducted on Applied Biosystems 7500 Real-Time PCR Systems (Thermo Fisher Scientific) using SYBR Green Master Mix and normalized with U6 for the detection of miR-204-5p and β-actin for the detection of the whole protein lysate and analyzed with Image J 1.52a .Total cellular protein was extracted from cell lines or tumor tissues with radio-immunoprecipitation assay lysis buffer (P0013C) purchased from Beyotime Biotechnology . Nuclear proteins were separated with Nuclear Plasma Separation Kit . The concentration of protein was quantified using a BCA Kit . The cell lysate was then mixed with 5× loading buffer  and separated using SDS-PAGE with gels ranging from 8% to 12%. After transferring the proteins onto poly(vinylidene fluoride) membranes  and blocking with 5% BSA at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The membranes were then incubated with secondary antibodies for 1 h and visualized in Tanon 5200 Chemiluminescent Imaging System  using an ECL Detection Kit (Millipore). Relative protein expression was normalized with Lamin B1 for the detection of NF-κB p65 or Cell lysates were collected at the indicated time and then extracted and quantified with the kit purchased from Beyotime Biotechnology, as described above. NF-κB transcription factor activation was detected using the kit from Abcam (ab176648), according to the manufacturer’s instructions.p-values < 0.05 were considered to be statistically significant.Data were analyzed using GraphPad Prism 8.01  and presented in the form of mean ± SEM. One-way analysis of variance (ANOVA) and two-way ANOVA followed by Tukey’s multiple comparison test was performed to analyze the differences between groups. α induces cell death, while a low dose of TNF-α induces cell survival instead of cell death  for further experiments. Time-dependent results revealed that the combination of TP and TNF-α decreased the cell viability at 24-h time interval  for different time intervals. The result illustrated that NF-κB activity was increased in a time-dependent manner, restored to normal at the 6-h time interval, and peaked at the 30-min time interval  for 21 days. Two doses of TP inhibited the tumor volume and tumor weight of AGS and MKN45 cells in a dose-dependent manner (α (5 μg/kg) twice a week. Three weeks after TP administration, we found that TNF-α alone has little effect on the tumor weight and volume. However, TP/TNF-α cotreatment dramatically inhibited the tumor growth of AGS and MKN45 cells and firmly proved that TP treatment increased the sensitivity of AGS and MKN45 cells to TNF-α in vivo  into the tail vein, although there might be some differences in the levels of miR-204-5p and H19 between the TP/TNF-α and TNF-α-treatment groups. Moreover, iv injection of TNF-α into the tail vein slightly upregulated NF-κB target genes (compared with the published articles), NFKBIA, and TNFAIP3, which were observed to examine the effect of TNF-α on the tumor NF-κB activity (α, intratumoral injection of TNF-α was administered in vivo. The result proved that TP/TNF-α inhibited the growth of AGS and MKN45 cells. Considering the poor accessibility of intratumoral injection, we supposed that exosome-based antibodies or other formulations for TNF-α delivery might be the optional plan (α delivery can enhance the antitumor activity of antibody-dependent cell–mediated cytotoxicity of an anti-Melanoma Immunoglobulin, peptide anticancer vaccine, and cancer cell membrane targeting therapy (α nanomaterials for targeting tumor tissues can be a better solution for the antitumor effects of TP.In this hypothesis, we proved that TP–induced sensitivity of AGS and MKN45 cells to the TNF-activity . Due to nal plan . In addi therapy . We beliα treatment. The inhibition of FLIP by lncRNA H19 and miR-204-5p interaction lying upstream of NF-κB is indispensable for inflammatory sensitization (α nanomaterial might be the better solution for the antitumor effects of TP.Pretreatment with a nontoxic dose of TP promoted apoptosis in AGS and MKN45 cells upon TNF-tization . In addi