The Potential of Clostridium butyricum to Preserve Gut Health, and to Mitigate Non-AIDS Comorbidities in People Living with HIV.

A dramatic reduction in mortality among people living with HIV (PLWH) has been achieved during the modern antiretroviral therapy (ART) era. However, ART does not restore gut barrier function even after long-term viral suppression, allowing microbial products to enter the systemic blood circulation and induce chronic immune activation. In PLWH, a chronic state of systemic inflammation exists and persists, which increases the risk of development of inflammation-associated non-AIDS comorbidities such as metabolic disorders, cardiovascular diseases, and cancer. Clostridium butyricum is a human butyrate-producing symbiont present in the gut microbiome. Convergent evidence has demonstrated favorable effects of C. butyricum for gastrointestinal health, including maintenance of the structural and functional integrity of the gut barrier, inhibition of pathogenic bacteria within the intestine, and reduction of microbial translocation. Moreover, C. butyricum supplementation has been observed to have a positive effect on various inflammation-related diseases such as diabetes, ulcerative colitis, and cancer, which are also recognized as non-AIDS comorbidities associated with epithelial gut damage. There is currently scant published research in the literature, focusing on the influence of C. butyricum in the gut of PLWH. In this hypothesis review, we speculate the use of C. butyricum as a probiotic oral supplementation may well emerge as a potential future synergistic adjunctive strategy in PLWH, in tandem with ART, to restore and consolidate intestinal barrier integrity, repair the leaky gut, prevent microbial translocation from the gut, and reduce both gut and systemic inflammation, with the ultimate objective of decreasing the risk for development of non-AIDS comorbidities in PLWH.

miR-15a-5p up-regulates TLR4 and induces the formation of hypertrophic scars and keloids.

The formation of hypertrophic scar and keloid is considered to be a very complex pathological process. Our previous studies have shown that miR-15a-5p is an important miRNA in HTS tissues, and its expression level is significantly increased. Therefore, the potential mechanism of action of miR-15a-5p in scarring arouses our interest. This study preliminarily investigated the expression level of miR-15a-5p in HTS tissue and normal skin tissue and further explored the molecular mechanism. The results of this study once again confirmed that the expression level of miR-15a-5p was increased in HTS tissues and cells, and the closely related mRNA and protein levels of MyD88 and TGF-ß were also highly expressed. The relative expression levels of fibrosis-related indicators in HTsFb cells were up-regulated, such as collagen-Ⅰ, collagen-III and α-SMA. We constructed the HTS cell model and BALB/c nude animal model, and down-regulating miR-15a-5p, the HTsFb cells proliferation was inhibited, and qRT-PCR results showed that the fibrosis index mRNA was also reduced, and significantly reduce the pathological state of scar tissue. In conclusion, miR-15a-5p may participate in the formation and development of HTS through TLR/MyD88 signaling pathway and TGF-ß1 signaling pathway.

RUNX1 overexpression triggers TGF-ß signaling to upregulate p15 and thereby blocks early hematopoiesis by inducing cell cycle arrest.

p15INK4b (cyclin-dependent kinase inhibitor 2B, CDKN2B, p15), a cyclin-dependent kinase inhibitor (CKI) belonging to the INK4 family, plays an important role in hematopoiesis. Its expression level was positively related to the blockage effects of RUNX1b at the early stage. Experiments using human embryonic stem cell (hESC) lines with inducible p15 expression suggested that p15 overexpression can significantly decrease the proportion of KDR+ cells in S and G2-M stages 4 days after induction from day 0. Moreover, p15 overexpression from the early stage can decrease production of CD34highCD43- cells and their derivative populations, but not CD34lowCD43- cells. These effects were weakened if induction was delayed and disappeared if induction started after day 6. All these effects were counteracted by inhibition of TGF-ß signaling. TGF-ß1 stimulation elicited similar effects as p15 overexpression. RUNX1 overexpression and activation of the TGF-ß signaling pathway upregulate the expression of p15, which is partially responsible for blockade of hematopoiesis and relevant to a change in the cell cycle status. However, it is possible that other mechanisms are involved in the regulation of hematopoiesis.

The distinct effects of P18 overexpression on different stages of hematopoiesis involve TGF-ß and NF-κB signaling.

Deficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. However, the effects of P18 overexpression, which is involved in the inhibitory effects of RUNX1b at the early stage of hematopoiesis, have not been examined in detail. In this study, we established inducible P18/hESC lines and monitored the effects of P18 overexpression on hematopoietic differentiation. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43- cells and derivative populations, but not that of CD34lowCD43- cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. By contrast, induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-ß or NF-κB signaling respectively. This is the first evidence that P18 promotes hematopoiesis, a rare property among cyclin-dependent kinase inhibitors (CKIs).

Overexpression of HOXA9 upregulates NF-κB signaling to promote human hematopoiesis and alter the hematopoietic differentiation potentials.

BACKGROUND: The HOX genes are master regulators of embryogenesis that are also involved in hematopoiesis. HOXA9 belongs to a cluster of HOX genes that play extensively studied roles in hematopoiesis and leukemogenesis. METHODS: We established HOXA9-inducible human embryonic stem cells (HOXA9/hESCs) with normal pluripotency and potential for hematopoiesis, which could be used to analyze gene function with high accuracy. HOXA9/hESCs co-cultured with aorta-gonad-mesonephros-derived stromal cells (AGM-S3) were induced to overexpress HOXA9 with doxycycline (DOX) at various times after hematopoiesis started and then subjected to flow cytometry. RESULTS: Induction of HOXA9 from Day 4 (D4) or later notably promoted hematopoiesis and also increased the production of CD34+ cells and derived populations. The potential for myelogenesis was significantly elevated while the potential for erythrogenesis was significantly reduced. At D14, a significant promotion of S phase was observed in green fluorescent protein positive (GFP+) cells overexpressing HOXA9. NF-κB signaling was also up-regulated at D14 following induction of HOXA9 on D4. All of these effects could be counteracted by addition of an NF-κB inhibitor or siRNA against NFKB1 along with DOX. CONCLUSIONS: Overexpression of HOXA9 starting at D4 or later during hematopoiesis significantly promoted hematopoiesis and the production of myeloid progenitors while reduced the production of erythroid progenitors, indicating that HOXA9 plays a key role in hematopoiesis and differentiation of hematopoietic lineages.

Overexpression of p21 Has Inhibitory Effect on Human Hematopoiesis by Blocking Generation of CD43＋ Cells via Cell-Cycle Regulation.

BACKGROUND AND OBJECTIVES: p21, an important member of the Cip/Kip family, is involved in inhibitory effects of RUNX1b overexpression during the early stage of human hematopoiesis. METHODS AND RESULTS: We established a human embryonic stem cell (hESC) line with inducible expression of p21 (p21/hESCs). Overexpression of p21 did not influence either mesoderm induction or emergence of CD34＋ cells, but it significantly decreased the production of CD43＋ cells and changed the expression profile of hematopoiesis-related factors, leading to the negative effects of p21 on hematopoiesis. CONCLUSIONS: In RUNX1b/hESC co-cultures when RUNX1b was induced from D0, perturbation of the cell cycle caused by upregulation of p21 probably prevented the appearance of CD43＋ cells, but not CD34＋ cells. The mechanisms via which CD34＋ cells are blocked by RUNX1b overexpression remain to be elucidated.

HOXC4 up-regulates NF-κB signaling and promotes the cell proliferation to drive development of human hematopoiesis, especially CD43+ cells.

The hematopoietic function of HOXC4 has not been extensively investigated. Our research indicated that induction of HOXC4 in co-culture system from D10 significantly promoted productions of most hematopoietic progenitor cells. CD34-CD43+ cells could be clearly classified into CD34-CD43low and CD34-CD43high sub-populations at D14. The former cells had greater myelogenic potential, and their production was not significantly influenced by induction of HOXC4. By contrast, the latter cells had greater potential to differentiate into megakaryocytes and erythroid cells, and thus had properties of erythroid-megakaryocyte common progenitors, which abundance was increased by ∼2-fold when HOXC4 was induced from D10. For CD34-CD43low, CD34+CD43+, and CD34-CD43high sub-populations, CD43 level served as a natural index for the tendency to undergo hematopoiesis. Induction of HOXC4 from D10 caused more CD43+ cells sustain in S-phase with up-regulation of NF-κB signaling, which could be counteracted by inhibition of NF-κB signaling. These observations suggested that promotion of hematopoiesis by HOXC4 is closely related to NF-κB signaling and a change in cell-cycle status, which containing potential of clinical applications.

MicroRNA-1224 Splicing CircularRNA-Filip1l in an Ago2-Dependent Manner Regulates Chronic Inflammatory Pain via Targeting Ubr5.

Dysfunctions of gene transcription and translation in the nociceptive pathways play the critical role in development and maintenance of chronic pain. Circular RNAs (circRNAs) are emerging as new players in regulation of gene expression, but whether and how circRNAs are involved in chronic pain remain elusive. We showed here that complete Freund's adjuvant-induced chronic inflammation pain significantly increased circRNA-Filip1l (filamin A interacting protein 1-like) expression in spinal neurons of mice. Blockage of this increase attenuated complete Freund's adjuvant-induced nociceptive behaviors, and overexpression of spinal circRNA-Filip1l in naive mice mimicked the nociceptive behaviors as evidenced by decreased thermal and mechanical nociceptive threshold. Furthermore, we found that mature circRNA-Filip1l expression was negatively regulated by miRNA-1224 via binding and splicing of precursor of circRNA-Filip1l (pre-circRNA-Filip1l) in the Argonaute-2 (Ago2)-dependent manner. Increase of spinal circRNA-Filip1l expression resulted from the decrease of miRNA-1224 expression under chronic inflammation pain state. miRNA-1224 knockdown or Ago2 overexpression induced nociceptive behaviors in naive mice, which was prevented by the knockdown of spinal circRNA-Filip1l. Finally, we demonstrated that a ubiquitin protein ligase E3 component n-recognin 5 (Ubr5), validated as a target of circRNA-Filip1l, plays a pivotal role in regulation of nociception by spinal circRNA-Filip1l. These data suggest that miRNA-1224-mediated and Ago2-dependent modulation of spinal circRNA-Filip1l expression regulates nociception via targeting Ubr5, revealing a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.SIGNIFICANCE STATEMENT circRNAs are emerging as new players in regulation of gene expression. Here, we found that the increase of circRNA-Filip1l mediated by miRNA-1224 in an Ago2-dependent way in the spinal cord is involved in regulation of nociception via targeting Ubr5 Our study reveals a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.

DNA Hydroxymethylation by Ten-eleven Translocation Methylcytosine Dioxygenase 1 and 3 Regulates Nociceptive Sensitization in a Chronic Inflammatory Pain Model.

BACKGROUND: Ten-eleven translocation methylcytosine dioxygenase converts 5-methylcytosine in DNA to 5-hydroxymethylcytosine, which plays an important role in gene transcription. Although 5-hydroxymethylcytosine is enriched in mammalian neurons, its regulatory function in nociceptive information processing is unknown. METHODS: The global levels of 5-hydroxymethylcytosine and ten-eleven translocation methylcytosine dioxygenase were measured in spinal cords in mice treated with complete Freund's adjuvant. Immunoblotting, immunohistochemistry, and behavioral tests were used to explore the downstream ten-eleven translocation methylcytosine dioxygenase-dependent signaling pathway. RESULTS: Complete Freund's adjuvant-induced nociception increased the mean levels (± SD) of spinal 5-hydroxymethylcytosine (178 ± 34 vs. 100 ± 21; P = 0.0019), ten-eleven translocation methylcytosine dioxygenase-1 (0.52 ± 0.11 vs. 0.36 ± 0.064; P = 0.0088), and ten-eleven translocation methylcytosine dioxygenase-3 (0.61 ± 0.13 vs. 0.39 ± 0.08; P = 0.0083) compared with levels in control mice (n = 6/group). The knockdown of ten-eleven translocation methylcytosine dioxygenase-1 or ten-eleven translocation methylcytosine dioxygenase-3 alleviated thermal hyperalgesia and mechanical allodynia, whereas overexpression cytosinethem in naïve mice (n = 6/group). Down-regulation of spinal ten-eleven translocation methylcytosine dioxygenase-1 and ten-eleven translocation methylcytosine dioxygenase-3 also reversed the increases in Fos expression (123 ± 26 vs. 294 ± 6; P = 0.0031; and 140 ± 21 vs. 294 ± 60; P = 0.0043, respectively; n = 6/group), 5-hydroxymethylcytosine levels in the Stat3 promoter (75 ± 16.1 vs. 156 ± 28.9; P = 0.0043; and 91 ± 19.1 vs. 156 ± 28.9; P = 0.0066, respectively; n = 5/group), and consequent Stat3 expression (93 ± 19.6 vs. 137 ± 27.5; P = 0.035; and 72 ± 15.2 vs. 137 ± 27.5; P = 0.0028, respectively; n = 5/group) in complete Freund's adjuvant-treated mice. CONCLUSIONS: This study reveals a novel epigenetic mechanism for ten-eleven translocation methylcytosine dioxygenase-1 and ten-eleven translocation methylcytosine dioxygenase-3 in the modulation of spinal nociceptive information via targeting of Stat3.

Effects of propofol and sevoflurane on perioperative immune response in patients undergoing laparoscopic radical hysterectomy for cervical cancer.

The aim of this study is to compare the effects of propofol and sevoflurane anesthesia on perioperative immune response in patients undergoing laparoscopic radical hysterectomy for cervical cancer.Sixty patients with cervical cancer scheduled for elective laparoscopic radical hysterectomy under general anesthesia were randomized into 2 groups. TIVA group received propofol induction and maintenance and SEVO group received sevoflurane induction and maintenance. Blood samples were collected at 30 min before induction (T0); the end of the operation (T1); and 24 h (T2), 48 h (T3), and 72 h (T4) after operation. The T lymphocyte subsets (including CD3+ cells, CD4+ cells, and CD8+ cells) and CD4+/CD8+ ratio, natural killer (NK) cells, and B lymphocytes were analyzed by flow cytometry.After surgery, all immunological indicators except CD8+ cells were significantly decreased in both groups compared to basal levels in T0, and the counts of CD3+ cells, CD4+ cells, NK cells, and the CD4+/CD8+ ratios were significantly lower in the SEVO groups than that in the TIVA group. However, the numbers of B cells were comparable at all the time points between 2 groups.Laparoscopic radical hysterectomy for cervical cancer is associated with postoperative lymphopenia. In terms of protecting circulating lymphocytes, propofol is superior to sevoflurane.

Hydroxymethylation of microRNA-365-3p Regulates Nociceptive Behaviors via Kcnh2.

DNA 5-hydroxylmethylcytosine (5hmC) catalyzed by ten-eleven translocation methylcytosine dioxygenase (TET) occurs abundantly in neurons of mammals. However, the in vivo causal link between TET dysregulation and nociceptive modulation has not been established. Here, we found that spinal TET1 and TET3 were significantly increased in the model of formalin-induced acute inflammatory pain, which was accompanied with the augment of genome-wide 5hmC content in spinal cord. Knockdown of spinal TET1 or TET3 alleviated the formalin-induced nociceptive behavior and overexpression of spinal TET1 or TET3 in naive mice produced pain-like behavior as evidenced by decreased thermal pain threshold. Furthermore, we found that TET1 or TET3 regulated the nociceptive behavior by targeting microRNA-365-3p (miR-365-3p). Formalin increased 5hmC in the miR-365-3p promoter, which was inhibited by knockdown of TET1 or TET3 and mimicked by overexpression of TET1 or TET3 in naive mice. Nociceptive behavior induced by formalin or overexpression of spinal TET1 or TET3 could be prevented by downregulation of miR-365-3p, and mimicked by overexpression of spinal miR-365-3p. Finally, we demonstrated that a potassium channel, voltage-gated eag-related subfamily H member 2 (Kcnh2), validated as a target of miR-365-3p, played a critical role in nociceptive modulation by spinal TET or miR-365-3p. Together, we concluded that TET-mediated hydroxymethylation of miR-365-3p regulates nociceptive behavior via Kcnh2. SIGNIFICANCE STATEMENT: Mounting evidence indicates that epigenetic modifications in the nociceptive pathway contribute to pain processes and analgesia response. Here, we found that the increase of 5hmC content mediated by TET1 or TET3 in miR-365-3p promoter in the spinal cord is involved in nociceptive modulation through targeting a potassium channel, Kcnh2. Our study reveals a new epigenetic mechanism underlying nociceptive information processing, which may be a novel target for development of antinociceptive drugs.

Epigenetic modification of spinal miR-219 expression regulates chronic inflammation pain by targeting CaMKIIγ.

Emerging evidence has shown that miRNA-mediated gene expression modulation contributes to chronic pain, but its functional regulatory mechanism remains unknown. Here, we found that complete Freund's adjuvant (CFA)-induced chronic inflammation pain significantly reduced miRNA-219 (miR-219) expression in mice spinal neurons. Furthermore, the expression of spinal CaMKIIγ, an experimentally validated target of miR-219, was increased in CFA mice. Overexpression of spinal miR-219 prevented and reversed thermal hyperalgesia and mechanical allodynia and spinal neuronal sensitization induced by CFA. Concurrently, increased expression of spinal CaMKIIγ was reversed by miR-219 overexpression. Downregulation of spinal miR-219 in naive mice induced pain-responsive behaviors and increased p-NMDAR1 expression, which could be inhibited by knockdown of CaMKIIγ. Bisulfite sequencing showed that CFA induced the hypermethylation of CpG islands in the miR-219 promoter. Treatment with demethylation agent 5'-aza-2'-deoxycytidine markedly attenuated pain behavior and spinal neuronal sensitization, which was accompanied with the increase of spinal miR-219 and decrease of CaMKIIγ expression. Together, we conclude that methylation-mediated epigenetic modification of spinal miR-219 expression regulates chronic inflammatory pain by targeting CaMKIIγ.