Spinal cord injury (SCI) results in a wide range of disabilities. Its complex pathophysiological process limits the effectiveness of many clinical treatments. Betulinic acid (BA) has been shown to be an effective treatment for some neurological diseases, but it has not been studied in SCI. In this study, we assessed the role of BA in SCI and investigated its underlying mechanism. We used a mouse model of SCI, and functional outcomes following injury were assessed. Western blotting, ELISA, and immunofluorescence techniques were employed to analyze levels of autophagy, mitophagy, pyroptosis, and AMPK-related signaling pathways were also examined. Our results showed that BA significantly improved functional recovery following SCI. Furthermore, autophagy, mitophagy, ROS level and pyroptosis were implicated in the mechanism of BA in the treatment of SCI. Specifically, our results suggest that BA restored autophagy flux following injury, which induced mitophagy to eliminate the accumulation of ROS and inhibits pyroptosis. Further mechanistic studies revealed that BA likely regulates autophagy and mitophagy via the AMPK-mTOR-TFEB signaling pathway. Those results showed that BA can significantly promote the recovery following SCI and that it may be a promising therapy for SCI. Spinal cord injury (SCI) is a disabling disease that often results in significant motor and sensory dysfunctions Pyroptosis, an inflammatory form of programmed cell death, is known to play a detrimental role in acute central nervous system (CNS) injuries such as traumatic brain injury, cerebral ischemia, and SCI Autophagy, key cellular process for removing cytosolic waste and damaged organelles Betulinic acid (BA), a natural pentacyclic triterpenoid, is known to be found in Chinese herbal medicine extensively Wenzhou Medical University's Experimental Animal Center  supplied female C57BL/6 mice . All laboratory procedures involving animals followed the China National Institutes of Health's Guide for the Care and Use of Laboratory Animals, and were approved by Wenzhou Medical University's Animal Research Committee (wydw2017-0022). Best efforts were undertaken to minimize the number of animals used and pain inflicted. Standard experimental cages with a 12-h light/dark cycle were applied to house all mice individually, and unrestricted access to food and water was provided. 105 mice were divided randomly into five groups: Sham (n = 25), SCI (n = 25), Betulinic Acid , BA +3-methyladenine  and BA+ Compound C .All reagents, antibodies and their suppliers are listed as follows: Betulinic Acid  was obtained from MedChemExpress . HE staining kit, Masson staining kit, Diaminobenzidine (DAB) developer, and pentobarbital sodium, were purchased from Solarbio Science & Technology . Dorsomorphin  was purchased from Med Chem Express . Primary antibody against TFEB was obtained from Bethyl Laboratories . Rabbit monoclonal anti-GAPDH was purchased from Biogot Technology . The rabbit monoclonal anti-Caspase-1, anti-phosphoinositide- 3-kinase (Vps34), and anti-cathepsin D (CTSD) antibodies were purchased from Proteintech Group . Rabbit monoclonal 3-methyladenine (3MA) and anti-microtubule-associated 1 protein light chain 3 (LC3) antibodies were obtained from Sigma-Aldrich Chemical Co . NLRP3, ASC, AMPK, Beclin1, mTOR, p-AMPK and p-mTOR were obtained from Cell Signaling Technology . Mouse monoclonal Synaptophysin, anti-SQSTM1/ p62, NeuN, goat anti-rabbit IgG H&L (Alexa Fluor® 488), MAP2 and goat anti-mouse IgG H&L (Alexa Fluor® 647) antibodies were acquired from Abcam . IL-1β. Antibodies against IL-18, Bnip3, GSDMD, Nix and Parkin were obtained from Affinity Biosciences (OH.USA). Santa Cruz Biotechnology  supplied the secondary antibody Horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG). Beyotime Biotechnology  provided the 4′,6-diamidino- 2-phenylindole (DAPI), and Boyun Biotechnology  supplied the Fluorescein isothiocyanate (FITC)-conjugated IgG secondary. ThermoFisher Scientific  provided the BCA kit. PerkinElmer Life Sciences  supplied the Electrochemiluminescence (ECL) Plus Reagent Kit.Prior to procedure, anesthesia was administered to all animals with intraperitoneal injections of one percent (w/v) pentobarbital sodium (50 mg/kg). Then, standard laminectomy was performed at the T11-T12 vertebra to expose a circle of the dura. Then, a weight-drop injury model was employed to cause a spinal contusion injury as previously described All drugs were dissolved in 2% DMSO in normal saline and administered intraperitoneally. Daily injections of 20 mg/kg BA after SCI were administered to the BA group. 3MA (15 mg/kg) and Dorsomorphin  were injected 30 min before BA administration (20 mg/kg) for BA + 3MA and BA+ CC groups. The dose and time of BA administration were used according to the previous study The Basso mouse scale (BMS) locomotion scale and the footprint test were administered at 0, 1, 7, 14, 21, and 28 days after SCI to evaluate functional recovery On postoperative day 14, mice in the Sham, SCI, BA, and BA +3MA groups were re-anesthetized with 2% (w/v) pentobarbital sodium and perfused with normal saline, followed by addition of 4% (w/v) paraformaldehyde in phosphate-buffered saline. Then, we separated the whole segments , then post-fixed them for 24 h in 4% (w/v) paraformaldehyde. Subsequently, each specimen was prepared for longitudinal paraffin sections after being embedded in paraffin. Using a microtome, 4 μm sections were cut and mounted on poly-L-lysine-coated slides for histopathological examination by HE staining as previously described After mice had been euthanized on day 3 following SCI, the spinal cord segments in mice  were dissected and stored for Western blot analyses at -80 °C. A part of the samples was processed by extracting proteins with a lysis buffer. Other samples were processed by extracting cytoplasmic protein and nuclear protein with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents. We used protein extraction reagents to purify total proteins from spinal cord specimens and used the BCA assay to measure the proteins. 12% (w/v) gel electrophoresis was used to separate an equal amount of 60 μg protein, which were then transferred to polyvinylidene fluoride membranes . After blocking for 2 h at room temperature with 5% (w/v) non-fat milk, the membranes were then incubated overnight at 4 °C with the subsequent primary antibodies: mTOR , p-mTOR , AMPK , p-AMPK , NLRP3 , TFEB , IL-1β , IL-18 , GSDMD , Nix , ASC , Parkin , Bnip3 (1:1000), Beclin1 , Caspase 1 , LC3 (1:500), p62 , CTSD , Vps34 , and GAPDH . Finally, membranes were incubated for 2 h at room temperature with HRP-conjugated IgG secondary antibody . ECL Plus Reagent Kit was used to visualize band signals, and Image Lab 3.0 software  was used to quantify band intensities.We homogenized, frozen and thawed spinal cord specimens repeatedly in liquid nitrogen. Then, we centrifuged  the homogenate at 4 °C for 10 minutes, and collected the supernatant for ELISA. ELISA kits were used to evaluate the levels of AOPP, 8-OHdG, and MDA in spinal cord specimens according to manufacturer's protocols . Finally, the quantifications of AOPP, 8-OHdG, and MDA were performed using a microplate reader at 550 nm with a correction wavelength of 450 nm.Immunofluorescence staining for tissue sides from the rostral spinal cord segments  were performed as previously described P < 0.05 was considered statistically significant.All statistical analyses were performed on SPSS 19.0 . All data are presented as mean ± Standard Error of Mean (SEM). Comparisons between two independent groups were performed using independent-sample t-test. Comparisons among more than three groups was performed using one-way ANOVA with LSD . p<0.001), down-regulated MAP2 (p < 0.001), and less SYN-positive synapses onto ventral motor neurons (p < 0.001). With BA treatment, animals had less glial scar, higher neuronal MAP2 expression, and more SYN-positive synapses onto ventral motor neurons compared with the untreated SCI group . Moreover, footprint analysis showed that the BA group fared better than SCI group for functional recovery at 28 days after injury . For the Sham group, the BMS scores were notably higher compared to the SCI group at days 1, 7, 14, 21 and 28 post-procedure . Similarly, the BA group also had higher BMS scores at days 1, 7, 14, 21 and 28 post-procedure compared with the SCI group . Together, these results demonstrate that BA promotes functional recovery following SCI.Compared to sham control, the SCI group showed an expanded area of glial scar , while BA decreased the densities of Caspase-1 and GSDMD compared with the SCI group . Western blots for ASC, GSDMD, Caspase-1, NLRP3, IL-1β, and IL-18 expressions levels were also assessed . Results demonstrated that the OD values for ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 were higher in the SCI group compared with the Sham group , and that BA decreases in the OD values for these markers relative to the SCI group . These results suggest that BA reduces pyroptosis-related markers, suggestive of an inhibitory effect on pyroptosis following SCI.ASC, GSDMD, Caspase-1, NLRP3, IL-1β, and IL-18 were assessed in the spinal cord after SCI to assess pyroptotic activity in Sham, SCI and BA groups. As shown in Fig. , immunofluorescence staining revealed p62 levels in the neurons at the lesion; a green label was used to mark p62 was labeled green, a red label for neurons, and a blue label for nuclei. Quantitative analyses showed that after SCI, the percentage of p62-positive neurons significantly increased (p < 0.001); however, the BA group had a lower percentage of p62-positive neurons compared with the SCI group . To assess LC3II levels, a green label was applied for LC3II, a red label for neurons (NeuN), and blue label for nuclei (DAPI). As shown in Fig. , the spinal cord exhibited higher percentage of LC3Ⅱ positive neurons in the SCI group than in the Sham group (p < 0.001); BA treatment further increased LC3Ⅱ positive neurons compared with the SCI group . The amount of p62, LC3II, Beclin1, Vps34, and CTSD proteins were measured by Western blot . Results showed that the OD of p62, LC3II, Beclin1, Vps34, were higher in the SCI group than in the Sham group , with lower OD values for CTSD in the SCI group (p < 0.001). BA enhanced the level of LC3II, Beclin1, Vps34, and CTSD as well as decreased level of p62 in the BA group compared with the SCI group . These results recapitulate the known phenomenon that following SCI, autophagy substrates accumulate despite an upregulation autophagosome- and autolysosome-related markers. These results also demonstrate that BA is not only able to increase autophagosome- and autolysosome-related markers, it also alleviates autophagy substrate burden, likely due to an inducing an overall increase in autophagic activity following SCI.To assess autophagic activity in the spinal cord lesion after SCI, we measured protein levels of autophagosomal markers , an autolysosome-related marker (CTSD), and an autophagic substrate protein (p62). As shown in p < 0.001, p < 0.001, respectively; Fig. ). The expression levels of p62, LC3II, Beclin1, Vps34, and CTSD were detected by Western blot . Results showed that the OD values for LC3II, Beclin1, Vps34, and CTSD were lower in the BA+3MA group than in the BA group , with a higher OD value for p62 in the BA+3MA group . These results demonstrate that 3MA was effective in inhibiting autophagy when co-administered with BA. Next, pyroptotic activity was assessed in BA-treated animals via immunofluorescence staining and Western blotting after co-administration of 3MA. Immunofluorescence showed that Caspase-1 and GSDMD densities in neurons were higher in the BA+3MA group than in the BA group . The expression levels of ASC, GSDMD, Caspase-1, NLRP3, IL-1β, and IL-18 were also measured by Western blot . Results revealed that the OD values for ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 were higher in the BA+3MA group than in the BA group . These results show that co-administration of 3MA with BA leads to a reduction in BA's effect on reducing pyroptosis, suggesting that the autophagy-enhancing effects of BA may underly the mechanism by which it inhibits pyroptosis.3MA, an autophagy inhibitor, was co-administered with BA to assess whether BA's beneficial effects on outcomes following SCI is due to autophagy activation. Immunofluorescence and neuron co-localization analyses revealed increased p62 density and decreased LC3II signals BA+3MA group compared with the BA group , decreased MAP2 levels  and lower number of SYN-positive synapses onto ventral motor neurons following SCI . At day 28 after injury, the BA group showed a significant restoration of hind legs movement with coordinated crawling, whereas the BA+3MA group was still dragging their hind legs . In the BA+3MA group, the BMS scores were significantly lower than those in the BA group after SCI at days 1, 7, 14, 21 and 28 . These results suggest that BA's autophagy enhancing effects may be responsible for improved outcomes with BA treatment following SCI.Compared to the BA group, the BA+3MA group showed an increased area of glial scar . BA treatment decreased the levels of AOPP, 8-OHdG, and MDA , and the levels of AOPP, 8-OHdG, and MDA were all higher in the BA+3MA group compared with the BA group . Biomarkers Bnip3, Nix and Parkin assessed to measure mitophagy in the Sham, SCI, BA and BA+3MA groups. Immunofluorescence showed that there were significantly more Nix-positive neurons in the SCI group relative to the Sham group (p = 0.026), and more Nix-positive neurons were observed in the BA group compared with the SCI group . Furthermore, compared to BA group, there were less Nix-positive neurons in the BA+3MA group . The expression levels of Bnip3, Nix and Parkin were also measured by Western blot . Results showed that Bnip3, Nix and Parkin levels were significantly higher in the SCI group compared to the Sham group , and these levels were even higher in the BA group compared with the SCI group . Finally, compared to BA group, the expression levels of Bnip3, Nix and Parkin in the BA+3MA group were all lower . Together, these results indicate that BA reduces ROS accumulation and augments mitophagy in SCI, and that these effects are likely due to its autophagy enhancing effects.ROS oxidation products -AOPP, 8-OHdG, and MDA - were measured by ELISA to evaluate changes in ROS levels after SCI. The levels of AOPP, 8-OHdG, and MDA were all higher in the SCI group compared with the Sham group , while p-mTOR was lower (p = 0.005). BA increased the levels of p-AMPK expression and TFEB nuclear translocation and decreased p-mTOR level . Next, we explored the effects of compound C (CC), an AMPK blocker, on the effects of BA. Here, p-AMPK and TFEB nuclear translocation levels were lower in the BA+CC group compared with the BA group , while p-mTOR levels in the BA+CC group was higher . We also evaluated whether the AMPK-mTOR -TFEB axis is also involved in the mechanism by which BA modulates pyroptosis, autophagy, and mitophagy related proteins. Our results demonstrated that Caspase-1, NLRP3 and GSDMD levels were higher in the BA+CC group when compared to the BA group ; p62 levels were higher in the BA+CC group when compared to the BA group (p = 0.004), with a lower level of LC3II (P = 0.009). Finally, Bnip3, Nix and Parkin were lower in the BA+CC group when compared to the BA group . Together, these findings suggest that BA activates autophagy, inhibits pyroptosis, and augments mitophagy via the AMPK-mTOR-TFEB pathway.We sought to investigate the mechanism underlying how BA modulates pyroptosis, autophagy and mitophagy. We assessed whether TFEB, a known activator of autophagy, is involved. Western blot analysis revealed that p-AMPK expression and TFEB nuclear translocation levels were higher in the SCI group compared with the Sham group (Betulinic acid (BA), a natural pentacyclic triterpenoid, has gained considerable attention in recent years for its strong biological and medicinal properties Autophagy, a lysosomal-dependent degradation pathway for intracellular proteins and organelles, plays an important role in human health and disease Pyroptosis is another cellular pathway that has also been implicated in a variety of pathologies such as colitis, myocardial dysfunction, and neuronal damage Increasing evidence demonstrates that the secondary injury stage after SCI is closely related to mitochondrial injury and excessive ROS generation 2+ through TRPML1 channels, which activate calcineurin In order to further to elucidate the underlying mechanism of how BA promotes autophagy in SCI, we also examined upstream mechanisms of autophagy. Transcription factor EB (TFEB) is a central regulator of autophagy + and CTSB in SCI. Finally, while these preclinical findings are promising, more work is needed to be done to explore optimal dosing and toxicities of BA in the treatment of SCI prior to clinical translation.Naturally, there are several limitations in our study that need to be further investigated. For example, previous studies revealed that AMPK-SPK2-CARM1 signaling pathway is another important pathway regulating TFEB in the nucleus Fig. 8. Overall, these results provide novel preclinical evidence demonstrating the therapeutic benefit of BA in SCI. Future investigations are now eagerly awaited to further the clinical translation of BA as a treatment for SCI patients.Taken together, our findings demonstrate that BA activates the AMPK-mTOR-TFEB signaling pathway, which enhances autophagy in SCI. Increased autophagy induces mitophagy and reduces ROS accumulation, subsequently inhibiting pyroptosis. Ultimately, these effects of BA culminate in improved outcomes following SCI. A schematic illustration of our findings is presented in


Low physical activity correlates with increased cancer risk in various cancer types, including colorectal cancer (CRC). However, the ways in which swimming can benefit CRC remain largely unknown. In this study, mice bearing tumors derived from CT-26 cells were randomly divided into the control and swimming groups. Mice in the swimming group were subjected to physical training (swimming) for 3 weeks. Compared with the control group, swimming clearly attenuated tumor volume and tumor weight in CT-26 tumor-bearing mice. RNA sequencing (RNA-seq) identified 715 upregulated and 629 downregulated transcripts (including VEGFA) in tumor tissues of mice in the swimming group. KEGG pathway analysis based on differentially expressed transcripts identified multiple enriched signaling pathways, including angiogenesis, hypoxia, and vascular endothelial growth factor (VEGF) pathways. Consistently, IHC analysis revealed that swimming significantly downregulated CD31, HIF-1α, VEGFA, and VEGFR2 protein expression in tumor tissues. In conclusion, swimming significantly attenuates tumor growth in CT-26 tumor-bearing mice by inhibiting tumor angiogenesis via the suppression of the HIF-1α/VEGFA pathway. Therefore, this disease ranks third in terms of incidence and second in mortality .33.1P < 0.05). Consistently, a significant decrease in the tumor weight was observed in the swimming group compared to that in the control group (P < 0.05). Moreover, monitoring of body weights did not reveal any obvious differences between the control and swimming groups (P > 0.05). These data indicate that swimming significantly attenuates the growth of CT-26 cell-derived tumors in vivo.To assess the benefits of exercise on tumor growth, CT-26 cells were transplanted into BALB/c mice, followed by quantitative daily swimming. Tumor volume was monitored and clearly indicated that it was decreased in the swimming group compared with the control group . Furthermore, as essential downstream effectors of the HIF-1α pathway, VEGFA and VEGFR2 expressions were analyzed from RNA-Seq results and confirmed by IHC analysis. As shown in P < 0.05; vs control group), while the mRNA of VEGFR2 remained unchanged in tumor tissues between the control and swimming groups (P > 0.05). Moreover, expression of both VEGFA and VEGFR2 proteins was obviously decreased in tumor tissues of the swimming group . These results suggest that the suppression of the HIF-1α/VEGFA/VEGFR2 signaling pathway might be an important underlying mechanism by which swimming attenuates tumor growth in vivo.Owing to the essential role of HIF-1α in tumor angiogenesis, we further detected the expression of HIF-1α. As shown in 4in vivo. Mechanistic studies identified 715 upregulated and 629 downregulated transcripts (including VEGFA) in tumor tissues derived from CT-26 cells in vivo after swimming. Further pathway analysis revealed significant enrichment of multiple pathways, including angiogenesis, hypoxia, and VEGF signaling pathways. Consistently, swimming also reduced the protein expression of CD31, HIF-1α, VEGFA, and VEGFR2, which suggests that the essential role of swimming is the suppression of tumor angiogenesis and that the inhibition of the HIF-1α/VEGFA/VEGFR2 axis might be an underlying mechanism by which swimming attenuates CRC tumor growth.Increasing evidence has revealed that exercise results in multiple benefits, including suppression of tumor growth , improvePrevious studies in mice demonstrated that 30–60 min/day of swimming leads to a protective effect against cancer in mice ,23. ConsAlthough it has been reported that exercise protects against the development of certain cancers and lowers the risk of recurrence, the underlying mechanism by which swimming achieves this is largely unknown. To explore the complicated mechanism of swimming on CRC, identification of DETs using RNA-seq technology revealed that swimming led to 715 upregulated and 629 downregulated transcripts. Among these DETs, multiple genes, including STAT3 , PDGFA , PLD2 2, and PIKAs a central mechanism for tumor growth, angiogenesis plays an essential role in tumor development and growth of human CRC ,27. TherMoreover, as a transcription factor, accumulated HIF-1α mediates the expression of more than 200 target genes, including VEGFA ,36, whicin vivo, reduces tumor angiogenesis, and downregulates the expression of HIF-1α, VEGFA, and its receptor VEGFR2. These studies suggest that swimming is a safe and viable intervention strategy for cancer patients. However, the effect of swimming on tumor vascular normalization, perfusion, oxygen transport, and anaerobic glucose metabolism, as well as its underlying mechanisms, should be further explored.In conclusion, swimming significantly attenuates the growth of CT-26 cell-derived tumors


Respirovirus, including a conserved motif 323FAPGNYALSYAM336 in the N protein, RNA editing sites 5'-717AAAAAAGGG725-3', and 5'-1038AGAAGAAAGAAAGG1051-3' (mRNA sense) in the P gene with multiple polypeptides coding capacity, a nuclear localization signal sequence 245KVGRMYSVEYCKQKIEK261 in the M protein, a conserved sialic acid binding motif 252NRKSCS257 in the HN protein, conserved lengths of the leader (55nt) and trailer (51nt) sequences, total coding percentages (92.6–93.4%), gene-start (AGGANNAAAG), gene-end (NANNANNAAAAA), and trinucleotide intergenic sequences . Phylogenetic analysis of their complete genomes showed that they were most closely related to bovine parainfluenza virus 3 (PIV3) genotype C strains. In the phylogenetic tree constructed using the complete L protein, the branch length between dromedary camel PIV3 (DcPIV3) and the nearest node is 0.04, which is >0.03, the definition used for species demarcation in the family Paramyxoviridae. Therefore, we show that DcPIV3 is a novel species of the genus Respirovirus that co-circulated with MERS-CoV in a dromedary herd in the Middle East.Since the emergence of Middle East Respiratory Syndrome (MERS) in 2012, there have been a surge in the discovery and evolutionary studies of viruses in dromedaries. Here, we investigated a herd of nine dromedary calves from Umm Al Quwain, the United Arab Emirates that developed respiratory signs. Viral culture of the nasal swabs from the nine calves on Vero cells showed two different types of cytopathic effects (CPEs), suggesting the presence of two different viruses. Three samples showed typical CPEs of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in Vero cells, which was confirmed by partial RdRp gene sequencing. Complete genome sequencing of the three MERS-CoV strains showed that they belonged to clade B3, most closely related to another dromedary MERS-CoV isolate previously detected in Dubai. They also showed evidence of recombination between lineages B4 and B5 in ORF1ab. Another three samples showed non-typical CPEs of MERS-CoV with cell rounding, progressive degeneration, and detachment. Electron microscopy revealed spherical viral particles with peplomers and diameter of about 170nm. High-throughput sequencing and metagenomic analysis showed that the genome organization (3'-N-P-M-F-HN-L-5') was typical of paramyxovirus. They possessed typical genome features similar to other viruses of the genus  Camelus dromedarius (dromedary or one-humped camel), which inhabits the Middle East, North, and Northeast Africa; Camelus bactrianus (Bactrian or two-humped camel) and Camelus ferus (the wild camel), both, inhabitants of Central Asia. Among the 20million camels on earth, 90% are dromedaries. Before the emergence of the Middle East Respiratory Syndrome (MERS) in 2012, viruses of at least eight families, including Paramyxoviridae, Flaviviridae, Herpesviridae, Papillomaviridae, Picornaviridae, Poxviridae, Reoviridae, and Rhabdoviridae, were known to infect dromedaries , suggesting the presence of two different viruses. To confirm the identities of these viruses, complete genome sequencing, phylogenetic, and comparative genome analyses were conducted.2 and inspected daily for CPEs for 7days by inverted microscopy. Two different types of CPEs were observed. All cultures with CPEs were screened for the presence of MERS-CoV using RT-PCR assay as described below.In May 2015, nine camel calves (4–8months old) of the same herd developed respiratory signs with clear nasal discharge and fever. Nasal swab samples were collected and sent to the Central Veterinary Research Laboratory in Dubai, the United Arab Emirates for investigations. Nasal swab samples from the nine camel calves were inoculated into Vero cells, respectively, for isolation of MERS-CoV as previously described . BrieflyViral RNA was extracted from the nine nasal swab samples and the corresponding culture samples using EZ1 Virus Mini Kit v2.0 . RNA was eluted in 60μl of AVE buffer  and was used as template for RT-PCR.Screening of MERS-CoV was performed by amplifying a 440-bp fragment of the RdRp gene of CoVs using conserved primers (5'-GGTTGGGACTATCCTAAGTGTGA-3' and 5'-ACCATCATCNGANARDATCATNA-3') as described previously .Three MERS-CoVs , which showed typical CPE in Vero cells, isolated from three nasal swab samples from three dromedary calves were included in this study. One complete genome of MERS-CoV strain D1189.1 was sequenced in our previous study . The comBootscan analysis was performed to detect possible recombination by using the complete nucleotide alignment of the genome sequences of MERS-CoV and Simplot version 3.5.1, as previously described . The anaThree culture isolates , which showed non-typical CPE in Vero cells, isolated from three nasal swab samples from three dromedary calves were negative for MERS-CoV using RT-PCR assay and subjected to further investigation by deep sequencing and microscopic analysis as described below. RNA was individually extracted from the three cultures, and the RNA samples were subjected to library preparation and Illumina sequencing, respectively, using NovaSeq 6000 (Pair-End sequencing of 151bp) at University of Hong Kong, Centre for Genomic Sciences , as described previously .de novo assembled into contigs using MIRA version 4.9.6 in accurate mode . Trimmed paired-end reads were analyzed as described previously . The taxate mode . The assThe putative open reading frames (ORFs) and their deduced amino acid sequences of the assembled genomes were predicted using ORF Finder.g at 4°C, after which the solution was fixed with GTA at a final concentration of 2.5% overnight. The sample was mounted into a carbon-formvar coated copper grid and stained with 3% urabyl acetate. The grid was then dried and irradiated with UV  for 15min with a Philips CM100 transmission electron microscope .Dromedary camel parainfluenza virus 3 isolated from the sample D1189.8 was subjected to negative-contrast electron microscopy analysis as described previously . BrieflyThe nucleotide sequences of the two dromedary MERS-CoV genomes and the three DcPIV3 genomes sequenced in this study have been submitted to GenBank sequence database under accession numbers MW545527, MW545528, MW504257, MW504258, and MW504259. Raw data have been submitted to Sequence Read Archive (SRA) under accession numbers SRR13442189, SRR13442188, and SRR13442187.Nasal swab samples from nine camel calves of the same herd were collected and cultured on Vero cells for MERS-CoV screening. Three samples, D1189.1, D1189.5, and D1189.6, showed typical CPEs of MERS-CoV in Vero cells on day 4. RT-PCR targeting the 440-bp fragment of the RdRp gene confirmed the presence of MERS-CoV in these three nasal swab samples and their corresponding culture samples.Another three cultures inoculated with samples D1189.2, D1189.4, and D1189.8 showed non-typical CPEs of MERS-CoV on day 4 with cell rounding, progressive degeneration, and detachment ,B. ElectAnalysis of the complete genomes of the three isolated dromedary MERS-CoV strains  showed that these sequences were 30,103 bases in length with G+C content of 41.1%. The size, G+C content, and genome structure of the three isolated dromedary MERS-CoV strains are similar to other dromedary MERS-CoVs. The genome sequence analysis showed that the three MERS-CoV isolates were closely related among each other, sharing 99.88–99.95% nucleotide identities. Phylogenetic analysis of the complete genomes of the three MERS-CoV isolates showed that they belonged to clade B3, being most closely related to another dromedary isolate, D1271 (GenBank accession number KX108945), which was previously detected in Dubai and they shared 99.84–99.93% nucleotide identities . CompariBootscan analysis showed high bootstrap frequencies (80–100%) for clustering between the three strains and lineage 4 MERS-CoV in their genomes ; but for position 15,000–24,000, bootscan analysis showed high bootstrap frequencies (75–100%) for clustering between the three strains and lineage 5 MERS-CoV . AdditioParamyxoviridae . De novo assembly of reads of Paramyxoviridae revealed a complete genome of parainfluenza virus 3 (PIV3) of the genus Respirovirus in all three samples, tentatively named dromedary camel parainfluenza virus 3 (DcPIV3), where DcPIV3-1189.2, DcPIV3-1189.4, and DcPIV3-1189.8 represented the parainfluenza virus 3 discovered in the sample D1189.2, D1189.4, and D1189.8, respectively. RT-PCR targeting 400-bp fragment of the F gene confirmed the presence of DcPIV3 in these three nasal swab samples and their corresponding culture samples.The three culture isolates  showed non-typical CPEs of MERS-CoV on Vero cells were subject to high-throughput sequencing, respectively, generating 5,621,614–6,500,327 paired-end 151-bp reads. After trimming adapter sequences and filtering the rRNA sequences, bacterial, and host genomes, a total of 4,377,997–5,432,096 clean reads remained and were used for downstream BLASTx analysis. Among these clean reads, 82,373–94,948 reads matched to viruses. The largest portion of these viral sequences was assigned to the family Respirovirus, including a conserved motif 323FAPGNYALSYAM336 in the N protein, RNA editing sites 5'–717AAAAAAGGG725–3' and 5'-1038AGAAGAAAGAAAGG1051-3' (mRNA sense) in the P gene with multiple polypeptides coding capacity, a nuclear localization signal sequence 245KVGRMYSVEYCKQKIEK261 in the M protein, a conserved sialic acid binding motif 252NRKSCS257 in the HN protein, conserved lengths of the leader  and trailer  sequences, total coding percentages (92.6–93.4%), gene-start (consensus: AGGANNAAAG), gene-end (consensus: NANNANNAAAAA), and trinucleotide intergenic sequences  strains, being most closely related to BPIV3c strain TVMDL20 (GenBank accession number KJ647287), sharing 85.2–85.5% nucleotide identities , porcine parainfluenza virus 1 (PPIV1), caprine parainfluenza virus 3 (CPIV3), and Sendai virus  can be found at: JT, UW, and PW conceived and designed the experiment. JT, HL, JF, SJ, KL, SE, and K-HC performed the experiment. JT, UW, HL, JYHF, HC, SL, and PW contributed to analysis. JT, UW, HL, and PW drafted the manuscript. All authors reviewed and revised the first and final drafts of this manuscript. PW and UW are co-corresponding authors who contributed equally to this article.This work was partly supported by the Health and Medical Research Fund-Commissioned Research on Control of Infectious Diseases .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Various vaccines against COVID-19 have been developed and proven to be effective, but their side effects, especially on kidney function, are not yet known in detail. In this study, we report the clinical courses and histopathologic findings of new-onset kidney diseases after COVID-19 vaccination as confirmed via kidney biopsy. Five patients aged 42 to 77 years were included in this study, and baseline kidney function was normal in all patients. The biopsy-proven diagnosis indicated newly developed kidney diseases: (1) IgA nephropathy presenting with painless gross hematuria, (2) minimal change disease presenting with nephrotic syndrome, (3) thrombotic microangiopathy, and (4) two cases of acute tubulointerstitial nephritis presenting with acute kidney injury. Individualized treatment was applied as per disease severity and underlying pathology, and the treatment outcomes of all patients were improved. Since this is not a controlled study, the specific pathophysiologic link and causality between the incidence of kidney diseases and COVID-19 vaccination are difficult to confirm. However, clinicians need to consider the possibility that kidney diseases may be provoked by vaccines in patients who have renal symptoms. The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is still ongoing globally. Although therapeutic agents that successfully suppress COVID-19 have not yet been developed, several vaccines against SARS-CoV-2 can help in the prevention of COVID-19 and alleviation of the disease’s severity.Two main types of vaccine are currently used to prevent COVID-19: (1) mRNA vaccines, such as BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna); and (2) vectored vaccines, such as AZD1222 (Oxford-AstraZeneca) and Ad26.COV2.S (Janssen). Several clinical studies have demonstrated that these vaccines have excellent protective effects against COVID-19, but their side effects are not yet well-known [Several studies have reported on kidney diseases that occurred after COVID-19 vaccinations, but the number of cases was few, and the histopathologic abnormalities and clinical courses were heterogeneous ,4. This A healthy 42-year-old woman presented with first-onset painless gross hematuria and proteinuria. She had no family history of kidney diseases. She had received two doses of the mRNA-1273 vaccine with a 4-week interval. One day after receiving the second dose, she developed dark reddish urine. Gross hematuria disappeared within several days, but follow-up urinalysis showed persistent microscopic hematuria and proteinuria. Based on the laboratory data, the serum creatinine level was 0.47 mg/dL, and the 24 h urinary protein level was 1.7 g/day. Serologic evaluation revealed no specific abnormalities in viral markers or autoantibodies. A kidney biopsy was performed 8 weeks after the vaccination, and the results are shown in A healthy 51-year-old man with no significant past medical history was referred to our clinic because of severe generalized edema. The edema had occurred 7 days after receiving the first dose of Ad26.COV.2 vaccine, and he gained 14 kg for 3 weeks after vaccination. The laboratory examinations revealed a serum creatinine level of 1.54 mg/dL, an albumin level of 1.6 g/dL, and a 24 h urinary protein level of 8.6 g/day. The serologic markers for glomerulonephritis, including viral markers, were all negative. A kidney biopsy was performed 33 days after the vaccination, and the results are shown in 9/L, serum creatinine level 3.69 mg/dL, and spot urine protein-to-creatinine ratio 2.7 g/g. The serologic evaluation for glomerulonephritis was all-negative. A kidney biopsy was performed 2 weeks after the vaccination, and the results are shown in A 69-year-old woman with type 2 diabetes mellitus visited the emergency room because of azotemia and thrombocytopenia. She received her first dose of the AZD1222 vaccine 5 days prior to her visit, and general weakness and gastrointestinal discomfort were observed 2 days later. Based on the laboratory examination, the hemoglobin level was 8.5 g/dL, platelet count 38 × 10A 44-year-old man with a chronic hepatitis B infection and type 2 diabetes mellitus visited the nephrology clinic because of decreased renal function. His baseline serum creatinine level was 0.91 mg/dL; however, it increased to 4.94 mg/dL upon admission. He received the first dose of the mRNA-1273 vaccine 3 weeks prior to his visit, and gastrointestinal discomfort and anorexia occurred 1 day after vaccination. His general condition did not improve, so he visited a local clinic and exhibited a serum creatinine level of 4.13 mg/dL 1 week after vaccination. He did not take any nephrotoxic agents and drank enough water, but his azotemia worsened. The laboratory examinations revealed a spot urine protein-to-creatinine ratio of 1.0 g/g and no microscopic hematuria, and the serologic markers for glomerulonephritis were all negative except for hepatitis B. A kidney biopsy was performed 28 days after the vaccination, and the results are shown in A 77-year-old woman with a chronic hepatitis B infection, hepatocellular carcinoma, and type 2 diabetes mellitus visited the emergency room, presenting with anorexia and nausea following vaccination. She received the second dose of the BNT162b2 vaccine 7 days prior to her visit. The day after vaccination, she had experienced severe nausea and vomiting, so she received intravenous fluid therapy. Her symptoms did not improve, and 1 week after the vaccination, her serum creatinine level was increased from 0.98 mg/dL to 10.75 mg/dL, and her bicarbonate level was decreased to 8.2 mmol/L. Based on the serologic examination, her muscle enzyme levels were slightly increased , and the serologic markers were all negative except for hepatitis B surface antigen. Hemodialysis was initiated, and a kidney biopsy was performed 2 weeks after vaccination . Light mIn this report, we reviewed the clinical course, treatment, and histopathologic findings of various new-onset kidney diseases following COVID-19 vaccination. This is not a controlled study, so causality between vaccinations and these diseases cannot be concluded. However, COVID-19 vaccines are known to cause new-onset or relapsing glomerular diseases due to potent immune dysregulation, and various therapeutic responses have been reported ,5. ThereIgA nephropathy is the most common glomerulonephritis identified in kidney biopsy, and it is an immune complex disease caused by mesangial IgA1 deposition with or without concurrent IgG and C3 deposits ,7. AlthoMinimal change disease (MCD) is a glomerular disease typically characterized by nephrotic-range proteinuria, diffuse foot process effacement in electron microscopy, and no specific abnormalities in light microscopy and immunofluorescence. It has also been confirmed that podocyte injury is caused by various circulating cytokines, such as interleukin (IL)-4, 5, 9, 10, and 13, which are released by activated T lymphocytes ,12,13. HTMA refers to a spectrum of similar disease entities featured by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury, and can be caused by various etiologies . TMA is Acute tubulointerstitial nephritis (ATIN) can be caused by a hypersensitivity reaction from the use of various vaccines or drugs ,19,20. TThis case series provides information on new-onset renal histopathology after COVID-19 vaccination. The need for COVID-19 vaccination, including booster shots, is being emphasized globally. Although we could not confirm causality between vaccinations and these phenomena, in this time of mass vaccination, clinicians need to consider the possibility that vaccines may have provoked kidney diseases in patients who have renal symptoms.


The cluster of differentiation 36 (CD36) is a scavenger receptor present on various types of cells and has multiple biological functions that may be important in inflammation and in the pathogenesis of metabolic diseases, including diabetes. Here, we consider recent insights into how the CD36 response becomes deregulated under metabolic conditions, as well as the therapeutic benefits of CD36 inhibition, which may provide clues for developing strategies aimed at the treatment or prevention of diabetes associated with metabolic diseases. To facilitate this process further, it is important to pinpoint regulatory mechanisms that are relevant under physiological and pathological conditions. In particular, understanding the mechanisms involved in dictating specific CD36 downstream cellular outcomes will aid in the discovery of potent compounds that target specific CD36 downstream signaling cascades. CD36 is a transmembrane glycoprotein found in platelets, mononuclear phagocytes, adipocytes, hepatocytes, myocytes, taste bud cells, and a variety of other cell types. It plays a role in lipid accumulation, inflammatory signaling, energy reprogramming, oxidative stress, and apoptosis, all of which contribute to metabolic dysfunction ,2,3,4,5.The cellular fatty acid uptake rate is determined by the presence of CD36 at the cell surface, which is regulated by subcellular vesicular recycling from endosomes to the sarcolemma (lipid raft). More specifically, VAMP2, a vesicle-associated membrane protein (VAMP) isoform, participates in CD36 translocation to the plasma membrane upon stimulation with insulin ,13,14,15O-GlcNAcylation of CD36 via translocation to the sarcolemma [O-GlcNAcylation is the dynamic regulatory post-translational modification of proteins stimulated by the addition of O-linked β-N-acetyl glucosamine (O-GlcNAc) to serine and threonine residues on target proteins [O-GlcNAcylation levels of CD36 promote high-fat uptake by gastric cancer cells, which is needed for their metastasis [O-GlcNAcylation has also been linked to the reprogramming of cardiac fatty acid and glucose metabolism during acute or chronic stresses [O-GlcNAcylation. CD36 is also palmitoylated and depalmitoylated by palmitoyl-transferases (PATs) and palmitoyl-protein thioesterases respectively. CD36, N-, and C-terminal tails contain cysteine residues that are palmitoylated by PATs, which anchors the protein to the plasma membrane in a reversible process. Under palmitic acid stimulation, CD36 is palmitoylated by PATs in the endoplasmic reticulum (ER) in a reversible thioester linkage between palmitate and cysteine residues [N-hydroxysuccinimidyl esters in the fatty acid-binding pocket, leading to the inhibition of fatty acid intake and/or fatty acid-induced signaling [CD36 is also known as fatty acid translocase (FAT), glycoprotein IIIb (GPIIIb), or platelet glycoprotein IV, PAS4, and SR-B2 . The humrcolemma . O-GlcNAproteins ,41. Howetastasis . Increasstresses , althougresidues ,45. Howeresidues . CD36 haresidues . Recentlresidues . CD36 caresidues . Notablyresidues ,51. Alonresidues . Sulfosuignaling . Thus, fCD36 exerts fundamental biological functions at the cellular and tissue levels in multiple homeostatic and pathological processes by its distinct binding sites. Pancreatic β-cells play a central role in regulating glucose metabolism to sustain energy homeostasis by mediating a balance between insulin, an anabolic hormone, and glucagon, a catabolic hormone. Pancreatic β-cells require suitable sensors and signaling molecules that are integrated to modulate insulin secretion and maintain homeostasis. However, type 2 diabetes (T2D) is based on the inability of pancreatic β-cells to sustain a compensatory secretory response, leading to insulin secretory dysfunction and the pathogenesis of T2D. CD36 is the most generously expressed transporter among fatty acid transporters in human islets. It is located in the plasma membrane and co-localizes with insulin granules . Interes2O2, via the activation of NADPH oxidase (NOX), which might trigger oxidative stress linked to β-cell death in T2D [N-terminal kinase (JNK) in macrophages and platelets, whereas Fyn and p38 are the primary mediators of endothelial cells [On the other hand, evidence suggests that hyperglycemia leads to the generation of reactive oxygen species (ROS), resulting in increased oxidative stress in β-cells ,74. The h in T2D . It was h in T2D . We alsoh in T2D . Howeverh in T2D . Nonetheh in T2D . In addial cells ,80,81. Wal cells ,83. Cellal cells . It is cHyperglycemia with elevated FFAs plays a significant role in insulin resistance and β-cell dysfunction in T2D ,86,87. M2O2, which promotes cysteine sulfenylation, a post-translational modification important to the augmentation of platelet activation and aggregation [A recent study showed that p66Shc mediates lipotoxicity-induced impaired metabolic changes that promote pancreatic β-cell dysfunction and apoptosis in diabetes . Earlierregation . Peroxirregation ,108. Theregation . Importaregation . This fi2+ from the ER to the cytoplasm, driving a rise in the cytosolic Ca2+ concentration that reflects expanded mitochondrial Ca2+ uptake. Increased mitochondrial Ca2+ uptake improves local buffering capacity and the discharge of proteins competent in apoptosis induction. Hence, Ca2+ activates the phosphatase calcineurin, which dephosphorylates and inactivates dynamin-related protein 1 (Drp1), a master controller of mitochondrial fission [2+. Recently, the presence of inositol trisphosphate receptor (IP3Rs) has been implicated in proapoptotic Ca2+ transfer between the ER and mitochondria [2+ exchange by means of IP3R3 and ensures against Ca2+ intervened apoptosis [Under normal conditions, mitochondria in β-cells persistently undergo fusion and fission. These processes may function to refute the negative impacts of the long-term presentation of β-cells to palmitate under high glucose conditions, causing mitochondrial fragmentation and impeding network dynamics by abolishing fusion and fission activity . There a fission . It can chondria . An impopoptosis . Interespoptosis . These e2O2, which in turn can react with PRDX3 to cause toxic mitochondrial dysfunction and apoptosis. CD36 can generate cell-specific reactions to multiple ligands through the binding of context-specific binding partners that contribute to the development of β-cell dysfunction. As described above, it has been shown that ER stress is connected to insulin resistance in diabetes, and, conjointly, an expansion of ER was recognized in β-cells from patients with T2D ,82. OxidRegarding the molecular mechanisms involved, CD36 overexpression partners with the increased uptake of oxLDL without exerting additive effects on oxLDL toxicity . EvidencOn the other hand, β-cells have a lower abundance of antioxidant defense enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) ,125,126.Among the variety of proapoptotic factors present in pancreatic β-cells, islet amyloid polypeptide (IAPP) is thought to play a crucial role in β-cell apoptosis. The proapoptotic effects of IAPP are mediated through a complex sequence of signaling events that lead to defects in mitochondrial dysfunction, autophagy, local inflammation, oxidative stress, cytokine production, and the enactment of signaling pathways driving to apoptosis ,141,142.Insulin resistance could be a trademark of metabolic defects, and it paves the way for the development of T2D. Impaired glucose tolerance, alcoholism, smoking, hypercholesterolemia, hypertriglyceridemia, low HDL, and hypertension are some of the risk factors involved in insulin resistance ,145. Mod2+ exchange from the ER to the lysosome, and this causes lysosomal impairment and inflammation in adipocytes. The inhibition of CD36 palmitoylation reduces both FYN kinase activation and Tyr353 phosphorylation of IP3R1, which may contribute to reduced lysosomal Ca2+ overload and inflammation in adipocytes. A recent study by Wang et al. demonstrated that palmitoyl acyltransferases DHHC4 and DHHC5 control the palmitoylation of CD36 in directing fatty acid uptake in adipose tissues [In an unforeseen finding, CD36 signaling through lysosomal impairment was shown to promote inflammasome activation in adipose tissue from obese mice, suggesting that lysosomes may be vital in obesity-induced adipose tissue inflammation . In addi tissues . In addi tissues . In addi tissues . In supp tissues . Therefo tissues ,170,171. tissues ,174,175.Experimental and clinical studies have fortified the noteworthiness of CD36 in metabolic disorders. CD36 has shown to act as a gatekeeper of various intracellular signaling systems that induce cell dysfunction under an excessive nutrition milieu. CD36 initiating redox signaling revealed the core pathophysiology of metabolic disorders such as diabetes, and promising therapeutic targets are expected. However, several questions regarding its impact on metabolic disease progression will need to be answered in future studies. Understanding this complex situation and the therapeutic agents that modulate the CD36 function could have a considerable impact on the treatment of metabolic diseases such as insulin resistance and diabetes.


Uterine cervical and endometrial cancers are the two most common gynecological malignancies. As demonstrated in other types of solid malignancies, an increased number of circulating or tumor-infiltrating myeloid-derived suppressor cells (MDSCs) have also been observed in uterine cervical and endometrial cancers, and increased MDSCs are associated with an advanced stage, a short survival, or a poor response to chemotherapy or radiotherapy. In murine models of uterine cervical and endometrial cancers, MDSCs have been shown to play important roles in the progression of cancer. In this review, we have introduced the definition of MDSCs and their functions, discussed the roles of MDSCs in uterine cervical and endometrial cancer progression, and reviewed treatment strategies targeting MDSCs, which may exhibit growth-inhibitory effects and enhance the efficacy of existing anticancer treatments. Uterine cervical and endometrial cancers are the two most common gynecological malignancies. In the United States, 13,800 and 65,620 new cases of cervical and endometrial cancers, respectively, were reported in 2020 [Cervical cancer has been considered an immunogenic tumor, as it is induced by persistent infection with human papillomavirus. Due to the existence of polymerase epsilon–ultramutated and microsatellite instability–hypermutated tumors, endometrial cancer has also been considered immunogenic and a reasonable candidate for active and/or passive immunotherapy . AlthougSuppression of tumor immune surveillance is a main mechanism that prevents the destruction of tumor cells by the immune system and limits the efficacy of existing cancer treatments, including radiotherapy, chemotherapy, and immunotherapy . MyeloidIn this review, we have summarized the current knowledge on MDSC biology and its role in uterine cervical and endometrial cancers. In addition, we have discussed the utility of MDSCs as a predictive marker and highlighted the therapeutic targets of MDSCs in patients with uterine cervical and endometrial cancers.MDSCs were discovered in the late 1970s. At that time, they were regarded as formerly unknown immune cells that possess immunosuppressive features .MDSCs are a heterogeneous population of IMCs, and their number is increased in states of cancer, inflammation, or infection. MDSCs differ from terminally differentiated mature myeloid cells , or neutrophils) and can be subdivided into two major subsets based on their phenotypic and morphological features—monocytic–MDSCs (M–MDSCs) and polymorphonuclear (PMN) MDSCs  ,8.+Gr-1+ cells). However, as Gr-1 is a combination of lymphocyte antigen (Ly) 6C and Ly6G, M–MDSCs can be further defined as CD11b+Ly6ChighLy6G– cells, and PMN–MDSCs can be defined as CD11b+Ly6Cl°wLy6G+ cells [In mice, MDSCs are characterized by the expression of glutathione reductase (Gr-1) and CD11b myeloid lineage differentiation markers  and lineage markers . PMN–MDSCs express CD15 but not CD14; hence, they are defined as CD11b5− cells . HoweverUnder normal circumstances, IMCs differentiate into macrophages, neutrophils, and DCs. However, under pathological conditions such as infection, inflammation, or cancer, the differentiation of IMCs is impaired, leading to the formation of MDSCs ,8. The dThese factors in MDSCs trigger the activation of the following signaling pathways to stimulate their suppressive activities: signal transducer and activator of transcription 3 (STAT3), nuclear factor–kappa B, phosphoinositide 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR), and CCAAT/enhancer-binding protein β ,8. Of thChemokines are important factors in the direct migration of MDSCs. Accumulating evidence has demonstrated that multiple chemokines in the tumor microenvironment (TME), including C-X-C motif ligand (CXCL) 1, CXCL8, CXCL12, C-C motif ligand (CCL) 1, CCL2, CCL3, CCL5, CCL7, and their corresponding receptors on MDSCs (C-C chemokine receptor (CCR) 2, CCR5, and C-X-C chemokine receptor 4) differentially regulate the recruitment of MDSCs [Radiotherapy and surgery have been curative treatment options in patients with uterine cervical or endometrial cancer. Recent investigations have suggested that radiotherapy has two opposite effects on MDSC recruitment into TME: conventional fractionated radiotherapy increases MDSCs, while ablative hypofractionated radiotherapy decreases MDSCs. In a mouse model of prostate cancer, a fractionated radiotherapy (3 Gy × 5) has been shown to increase MDSC in the tumor, spleen, or lymph nodes via the production of colony stimulating factor 1 . On the MDSCs suppress T cells in both antigen-specific and antigen-nonspecific ways by utilizing several mechanisms. The most prominent factors include arginase-1 (Arg-1), nitric oxide (NO), and reactive oxygen species (ROS). Of these, ROS is responsible for antigen-specific suppression that requires close contact of MDSCs and T cells, as ROS are unstable and active only for a very short period. In contrast, NO and Arg-1 that have relatively longer half-life, are responsible for antigen-nonspecific suppression.Both PMN– and M–MDSCs produce Arg-1 , which cPMN–MDSCs have increased NADPH oxidase activity and produce large amounts of ROS, which lead to the production of peroxynitrite (PNT). As ROS and PNT are unstable and have very short half-life, PMN–MDSCs require close cell-to-cell contact to exert their effect on T cells. During the close interaction between MDSCs and CD8+ T cells via antigen recognition, PNT causes nitration and conformational changes of the TCR complex. CD8+ T cells consequently lose their binding ability to peptide–MHC class I complex and become nonresponsive to specific peptide presented by tumor cells. PNT may also induce nitration and structural changes of MHC class I molecules on tumor cells, leading to reduced capacity of antigenic peptide binding and impairment of recognition of tumor cells by CD8+ T cells ,17.M–MDSCs show low ROS production; however, they express high levels of iNOS, which produces NO that nitrates signaling molecules downstream of FcgRIIIA, resulting in the inhibition of the activities of T cells and NK cells ,8. NO alOther roles of MDSCs in immune suppression include the production of indoleamine-2,3-dioxygenase, which decreases tryptophan levels in the TME, leading to the induction of cell cycle arrest or apoptosis of T cells. MDSCs can also produce immunosuppressive cytokines such as IL-10 and TGF-β, affect NK cell function, and induce regulatory T cell (Treg) expansion . Lastly,In addition to immune-suppressive mechanisms, MDSCs promote cancer progression by stimulating tumor angiogenesis, promoting invasiveness by facilitating epithelial-to-mesenchymal transition or enhancing the metastatic activity of cancer cells by creating “premetastatic niches” ,22,23. TAn increased number of circulating MDSCs has been detected in various patients with cancers. In most cancers, including lung, breast, colon, renal, head and neck, and pancreatic cancers, PMN–MDSCs represent the major population of MDSCs. However, patients with melanoma, multiple myeloma, and prostate cancer have a substantially higher proportion of M–MDSCs in the peripheral blood than that of PMN–MDSCs .According to previous reports, an increased number of pretreatment MDSCs has been associated with advanced clinical stage, high probability of recurrence, and short survival ,6,8,19. + T cells [In preclinical investigations , cervica T cells ,30,31,35 T cells , contrib T cells ,23, and  T cells ,30, all  T cells , and res T cells ,28.In 2014, Mabuchi et al.  and VandIn studies investigating the cause of increased MDSC production in patients with uterine cervical and endometrial cancers, an increased number of MDSCs was found to be associated with increased serum E2 concentrations , serum P+ T cells [Consistent with the findings of in vitro and in vivo experiments, an increase in the number of circulating or tumor-infiltrating MDSCs was associated with a decrease in the number of tumor-infiltrating CD8 T cells ,31,35. A T cells ,35,38, d T cells ,28,29, h T cells ,28,29,31 T cells .However, abovementioned studies have limitations that warrant further investigation—small sample size, inconsistent histological subtypes, use of inconsistent MDSC surface markers, and limited clinical information.During pregnancy, maternal plasma levels of estradiol increase up to 100-fold compared to the nonpregnant status. A previous study demonstrated that the exogenous E2 treatment stimulated the mobilization of MDSC from bone marrow and directly augmented their suppressive activities, leading to the progression of cervical cancer . ConsistAccumulating preclinical evidence has shown that MDSC inhibition has therapeutic efficacy against various solid malignancies as a monotherapy or as in combination with existing anticancer treatments ,6. Altho+ T cells, which leads to the inhibition of tumor growth or metastasis [Various MDSC-targeting strategies have been evaluated in murine models of uterine cervical and endometrial cancers , such astastasis ,22,29,33tastasis , attenuatastasis  or radiotastasis . In additastasis  and endotastasis ,29,33,34In murine studies, anti-Gr-1 antibody has been widely used to eliminate MDSCs from the TME. However, anti-Gr-1 antibodies cannot be used clinically, owing to the absence of a Gr-1 homolog in humans. Currently, although no specific inhibitors of human MDSCs have been developed, various strategies to target MDSCs have been proposed; they have shown promising antitumor effects in preclinical models of solid cancers—(1) depletion of MDSCs, (2) MDSC deactivation, (3) inhibition of MDSC recruitment, and (4) promotion of the differentiation of MDSCs into mature cells  41,42,4,74,75,76In previous investigations including uterine cervical and endometrial cancer patients, an increased number of MDSCs were observed only in those who displayed tumor-related leukocytosis (TRL) ,22,28. IMoreover, recently, it was found that an increased number of MDSCs was associated with increased bone marrow FDG uptake in patients with uterine cervical cancer . Thus, bTheoretically, other tumor-derived substances  that stimulate the production of MDSC can also be predictive biomarkers. We hope the clinical utility of such biomarkers be evaluated preclinically and clinically in the future, which would enable physicians to identify patients who might benefit from MDSC-targeting therapy.+ effector T cells and decreased the number of MDSCs in PDAC tumors, suggesting that CXCR4 inhibition may enhance the therapeutic efficacy of PD-1 blockade in patients with PDAC and warrants confirmation in subsequent randomized trials.Although various MDSC-targeting strategies have been proposed in preclinical investigations , only a Activating mutation of PIK3CA and the resulting activation of PI3K is frequently observed in both uterine endometrial and cervical cancer, and, thus, PI3K-inhibition has been regarded as promising treatment . MoreoveAn increased number of MDSCs is observed in patients with uterine cervical and endometrial cancers. MDSCs play a significant role in disease progression. To inhibit their tumor-promoting effects, the efficacy of MDSC-targeting therapies (either as monotherapies or in combination with existing treatments) against uterine cervical and endometrial cancers is currently being evaluated preclinically. We believe that increasing our understanding of MDSC biology will aid in the development of optimal MDSC-targeting therapies for patients with uterine cervical and endometrial cancers.


We revealed that the stable gastric pentadecapeptide BPC 157, a useful peptide therapy against isoprenaline myocardial infarction, as well as against isoprenaline myocardial reinfarction, may follow the counteraction of the recently described occlusion-like syndrome, induced peripherally and centrally, which was described for the first time in isoprenaline-treated rats. BPC 157 , L-NAME (5 mg/kg i.p.), and L-arginine (200 mg/kg i.p.) were given alone or together at (i) 30 min before or, alternatively, (ii) at 5 min after isoprenaline (75 or 150 mg/kg s.c.). At 30 min after isoprenaline 75 mg/kg s.c., we noted an early multiorgan failure , thrombosis, intracranial  hypertension, portal and caval hypertension, and aortal hypotension, in its full presentation  via activation of the azygos vein). Further, we studied isoprenaline (75 or 150 mg/kg s.c.) myocardial infarction  and reinfarction  in rats (assessed at the end of the subsequent 24 h period). BPC 157 reduced levels of all necrosis markers, CK, CK-MB, LDH, and cTnT, and attenuated gross (no visible infarcted area) and histological damage, ECG (no ST-T ischemic changes), and echocardiography (preservation of systolic left ventricular function) damage induced by isoprenaline. Its effect was associated with a significant decrease in oxidative stress parameters and likely maintained NO system function, providing that BPC 157 interacted with eNOS and COX2 gene expression in a particular way and counteracted the noxious effect of the NOS-blocker, L-NAME. Isoprenaline myocardial infarction in rats is known to be a rapid, simple and non-invasive method that produces myocardial damage similar to that seen in acute myocardial infarction in humans . We hypoConsidering the particular points of the isoprenaline application , this maFor illustration, an activated azygos vein as a rescuing pathway, avoiding both the lung and liver, combines the inferior caval vein and superior caval vein via direct blood delivery. Thus, an activated azygos vein shunt could reorganize blood flow and instantly attenuate the consequences of maintained occlusion-induced vascular failure, both peripherally and centrally ,18,19,20Isoprenaline-induced lesions appeared due to damaging free radical induction . BPC 157In the present study, isoprenaline-treated rats received pentadecapeptide BPC 157, L-NAME, and L-arginine alone and/or in combination. Further assessments included eNOS, iNOS, COX-2 mRNA levels and lipid peroxidation in infarcted hearts . We also included investigations related to the necrosis markers commonly measured in routine practice , includiMale Wistar Albino rats (200–250 g) were used in all experiments and were randomly assigned, with 10 rats per experimental group, were used. Rats were fed with a standard rodent chow ad libitum. All experiments were approved by the local ethics committee.Pentadecapeptide BPC 157 , , dissolved in saline, was used in all experiments. The peptide BPC 157 is part of the sequence of human gastric juice protein BPC and is freely soluble in water at pH 7.0 and saline ,3,4. It The assessment was conducted by measuring ECG recordings, serum enzymes, troponin T, RNA extraction and RT-PCR , macro/microscopic lesion presentation, clinical presentation, echocardiography and oxidative stress. For the early isoprenaline-induced occlusion-like syndrome, we also assessed multiorgan failure , thrombosis, intracranial  hypertension, portal and caval hypertension, and aortal hypotension.We induced myocardial isoprenaline lesions  given at 5 min after isoprenaline, we assessed the early “occlusion-like” syndrome in the isoprenaline-treated rats as an early multiorgan failure , thrombosis, intracranial  hypertension, portal and caval hypertension, and aortal hypotension, full presentation.The gross lesions were recorded in deeply anaesthetized, laparatomized, craniotomized and thoracotomized rats, with a camera attached to a VMS-004 Discovery Deluxe USB microscope  for the early “occlusion-like” syndrome assessment. Features investigated in recorded photographs included the hemorrhagic lesions in the stomach assessed as the sum of the longest lesion diameters after isoprenaline administration and following medication at 30 min; the rats were then sacrificed. The other investigated features included the gross presentation of the brain, the superior mesenteric vein, the inferior vena cava, azygos vein and the heart assessment.For the group which underwent infarction and re-infarction induction at 24 h after first isoprenaline, at the point of 24 h after the second application , the rat’s chest cavities were opened to remove the heart shortly after blood samples were taken. Hearts were assessed for lesions expressed as percentage of the injured area.A2 is the surface area of the brain (or vein) in saline-treated animals, and A1 is the surface area of the brain (or veins) in BPC 157-treated animals. Starting from the square-cube law, Equations (1) and (2), an equation for the saline-to-BPC 157 brain (or vein) volume ratio proportional to the the saline-to-BPC 157 brain (or vein) surface area ratio Equation (6) was derived. For Equations (1)–(5), any arbitrary one-dimensional length on the photograph was defined . This was used only to define the one-dimensional proportion (l2/l1) between two observed brains or veins and as an inter-factor  for deriving the final Equation (6). The procedure was as follows:As described previously ,21,22,23(1) Square-cube law;(2) Square-cube law;A1;(3) From (1), after dividing both sides by (4) From (3), after taking the square root of both sides;V1;(5) From (2), after dividing both sides by (6) After incorporating (4) into (5).This measuring procedure, followed by the calculation of volume ratios, was performed separately for the brain and veins.Brain swelling was recorded in separate rats 15 min after complete calvariectomy. Briefly, six burr holes were drilled in three horizontal lines, all of them medial to the superior temporal lines and temporalis muscle attachments. The two rostral burr holes were placed just basal from the posterior interocular line, the two basal burr holes were placed just rostral to the lambdoid suture (and transverse sinuses) on both sides, respectively, and the middle two burr holes were placed in the line between the basal and rostral burr holes.After euthanasia at 30 min after isoprenaline, the superior sagittal sinus, the portal vein, the inferior vena cava, the superior mesenteric artery, and the abdominal aorta were removed from the rats, and clots were weighed ,21,22,23Recordings were made in deeply anaesthetized rats with a cannula (BD Neoflon Cannula) connected to a pressure transducer  inserted into the superior sagittal sinus, portal vein, inferior vena cava and abdominal aorta at the level of the bifurcation at 30 min after isoprenaline 75 mg/kg sc ; the rats were then sacrificed. Each recording lasted 1 min. For the superior sagittal sinus pressure recording, we made a single burr hole in the rostral part of the sagittal suture, above the superior sagittal sinus, and cannulated the anterior portion of the superior sagittal sinus by Braun intravenous cannulas. We then laparatomized the rats for the portal vein, inferior vena caval and abdominal aortal pressure recordings.Of note, normal rats exhibited a superior sagittal sinus pressure of −24 to −27 mmHg and a portal pressure of 3–5 mmHg, similar to that of the inferior vena cava, although this was at least 1 mmHg higher in the portal vein. By contrast, the abdominal aorta blood pressure was 100–120 mm Hg at the level of the bifurcation ,22,23.https://www.sakura.eu/Solutions/Staining-Coverslipping/H-E-Kit accessed on 19 October 2021): rehydration in distilled water, staining with hematoxylin, washing in running tap water, differentiation with 70% alcohol, staining with eosin, dehydration, clearing, and mounting.Organ tissue was fixed in 10% neutral buffered formalin (pH 7.4) at room temperature for 24 h . Representative tissue specimens were collected, dehydrated and embedded in paraffin blocks, sectioned at 4 μm, and stained with hematoxylin-eosin according to the following automated Sakura Tissue-Tek DRS 2000 Slide Stainer protocol  based on blind assessment. Histological examination was conducted using an Olympus BX51 microscope, and images of tissue were saved as uncompressed 24-bit RGB TIFF files using an Olympus 71 digital camera.In the assessment of the “occlusion-like” syndrome in isoprenaline-treated rats sacrificed at 30 min after isoprenaline 75 mg/kg s.c. , the brain was dissected according to NTP-7, at Level 3 and 6 with neuroanatomic subsites presented in certain brain sections using coronal sections with 3 mandatory sections and analyzed using a semiquantitative neuropathological scoring system, as previously described ,21,22,58In the assessment of the “occlusion-like” syndrome in isoprenaline-treated rats sacrificed at 30 min after administration of isoprenaline 75 mg/kg s.c. , we used a scoring system to grade the degree of lung injury, including observations of focal thickening of the alveolar membranes, congestion, pulmonary edema, intra-alveolar hemorrhage, interstitial neutrophil infiltration and intra-alveolar neutrophil infiltration. Each feature was assigned a score from 0 to 3 based on its absence (0) or presence to a mild (1), moderate (2) or severe (3) degree. A total cumulative histology score was determined ,22,23,59The renal injury was based on the degeneration of the Bowman’s space, glomeruli and proximal and distal tubules; vascular congestion; and interstitial edema. The criteria for liver injury were vacuolization of hepatocytes and pyknotic hepatocyte nuclei, activation of Kupffer cells and enlargement of sinusoids. Each specimen was scored using a scale from 0 to 3  for each criterion ,22,23,60The myocardium was graded for the severity of necrosis exhibited in the ventricles. The mean value of their scores is presented. The pathological criteria for grading the severity of necrosis were: score 1 (mild), one or two small foci; score 2 (slight), several small foci; score 3 (moderate), multiple small foci or several large foci; score 4 (severe), multiple large foci or diffuse area of necrosis ,22,23,61Intestinal tissue damage was analyzed using a histologic scoring scale adapted from Chui and co-workers  on a scaIn deeply anesthetized rats, an ECG was recorded continuously in all three main leads by positioning stainless steel electrodes on all four limbs using an ECG monitor by 2090 Medtronic programmer  connected to a digital oscilloscope LeCroywaverunnerLT342 , which enabled precise recordings, measurements and analysis of ECG parameters. At 30 min, 24 h and 48 h, the ECG changes were analyzed ,22,23.Serum enzymes  were measured using an auto-analyzer AU-800 , and troponin T was assessed by a rapid quantitative assay using the Cardiac Reader .RNA was extracted from 5 to 10 µm thick sections of paraffin-embedded heart septum tissue using the High Pure RNA Paraffin Kit . RNA density was measured using NanoDrop ND-1000 Spectrophotometer . A total of 150 ng of RNA from each sample was used in RT-PCRs performed in a total volume of 25 µL using the QIAGEN OneStep RT-PCR Kit . The RT-PCR mixture contained 5 µL 5xQIAGEN OneStep RT-PCR Buffer, 1 µL dNTP Mix, 1.5 µL of 10 µM sense primer, 1.5 µL of 10 µM antisense primer, 1 µL OneStep RT-PCR Enzyme Mix, RNA as described and RNase-free water. RT-PCR was performed in a thermocycler GeneAmp PCR System 9700 . The samples were incubated at 55 °C for 30 min to enable reverse transcription, followed by heating at 94 °C for 15 min to activate the Hot StartTaq DNA Polymerase. After that, the samples were incubated at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s for 40 cycles with GAPDH, iNOS, eNOS and COX-2 primers . At the Assessment included edema ) that may be prominent, scored from 0 to 3 , poor respiration as respiratory rate  and the Echocardiography (2D) was performed in all animals 24 h and 48 h after isoprenaline (75 and 150 mg/kg s.c. for the second time at 0 and 24 h) with a 7.5-MHz, phased-array transducer (SONOS 2500 Hewlett Packard). The image of the left ventricular cavity was obtained from a long parasternal view, and the M mode cursor was positioned between the papillary muscles, just below the mitral valve plane. M-mode measurements of the LV cavity size in systole and diastole were made, and global LV function was assessed by calculating ejection fraction (EF) from end-diastolic and end-systolic volumes of the left ventricle according to the following equations: diastolic volume (DV) (mL) = 0.001047 × left ventricle internal dimension in diastole (LVIDd (mm)) ; systolic volume (SV) (mL) = 0.001047 × left ventricle internal dimension in systole (LVIDs (mm)) .At the end of the 24 h period in isoprenaline-treated rats that received a higher dose of BPC 157 before or after the isoprenaline challenge, oxidative stress in the collected tissue samples was assessed by quantifying thiobarbituric acid-reactive species (TBARS) as malonedialdehyde (MDA) equivalents. The tissue samples were homogenized in PBS (pH 7.4) containing 0.1 mM butylated hydroxytoluene (BHT)  and sonicated for 30 sec in an ice bath . Trichloroacetic acid  was added to the homogenate, the mixture was centrifuged at 3000 rpm for 5 min, and the supernatant was collected. Then, 1% TBA was added, and the samples were boiled . The tubes were then kept on ice for 10 min. Following centrifugation , the absorbance of the mixture at the wavelength of 532 nm was determined. The concentration of MDA was read from a standard calibration curve plotted using 1,1,3,3′-tetraethoxy propane (TEP). The extent of lipid peroxidation was expressed as MDA using a molar extinction coefficient for MDA of 1.56 × 105 mol/L/cm. The protein concentration was determined using a commercial kit. The results are expressed in nmol per mg of protein.p value of 0.05 or less was considered statistically significant.Statistical analysis was performed using parametric two-way mixed model ANOVA (one factor is repeated-measures) and Student–Newman–Keuls test to compare the difference between groups. Fisher’s exact probability test was used to assess the number of dead and surviving rats. A To clarify the isoprenaline infarction/reinfarction and stable gastric pentadecapeptide BPC 157, we challenge the possibility that the stable gastric pentadecapeptide BPC 157, as a useful peptide therapy against isoprenaline myocardial infarction, as well as against isoprenaline myocardial reinfarction, should follow the activation of the collateral rescuing pathways seeable for the counteraction of the recently described occlusion-like syndrome, induced peripherally and centrally, that would also appear in the isoprenaline-treatedrats, being decisive for further full myocardial infarction development. At 30 min after isoprenaline 75 mg/kg s.c., we noted an early multiorgan failure , thrombosis, intracranial  hypertension, portal and caval hypertension, and aortal hypotension, full presentation  via activation of the azygos vein) .In further course, isoprenaline challenge(s) resulted in uniform severe myocardial lesions presentation . ContrarAt 30 min after isoprenaline 75 mg/kg s.c., as an occlusion-like syndrome, hearts regularly presented in isoprenaline control rats with congested blood vessels of the myocardium with areas of numerous subendocardial ishemic myocytes, while BPC 157 rats showed no congestion in the heart, only some subendocardial ischemic myocytes and normal T-wave .p ˂ 0.05, vs. control, at least).On ECG, isoprenaline control rats had giant T-waves (0.5 ± 0.02 mV), while BPC 157 rats showed normal T-waves (0.2 ± 0.02 (µg) 0.2 ± 0.03 (ng) mV) . An increased rate of congestion was found in the gastrointestinal tract of control animals, from the stomach to colon, while only mild congestion was found in the colon with no changes in the stomach and small intestine in BPC 157-treated rats , while BPC 157-treated rats had spared gastric mucosa  hypertension, portal and caval hypertension, and aortal hypotension. BPC 157 completely eliminated portal and caval hypertension, and markedly attenuated intracranial  hypertension and aortal hypotension .As previously reported, BPC 157 therapy  goes via activation of the azygos vein . Of noteIn conclusion, we established in the early isoprenaline course an early multiorgan failure , thrombosis, intracranial  hypertension, portal and caval hypertension, and aortal hypotension versus attenuation by BPC 157 therapy  via activation of the azygos vein. This may be essential for further isoprenaline challenge(s) resulting in the presentation of uniform severe myocardial lesions and for BPC 157’s counteracting potential.Specifically, in BPC 157-treated rats, biochemical assessment demonstrated decreased cardiospecific serum CK-MB, CK, LDH and troponin T .BPC 157-treated rats showed evidently improved clinical presentation   and markIn BPC 157-treated rats, ECG recordings exhibit reduced ST elevation/depression . AdditioIn particular, in BPC 157-treated rats, echocardiography presentation shows better preserved heart function, as shown by improved DV, SV, EF LV, using both the prophylactic and therapeutic regimen .Indicatively, in BPC 157-treated rats, there is markedly attenuated isoprenaline-induced oxidative stress .Thus, it is evident that BPC 157 markedly counteracts myocardial isoprenaline lesions. This is the case if one isoprenaline application of 75 mg/kg or 150 mg/kg s.c. is given for initial infarct induction and assessed at 24 h thereafter. Likewise, the counteraction includes the myocardial lesions after 2 isoprenaline applications , if the next application for re-infarct induction occurs at the point of 24 h and assessment after the end of the subsequent 24 h period. Specifically, this includes both 10 ng and 10 µg regimens. A sustained effect  and a rapid effect  were evidenced for counteraction of both isoprenaline regimens (75 mg/kg s.c. and 150 mg/kg s.c.). The salutary effect of BPC 157 therapy (10 ng and 10 µg/kg in pretreatment and post-treatment regimens) was also observed in terms of mortality, which was significantly lower in BPC 157 groups .Considering the NO agents’ effects, L-arginine was also beneficial, while L-NAME application further aggravated isoprenaline lesions given either before or after isoprenaline challenge(s). Further, we considered the effects of their mutual interaction. L-arginine brings down aggravation by NOS-blockade with L-NAME to the control levels. Illustratively, L-NAME appears to be more sensitive to BPC 157 application, providing that BPC 157 nullifies the effect of L-NAME and brings all isoprenaline (+L-NAME) lesions markedly below control values . With L-arginine, BPC 157 maintained its original beneficial effect.The relevance of the isoprenaline-induced myocardial infarct is supported by the evidence that catecholamines represent a common pathway in the evolution of myocardial changes in humans who develop myocardial lesions without narrowing or obstruction of coronary arteries . RecentlThe results demonstrated that BPC 157 caused a significant reduction in all of the necrosis markers commonly measured in routine practice  and attenuated gross (no visible infarcted area) and histological damage, including ECG (no ST-T ischemic changes) and echocardiography (preservation of systolic left ventricular function) damage induced by isoprenaline. Its effect was associated with a significant decrease in oxidative stress parameters and likely maintained NO system function, providing that BPC 157 interacted with eNOS and COX2 gene expression, in a particular way and counteracted the noxious effect of the NOS-blocker, L-NAME.Of note, as mentioned, BPC 157 may counteract the isoprenaline-induced damage since the very beginning  as it markedly attenuated the early isoprenaline-induced occlusion-like syndrome. Indicatively, the demonstrated leading role of rapid heart disturbances and general vascular failure in the major vessels-induced occlusive syndromes ,18,19,20It is possible that isoprenaline-induced myocardial infarction appeared as a NO system failure  that BPCFinally, BPC 157’s beneficial effect combined with the initial reduced activity of eNOS and COX2 may provide an additional clue. Possibly, reduced activity of eNOS and COX2 may be an effect of BPC 157, in particular substituting the NO effect, whether an additive and/or synergistic effect, related to the activity of BPC 157 administration in isoprenaline-treated rats with infarction and reinfarction. Namely, eNOS, iNOS and COX2 activity are thought to be specifically up-regulated in myocardial infarction . They arIn addition, these beneficial effects correlate with the counteraction of the isoprenaline-induced increased lipid peroxication products in the heart tissue and reduction of all of the necrosis markers commonly measured. Furthermore, BPC 157 acts as a stabilizer of cellular junctions  and a frFinally, this study should emphasize the general point that animal studies, per se, must be cautious, and isoprenaline studies, in particular, must be cautious regarding their results and the relative paucity of BPC 157 clinical data ,3,4. On