Traditional herbal pair Portulacae Herba and Granati Pericarpium alleviates DSS-induced colitis in mice through IL-6/STAT3/SOCS3 pathway.

BACKGROUND: Portulacae Herba and Granati Pericarpium pair (PGP) is a traditional Chinese herbal medicine treatment for colitis, clinically demonstrating a relatively favorable effect on relieving diarrhea and abnormal stools. However, the underlying mechanism remain uncertain. PURPOSE: The present study intends to evaluate the efficacy of PGP in treating colitis in mice and investigate its underlying mechanism. METHODS: The protective effect of PGP against colitis was determined by monitoring body weight, colon length, colon weight, and survival rate in mice. Colonic inflammation was assessed by serum cytokine levels, colonic H&E staining, and local neutrophil infiltration. The reversal of intestinal epithelial barrier damage by PGP was subsequently analyzed with Western blot and histological staining. Furthermore, RNA-seq analysis and molecular docking were performed to identify potential pathways recruited by PGP. Following the hints of the transcriptomic results, the role of PGP through the IL-6/STAT3/SOCS3 pathway in DSS-induced colitis mice was verified by Western blot. RESULTS: DSS-induced colitis in mice was significantly curbed by PGP treatment. PGP treatment significantly mitigated DSS-induced colitis in mice, as evidenced by improvements in body weight, DAI severity, survival rate, and inflammatory cytokines levels in serum and colon. Moreover, PGP treatment up-regulated the level of Slc26a3, thereby increasing the expressions of the tight junction/adherens junction proteins ZO-1, occludin and E-cadherin in the colon. RNA-seq analysis revealed that PGP inhibits the IL-6/STAT3/SOCS3 pathway at the transcriptional level. Molecular docking indicated that the major components of PGP could bind tightly to the proteins of IL-6 and SOCS3. Meanwhile, the result of Western blot revealed that the IL-6/STAT3/SOCS3 pathway was inhibited at the protein level after PGP administration. CONCLUSION: PGP could alleviate colonic inflammation and reverse damage to the intestinal epithelial barrier in DSS-induced colitis mice. The underlying mechanism involves the inhibition of the IL-6/STAT3/SOCS3 pathway.

SLC26A1 is a major determinant of sulfate homeostasis in humans.

Sulfate plays a pivotal role in numerous physiological processes in the human body, including bone and cartilage health. A role of the anion transporter SLC26A1 (Sat1) for sulfate reabsorption in the kidney is supported by the observation of hyposulfatemia and hypersulfaturia in Slc26a1-knockout mice. The impact of SLC26A1 on sulfate homeostasis in humans remains to be defined. By combining clinical genetics, functional expression assays, and population exome analysis, we identify SLC26A1 as a sulfate transporter in humans and experimentally validate several loss-of-function alleles. Whole-exome sequencing from a patient presenting with painful perichondritis, hyposulfatemia, and renal sulfate wasting revealed a homozygous mutation in SLC26A1, which has not been previously described to the best of our knowledge. Whole-exome data analysis of more than 5,000 individuals confirmed that rare, putatively damaging SCL26A1 variants were significantly associated with lower plasma sulfate at the population level. Functional expression assays confirmed a substantial reduction in sulfate transport for the SLC26A1 mutation of our patient, which we consider to be novel, as well as for the additional variants detected in the population study. In conclusion, combined evidence from 3 complementary approaches supports SLC26A1 activity as a major determinant of sulfate homeostasis in humans. In view of recent evidence linking sulfate homeostasis with back pain and intervertebral disc disorder, our study identifies SLC26A1 as a potential target for modulation of musculoskeletal health.

SLC26A6-selective inhibitor identified in a small-molecule screen blocks fluid absorption in small intestine.

SLC26A6 (also known as putative anion transporter 1 [PAT1]) is a Cl-/HCO3- exchanger expressed at the luminal membrane of enterocytes where it facilitates intestinal Cl- and fluid absorption. Here, high-throughput screening of 50,000 synthetic small molecules in cells expressing PAT1 and a halide-sensing fluorescent protein identified several classes of inhibitors. The most potent compound, the pyrazolo-pyrido-pyrimidinone PAT1inh-B01, fully inhibited PAT1-mediated anion exchange (IC50 ~350 nM), without inhibition of the related intestinal transporter SLC26A3 (also known as DRA). In closed midjejunal loops in mice, PAT1inh-B01 inhibited fluid absorption by 50%, which increased to >90% when coadministered with DRA inhibitor DRAinh-A270. In ileal loops, PAT1inh-B01 blocked fluid absorption by >80%, whereas DRAinh-A270 was without effect. In colonic loops, PAT1inh-B01 was without effect, whereas DRAinh-A270 completely blocked fluid absorption. In a loperamide constipation model, coadministration of PAT1inh-B01 with DRAinh-A270 increased stool output compared with DRAinh-A270 alone. These results provide functional evidence for complementary and region-specific roles of PAT1 and DRA in intestinal fluid absorption, with PAT1 as the predominant anion exchanger in mouse ileum. We believe that PAT1inh-B01 is a novel tool to study intestinal ion and fluid transport and perhaps a drug candidate for small intestinal hyposecretory disorders such as cystic fibrosis-related meconium ileus and distal intestinal obstruction syndrome.

Genome-Wide Identification and Expansion Patterns of SULTR Gene Family in Gramineae Crops and Their Expression Profiles under Abiotic Stress in Oryza sativa.

Sulfate transporters (SULTRs), also known as H+/SO42- symporters, play a key role in sulfate transport, plant growth and stress responses. However, the evolutionary relationships and functional differentiation of SULTRs in Gramineae crops are rarely reported. Here, 111 SULTRs were retrieved from the genomes of 10 Gramineae species, including Brachypodium disachyon, Hordeum vulgare, Setaria italica, Sorghum bicolor, Zea mays, Oryza barthii, Oryza rufipogon, Oryza glabbermia and Oryza sativa (Oryza sativa ssp. indica and Oryza sativa ssp. japonica). The SULTRs were clustered into five clades based on a phylogenetic analysis. Syntheny analysis indicates that whole-genome duplication/segmental duplication and tandem duplication events were essential in the SULTRs family expansion. We further found that different clades and orthologous groups of SULTRs were under a strong purifying selective force. Expression analysis showed that rice SULTRs with high-affinity transporters are associated with the functions of sulfate uptake and transport during rice seedling development. Furthermore, using Oryza sativa ssp. indica as a model species, we found that OsiSULTR10 was significantly upregulated under salt stress, while OsiSULTR3 and OsiSULTR12 showed remarkable upregulation under high temperature, low-selenium and drought stresses. OsiSULTR3 and OsiSULTR9 were upregulated under both low-selenium and high-selenium stresses. This study illustrates the expression and evolutionary patterns of the SULTRs family in Gramineae species, which will facilitate further studies of SULTR in other Gramineae species.

Possible mechanism of bamboo shoots (Bambusa balcooa) induced thyroid disruption - An in vitro study.

Endemic goitre and associated iodine deficiency disorders (IDDs) are a major concern in public health even in the period of post salt iodization in many regions. Among others the consumption of cyanogenic plants found responsible for the persistence of such diseases. Bamboo shoots (BS) is one such cyanogenic plant food that caused disruption of certain thyroid hormone synthesizing regulatory element as has already been reported in our earlier study. In this investigation the possible mechanism of thyrocytes disruption along with interruption of thyroid hormone biosynthesis by BS has been worked out. Commonly consumed BS, Bambusa Balcooa Roxb (BBR) water extract was analysed by GC MS; three doses below IC50 were administered to thyrocytes in culture with and without iodine. Expressions of thyroglobulin (Tg), pendrin (PDS) and monocarboxylate transporter 8 (MCT8) were evaluated in thyrocytes with cell cycle analysis, reactive oxygen species (ROS) generation, DNA oxidation and apoptotic regulation through Bax, Bcl-2 and p53. Phytochemical analysis of BBR extract revealed the presence of precursors and metabolic end products of cyanogenic glycosides. Dose dependent decrease in expression of Tg and PDS with concomitant decrease in gene expression of these with MCT8 were observed. Increased ROS, DNA oxidation and associated imbalance were found through increased Bax and p53 with decreased Bcl-2 that perturbed thyrocytes cell cycle. Cyanogenic constituents of BBR generates ROS associated oxidative changes in thyrocytes with DNA damage and oxidation and cell cycle disruption followed by inhibition of thyroid hormone synthesizing regulatory elements; addition of extra iodine showed partial prevention.

Regulation of renal pendrin activity by aldosterone.

PURPOSE OF REVIEW: Pendrin resides on the luminal membrane of type B intercalated cells in the renal collecting tubule system mediating the absorption of chloride in exchange for bicarbonate. In mice or humans lacking pendrin, blood pressure is lower, and pendrin knockout mice are resistant to aldosterone-induced hypertension. Here we discuss recent findings on the regulation of pendrin. RECENT FINDINGS: Pendrin activity is stimulated during alkalosis partly mediated by secretin. Also, angiotensin II and aldosterone stimulate pendrin activity requiring the mineralocorticoid receptor in intercalated cells. Angiotensin II induces dephosphorylation of the mineralocorticoid receptor rendering the receptor susceptible for aldosterone binding. In the absence of the mineralocorticoid receptor in intercalated cells, angiotensin II does not stimulate pendrin. The effect of aldosterone on pendrin expression is in part mediated by the development of hypokalemic alkalosis and blunted by K-supplements or amiloride. Part of the blood pressure-increasing effect of pendrin is also mediated by its stimulatory effect on the epithelial Na-channel in neighbouring principal cells. SUMMARY: These findings identify pendrin as a critical regulator of renal salt handling and blood pressure along with acid--base balance. A regulatory network of hormones fine-tuning activity is emerging. Drugs blocking pendrin are being developed.

Two Mineralocorticoid Receptor-Mediated Mechanisms of Pendrin Activation in Distal Nephrons.

BACKGROUND: Regulation of sodium chloride transport in the aldosterone-sensitive distal nephron is essential for fluid homeostasis and BP control. The chloride-bicarbonate exchanger pendrin in ß-intercalated cells, along with sodium chloride cotransporter (NCC) in distal convoluted tubules, complementarily regulate sodium chloride handling, which is controlled by the renin-angiotensin-aldosterone system. METHODS: Using mice with mineralocorticoid receptor deletion in intercalated cells, we examined the mechanism and roles of pendrin upregulation via mineralocorticoid receptor in two different models of renin-angiotensin-aldosterone system activation. We also used aldosterone-treated NCC knockout mice to examine the role of pendrin regulation in salt-sensitive hypertension. RESULTS: Deletion of mineralocorticoid receptor in intercalated cells suppressed the increase in renal pendrin expression induced by either exogenous angiotensin II infusion or endogenous angiotensin II upregulation via salt restriction. When fed a low-salt diet, intercalated cell-specific mineralocorticoid receptor knockout mice with suppression of pendrin upregulation showed BP reduction that was attenuated by compensatory activation of NCC. In contrast, upregulation of pendrin induced by aldosterone excess combined with a high-salt diet was scarcely affected by deletion of mineralocorticoid receptor in intercalated cells, but depended instead on hypokalemic alkalosis through the activated mineralocorticoid receptor-epithelial sodium channel cascade in principal cells. In aldosterone-treated NCC knockout mice showing upregulation of pendrin, potassium supplementation corrected alkalosis and inhibited the pendrin upregulation, thereby lowering BP. CONCLUSIONS: In conjunction with NCC, the two pathways of pendrin upregulation, induced by angiotensin II through mineralocorticoid receptor activation in intercalated cells and by alkalosis through mineralocorticoid receptor activation in principal cells, play important roles in fluid homeostasis during salt depletion and salt-sensitive hypertension mediated by aldosterone excess.

Clinical Features, Molecular Genetics, and Long-Term Outcome in Congenital Chloride Diarrhea: A Nationwide Study in Japan.

OBJECTIVE: To clarify clinical and genetic features of Japanese children with congenital chloride diarrhea (CCD). STUDY DESIGN: This was a multi-institutional, retrospective survey of 616 pediatric centers in Japan with identified patients with CCD between 2014 and 2018. Mutations involving SLC26A3 were detected by Sanger sequencing. RESULTS: Thirteen patients met all entry criteria including mutations in SLC26A3, and 14 patients satisfied clinical diagnostic criteria. Homozygous or compound heterozygous mutations in SLC26A3, including 6 novel mutations, were identified in 13 of these 14 patients (93%). The most common (detected in 7 of 13) was c.2063-1g>t. Median age at diagnosis was 1 day. Nine of the patients meeting all criteria were diagnosed as neonates (69%). Median follow-up duration was 10 years. When studied, 8 patients had <5 stools daily (62%), and all had fewer than in infancy. Only 1 patient had nephrocalcinosis, and 3 (23%) had mild chronic kidney disease. Neurodevelopment was generally good; only 1 patient required special education. Five patients (38%) received long-term sodium, potassium, and chloride supplementation. CONCLUSIONS: Early fetal ultrasound diagnosis and prompt long-term sodium, potassium, and chloride supplementation were common management features. Genetic analysis of SLC26A3 provided definitive diagnosis of CCD. In contrast with previously reported localities, c.2063-1g>t might be a founder mutation in East Asia.

SULTR3s Function in Chloroplast Sulfate Uptake and Affect ABA Biosynthesis and the Stress Response.

Plants are major sulfur reducers in the global sulfur cycle. Sulfate, the major natural sulfur source in soil, is absorbed by plant roots and transported into plastids, where it is reduced and assimilated into Cys for further metabolic processes. Despite its importance, how sulfate is transported into plastids is poorly understood. We previously demonstrated using single Arabidopsis (Arabidopsis thaliana) genetic mutants that each member of the sulfate transporter (SULTR) subfamily 3 was able to transport sulfate across the chloroplast envelope membrane. To resolve the function of SULTR3s, we constructed a sultr3 quintuple mutant completely knocking out all five members of the subfamily. Here we report that all members of the SULTR3 subfamily show chloroplast membrane localization. Sulfate uptake by chloroplasts of the quintuple mutant is reduced by more than 50% compared with the wild type. Consequently, Cys and abscisic acid (ABA) content are reduced to ∼67 and ∼20% of the wild-type level, respectively, and strong positive correlations are found among sulfate, Cys, and ABA content. The sultr3 quintuple mutant shows obvious growth retardation with smaller rosettes and shorter roots. Seed germination of the sultr3 quintuple mutant is hypersensitive to exogenous ABA and salt stress, but is rescued by sulfide supplementation. Furthermore, sulfate-induced stomatal closure is abolished in the quintuple mutant, strongly suggesting that chloroplast sulfate is required for stomatal closure. Our genetic analyses unequivocally demonstrate that sulfate transporter subfamily 3 is responsible for more than half of the chloroplast sulfate uptake and influences downstream sulfate assimilation and ABA biosynthesis.

SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation.

SLC26A3 (downregulated in adenoma; DRA) is a Cl-/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl- exchange with HCO3-, I-, and thiocyanate (SCN-), with an IC50 of ~0.2 µM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.