Topical bilirubin-deferoxamine hastens excisional wound healing by modulating inflammation, oxidative stress, angiogenesis, and collagen deposition in diabetic rats.

AIM OF THE STUDY: The study was performed to understand the detailed mechanism of diabetic wound healing by bilirubin-deferoxamine (DFO) combination on topical application. MATERIALS AND METHODS: There were two study groups, control, and treatment. The granulation tissues collected on different days (3, 7, 14, and 19) were studied in detail for inflammatory mediators, angiogenesis markers, epithelialization, and oxidative stress parameters. RESULTS: A significant increase in wound contraction percentage was observed from day 7 in the bilirubin-DFO treatment group. The combinatorial treatment significantly reduced tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß), and enhanced IL-10 levels. Upregulated mRNAs of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 alpha (HIF-1 α) along with CD31 immunohistochemistry showed the pro-angiogenesis potential of the combination. Hematoxylin and Eosin (H and E) staining and Masson's trichrome staining showed reduced inflammatory cell infiltration, enhanced fibroblast proliferation, well-organized collagen fibers, and the development of new blood vessels. Collagen deposition is further supported by immunohistochemistry studies and Masson's trichrome staining. Bilirubin-DFO combination also reduced lipid peroxidation and elevated antioxidative enzymes. CONCLUSION: Topical application of bilirubin-DFO showed immense potential in augmenting skin wound regeneration in diabetes by upregulating the antioxidant status as well as increasing angiogenesis, collagen deposition, and modulating cytokines.

Angiogenic and MMPs modulatory effects of icariin improved cutaneous wound healing in rats.

Icariin is a flavonoid from plant belonging to the genus Epimedium, commonly known as Horny goat weed or Yin Yang Huo. The compound possesses multiple biological activities which are associated with the modulation of many signalling pathways, like NF-κB, Erk-p38-JNK, and release of various cytokines and growth factors. The present study determined wound healing potential of icariin in male Wistar rats. Icariin ointment (0%, 0.004%, 0.02%, 0.1% and 0.5%), was applied daily (b.i.d.) for 14 days on ≈ 400 mm2 cutaneous wound in different groups of rats. On day 14 post-wounding, 0.1% and 0.5% icariin treatment significantly (P < 0.01 and P < 0.001, respectively) increased wound contraction, as compared to control. Western blots revealed upregulation of IL-10 and downregulation of NF-κB and TNF-α. Increased expression of CD-31 showed abundance of microvessels in healing tissues after treatment with icariin. The MMP-2 and MMP-9 activities were reduced in icariin treated groups. Masson's trichrome staining revealed relatively better completion of re-epithelisation as well as increased deposition of well organised collagen fibres in the healing tissues compared to control. It is concluded that icariin has potential to accelerate cutaneous wound healing in rats.

Endometrial transcript profile of progesterone-regulated genes during early pregnancy of Water Buffalo (Bubalus bubalis).

Progesterone (P4 ) plays a key role in the establishment and maintenance of pregnancy in most mammals. Unravelling the expression of progesterone-regulated genes can expand the understanding of the embryonic mortality. Accordingly, we studied the relative mRNA expression of the P4 -regulated genes in the buffalo. Uteri were collected from the abattoir and categorized into nonpregnant late luteal phase, stage I (28-38th days of gestation) and stage II (48-56th days of gestation) of pregnancy (n = 6/group). After extraction of total RNA from the endometrial tissues, we carried out qRT-PCR for determining the relative mRNA expression of the P4 -regulated genes using nonpregnant late luteal phase as calibrator group. The expression of LGALS3BP (essential for maternal recognition of pregnancy) gene was found to be significantly upregulated (p < 0.05), while MUC1 (important for embryo attachment) gene was downregulated in stage I and II of pregnancy. We observed no significant change in the expression of LGALS1, LGALS9 and CTSL genes. The SLC5A11 and SLC2A1 genes (involved in the transport of glucose to endometrium) in early pregnancy were upregulated in the pregnancy stage I (p < 0.05) relative to nonpregnant late luteal phase. The CST3 gene was significantly upregulated in pregnancy stage II (p < 0.01). These results provide molecular insights into the specific pathways involved in foeto-maternal communication during early pregnancy in buffaloes.

Differential expression of ten candidate genes regulating prostaglandin action in reproductive tissues of buffalo during estrous cycle and pregnancy.

Prostaglandins (PGs) are the key mediators of several female reproductive functions, including luteolysis, ovulation, fertilization, implantation, pregnancy, and parturition. The present study was conducted in buffalo endometrial and luteal tissues between nonpregnant and two stages of pregnancy (29-38 days of pregnancy, 48-56 days of pregnancy) tissue samples. The genes involved from synthesis upto receptor level effect of PGs (PGF2α and PGE2) were studied for their relative mRNA expression. We have collected the endometrial and luteal tissues from slaughtered animals and confirmed the stages by external examination and crown vertebral rump length measurement of the foetus. The mRNA expression of COX-2 and PGFS genes revealed high significant rise in the transcript at pregnancy stage I as compared to the late luteal phase of nonpregnant. However, EP2 and EP3 genes were highly upregulated in pregnancy stage II. The expression of PLA2G4A and PGT genes showed difference in their transcripts in pregnancy, however, the difference was nonsignificant as compared to the nonpregnant stage. The findings emerged from this study also suggested the strict regulation at COX-2 mRNA level than at synthase enzyme's level. Among the four subtypes of EP gene, we have observed highly significant expression difference in EP2 followed by EP3 after implantation.

Study of lysophosphatidic acid receptors (LPARs) in buffalo uterus demonstrated upregulation of LPAR1 and LPAR6 in early pregnancy.

Lysophosphatidic acid (LPA) is an important factor involved in embryo implantation and pregnancy establishment in humans and domestic livestock. LPA exerts its action through six G-protein-coupled receptors (LPA1-LPA6). We investigated the types of LPA receptors expressed in buffalo uterus and also their differential expression in the nonpregnant, and early-pregnant endometrium. The nonpregnant, and early-pregnant (<42 days) uteri were collected from the local slaughterhouse. RT-PCR experiments detected mRNAs of all the six LPA receptors (LPAR1-LPAR6) in both nonpregnant, and early-pregnant endometrial tissues. Their comparative profiling by real-time PCR revealed that the early pregnant endometrium expressed more mRNAs of LPAR1 and LPAR6. All the mRNA fragments were sequenced and submitted to Genbank, NCBI. Western blot studies also showed a similar expression pattern of these two receptor proteins, including higher expression of both LPA1 and LPA6 proteins during early pregnancy. And between these two receptors, LPA6 upregulation was more pronounced than LPA1. In immunohistochemistry, these receptors were found to be localized in the endometrial glandular epithelial cells of both types of uterus. Level of LPA was also higher in early pregnant endometrial tissues. In summary, our study demonstrated expression of all the six LPAR mRNAs in buffalo uterus, wherein the early-pregnant uterus did express comparatively higher mRNA as well as protein of LPA1 and LPA6, indicating their role in pregnancy. The more pronounced expression of LPA6 possibly indicates its greater contribution to mediating LPA signaling in early pregnancy (29-42 days) of buffalo.

Hypercholesterolemia impairs oxytocin-induced uterine contractility in late pregnant mouse.

High cholesterol is known to negatively affect uterine contractility in ex vivo conditions. The aim of the present study was to reveal the effect of in vivo hypercholesterolemia on spontaneous and oxytocin-induced uterine contractility in late pregnant mouse uterus. Female Swiss albino mice were fed with high cholesterol (HC) diet (0.5% sodium cholate, 1.25% cholesterol and 15% fat) for 6 weeks and then throughout the gestation period after mating. On day 19 of gestation, serum cholesterol level was increased more than 3-fold while triglycerides level was reduced in HC diet-fed animals as compared to control animals fed with a standard diet. In tension experiments, neither the mean integral tension of spontaneous contractility nor the response to CaCl2 in high K+-depolarized tissues was altered, but the oxytocin-induced concentration-dependent contractile response in uterine strips was attenuated in hypercholesterolemic mice as compared to control. Similarly, hypercholesterolemia dampened concentration-dependent uterine contractions elicited by a GNAQ protein activator, Pasteurella multocida toxin. However, it had no effect on endogenous oxytocin level either in plasma or in uterine tissue. It also did not affect the prostaglandin release in oxytocin-stimulated tissues. Western blot data showed a significant increase in caveolin-1 and GRK6 proteins but decline in oxytocin receptor, GNAQ and RHOA protein expressions in hypercholesterolemic mouse uterus. The results of the present study suggest that hypercholesterolemia may attenuate the uterotonic action of oxytocin in late pregnancy by causing downregulation of oxytocin receptors and suppressing the signaling efficacy through GNAQ and RHOA proteins.

Candesartan ameliorates arsenic-induced hypertensive vascular remodeling by regularizing angiotensin II and TGF-beta signaling in rats.

Arsenic exposure can cause several cardiovascular diseases, including hypertension, atherosclerosis and microvascular disease. Earlier, we reported that arsenic-mediated enhancement of angiotensin II (AngII) signaling can impair vascular physiology. Here, we investigated whether the AT1 receptor (AT1R) blocker candesartan can ameliorate the arsenic-induced hypertensive vascular remodeling in rats and whether the amelioration could relate to attenuation in vascular AngII and TGF-ß signaling. Rats were exposed to sodium arsenite (50ppm) through drinking water for 90 consecutive days. Candesartan (1mg/kg bw, orally) was administered once daily during the last 30days of arsenic exposure. Non-invasive blood pressure was recorded weekly in conscious rats, while AngII-induced change in mean arterial pressure in anaesthetized rats was measured by invasive method on the 91st day. On this day, blood was collected from other animals for measuring AngII level. Western blot of AT1, AT2 and TßRII receptors; ELISA of PTK, RasGAP, ERK-1/2, TGF-ß and CTGF; immunohistochemistry of phosphorylated Smad3, Smad4 and collagen III, hydroxyproline/total collagen estimation, collagen deposition by Masson's trichrome staining and histomorphometry were carried out in thoracic aorta. Arsenic increased non-invasive systolic, diastolic and mean arterial pressure. Further, AngII caused concentration-dependent incremental change in mean arterial pressure in the arsenic-exposed rats. Arsenic upregulated AT1 and TßRII receptor proteins; elevated the levels of PTK, ERK-1/2, TGF-ß and CTGF, decreased RasGAP level and augmented the immunoreactivities of Smad3, Smad4 and collagen III. Arsenic also increased hydroxyproline/total collagen level, proliferation of collagen fibres and thickness of aortic wall and collagenous adventitia. Candesartan normalized blood pressure, regularized receptor expressions, MAP kinase and TGF-ß signaling, restored collagen deposition and regressed aortic thickness. Our results demonstrate that candesartan can ameliorate the arsenic-mediated systemic hypertension and vascular remodeling in rats by regularizing the signaling pathways of AngII and TGF-ß.