Cellular and mitochondrial taurine depletion in bile duct ligated rats: a justification for taurine supplementation in cholestasis/cirrhosis.

Taurine (TAU) is a free amino acid abundant in the human body. Various physiological roles have been attributed to TAU. At the subcellular level, mitochondria are the primary targets for TAU function. Meanwhile, it has been found that TAU depletion is associated with severe pathologies. Cholestasis is a severe clinical complication that can progress to liver fibrosis, cirrhosis, and hepatic failure. Bile duct ligation (BDL) is a reliable model for assessing cholestasis/cirrhosis and related complications. The current study was designed to investigate the effects of cholestasis/cirrhosis on tissue and mitochondrial TAU reservoirs. Cholestatic rats were monitored (14 and 42 days after BDL surgery), and TAU levels were assessed in various tissues and isolated mitochondria. There was a significant decrease in TAU in the brain, heart, liver, kidney, skeletal muscle, intestine, lung, testis, and ovary of the BDL animals (14 and 42 days after surgery). Mitochondrial levels of TAU were also significantly depleted in BDL animals. Tissue and mitochondrial TAU levels in cirrhotic animals (42 days after the BDL operation) were substantially lower than those in the cholestatic rats (14 days after BDL surgery). These data indicate an essential role for tissue and mitochondrial TAU in preventing organ injury induced by cholestasis/cirrhosis and could justify TAU supplementation for therapeutic purposes.

Calcium alleviates fluoride-induced kidney damage via FAS/FASL, TNFR/TNF, DR5/TRAIL pathways in rats.

Long-term excessive intake of fluoride (F) can cause osseous and non-osseous damage. The kidney is the main fluoride excretion organ of the body. This study aimed to explore whether dietary calcium (Ca) supplementation can alleviate kidney damage caused by fluorosis and to further investigate the effects of Ca on the mitigation mechanism of renal cell apoptosis triggered by F. We evaluated the histopathological structure, renal function indicators, and gene and protein expression levels of death receptor-mediated apoptosis pathways in Sprague Dawley (SD) rats treated with sodium fluoride (NaF) and/or calcium carbonate (CaCO3) for 120 days. The results showed that 100 mg/L NaF induced kidney histopathological injury and apoptosis, increased the concentrations of Creatinine (CRE), uric acid (UA), blood urea nitrogen (BUN), potassium (K), phosphorus (P) and F (p < 0.05), and decrease the level of serum magnesium (Mg) (p < 0.05). Moreover, NaF increased the mRNA and protein expression levels of Fas cell surface death receptor (FAS), tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL), Caspase 8, Caspase 3 and poly ADP-ribose polymerase (PARP) (p < 0.01), which finally activated the death receptor pathway. Inversely, Ca supplementation reversed the decrease of CRE, BUN, UA, F and P levels induced by F, alleviated histopathological damage and apoptosis, and reduced the gene and protein expression levels of death receptor pathway-related markers. In conclusion, 1% Ca alleviates F-induced kidney apoptosis through FAS/FASL, TNFR/TNF, DR5/TRAIL signaling pathways.

Effects of Different Doses of Calcium on the Mitochondrial Apoptotic Pathway and Rho/ROCK Signaling Pathway in the Bone of Fluorosis Rats.

For this study, we investigate more deeply the effect calcium (Ca) develops on the mechanism underlying fluoride-triggered osteocyte apoptosis. We detected the morphology of osteocytes by HE staining, mitochondrial microstructure by using the transmission electron microscope, and the biochemical indexes related to bone metabolism and the expression of apoptosis-related genes. These results showed that NaF brought out the reduced osteocytes and ruptured mitochondrial outer membrane, with a significantly increased StrACP activity by 10.414 IU/L at the 4th week (P < 0.05), markedly upregulating the mRNA expression of Bax, Cyto-C, Apaf-1, caspase-7, ROCK-1, BMP-2, and BGP (P < 0.01), as well as caspase-6 (P < 0.05), while downregulating Bcl-2 by 61.3% (P < 0.01). Through immunohistochemical analysis, we also found that NaF notably increased the protein expression of ROCK-1 (P < 0.05) and Cyto-C, BMP-2, and BGP (P < 0.01), suggesting that NaF triggered the activation of the mitochondrial apoptotic pathway and Rho/ROCK signaling pathway. Nevertheless, 1% Ca supplementation in diet notably enhanced the mRNA expression of Bcl-2 by 39.3% (P < 0.01), thus blocking the increment of the expression of mitochondrial apoptotic pathway-related genes and ROCK-1. Meanwhile, Ca could attenuate the StrACP activity by 10.741 IU/L at the 4th week (P < 0.05) and protect the integrity of the mitochondrial outer membrane. These findings strongly suggest that 1% Ca abated the mitochondrial apoptosis pathway by increasing the anti-apoptotic gene Bcl-2 expression, and effectively inhibited the hyper-activation of ROCK-1, dually protecting the structural integrity of the mitochondrial outer membrane and maintaining normal cellular metabolic function.

Calcium relieves fluoride-induced bone damage through the PI3K/AKT pathway.

Bone is the main target of fluorosis, and it has been perfectly elaborated that a moderate dosage of calcium (Ca) can alleviate bone fluorosis. However, whether Ca can alleviate fluorosis through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling pathway has not yet been reported. Hence, we evaluated the histopathological structure, the imbalance of the biochemical index of bone metabolism, and the expression levels of PI3K/AKT apoptosis signaling pathway-related genes in rats treated with sodium fluoride (NaF, F) and/or calcium carbonate (CaCO3) for 120 days. Our results suggest that 100 mg L-1 NaF induced histopathological injury as alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (StrACP) activity increased, with a decrease in the serum Ca levels (p < 0.05). Moreover, the results of qRT-PCR and western blotting showed that F increased the expression levels of transglutaminase 2 (TGM2), focal adhesion kinase (FAK), PI3K, AKT, forkhead box O1 (Foxo1), Bcl-2 interacting mediator of cell death (BIM), Bcl2-associated x protein (Bax) and Caspase 3 (p < 0.05, p < 0.01). It also decreased the expression of AnnexinA5 (Anxa5), 3'-phosphoinositide-dependent kinase 1 (PDK1) and B-cell lymphoma-2 (Bcl-2) (p < 0.05, p < 0.01), which finally activated the PI3K/AKT pathway. On the other hand, CaCO3 supplementation reversed the histopathological injury along with the levels of ALP, StrACP and serum Ca, alleviating the gene expression levels of PI3K/AKT pathway-related markers. Altogether, we can conclude that CaCO3 supplementation mitigated F-induced bone damage via the PI3K/AKT signaling pathway.

Calcium Alleviates Fluoride-Induced Bone Damage by Inhibiting Endoplasmic Reticulum Stress and Mitochondrial Dysfunction.

Excessive fluoride mainly causes skeletal lesions. Recently, it has been reported that an appropriate level of calcium can alleviate fluorosis. However, the appropriate concentration and mechanism of calcium addition is unclear. Hence, we evaluated the histopathology and ultrastructure, DNA fragmentation, hormonal imbalances, biomechanical levels, and expression of apoptosis-related genes after treating the rats with 150 mg/L NaF and different concentrations of CaCO3. Our results suggested that NaF induced the histopathological and ultrastructural injury, with a concomitant increase in the DNA fragmentation (P < 0.05) and serum OC (17.5 ± 0.89 pmoL/L) at 120 days. In addition, the qRT-PCR and western blotting results indicated that NaF exposure upregulated the mRNA and protein expression of Bax, Calpain, Caspase 12, Caspase 9, Caspase 7, Caspase 3, CAD, PARP, and AIF while downregulated Bcl-2 (P < 0.01) and decreased the bone ultimate load by 27.1%, the ultimate stress by 10.1%, and the ultimate deformity by 23.3% at 120 days. However, 1% CaCO3 supplementation decreased the serum OC (14.7 ± 0.65 pmoL/L), bone F content (P < 0.01), and fracture and breakage of collagen fibers and changed the expression of endoplasmic reticulum pathway-related genes and proteins at 120 days. Further, 1% CaCO3 supplementation increased the bone ultimate load by 20.9%, the ultimate stress by 4.89%, and the ultimate deformity by 21.6%. In summary, we conclude that 1% CaCO3 supplementation alleviated fluoride-induced bone damage by inhibiting endoplasmic reticulum stress and mitochondrial dysfunction.

Influence of Calcium Supplementation against Fluoride-Mediated Osteoblast Impairment in Vitro: Involvement of the Canonical Wnt/ß-Catenin Signaling Pathway.

Fluoride (F) is capable of promoting abnormal proliferation and differentiation in primary cultured mouse osteoblasts (OB cells), although the underlying mechanism responsible remains rare. This study aimed to explore the roles of wingless and INT-1 (Wnt) signaling pathways and screen appropriate doses of calcium (Ca2+) to alleviate the sodium fluoride (NaF)-induced OB cell toxicity. For this, we evaluated the effect of dickkopf-related protein 1 (DKK1) and Ca2+ on mRNA levels of wingless/integrated 3a (Wnt3a), low-density lipoprotein receptor-related protein 5 (LRP5), dishevelled 1 (Dv1), glycogen synthase kinase 3ß (GSK3ß), ß-catenin, lymphoid enhancer binding factor 1 (LEF1), and cellular myelocytomatosis oncogene (cMYC), as well as Ccnd1 (Cyclin D1) in OB cells challenged with 10-6 mol/L NaF for 24 h. The demonstrated data showed that F significantly increased the OB cell proliferation rate. Ectogenic 0.5 mg/L DKK1 significantly inhibited the proliferation of OB cells induced by F. The mRNA expression levels of Wnt3a, LRP5, Dv1, LEF1, ß-catenin, cMYC, and Ccnd1 were significantly increased in the F group, while significantly decreased in the 10-6 mol/L NaF + 0.5 mg/L DKK1 (FY) group. The mRNA expression levels of Wnt3a, LRP5, ß-catenin, and cMYC were significantly decreased in the 10-6 mol/L NaF + 2 mmol/L CaCl2 (F+CaII) group. The protein expression levels of Wnt3a, Cyclin D1, cMYC, and ß-catenin were significantly increased in the F group, whereas they were decreased in the F+CaII group. However, the mRNA and protein expression levels of GSK3ß were significantly decreased in the F group while significantly increased in the F+CaII group. In summary, F activated the canonical Wnt/ß-catenin pathway and changed the related gene expression and ß-catenin protein location in OB cells, promoting cell proliferation. Ca2+ supplementation (2 mmol/L) reversed the expression levels of genes and proteins related to the canonical Wnt/ß-catenin pathway.

Effects of different Ca2+ level on fluoride-induced apoptosis pathway of endoplasmic reticulum in the rabbit osteoblast in vitro.

In reviewing the literature, the cellular mechanism of fluoride F-induced osteoblast OB cells apoptosis is diverse and perplexing, but detailed regulatory pathway, targets and role of extracellular Ca2+ remains still unclear. Hence, in the present study, we investigated the effects of F (9 mg/L F ion) and different Ca2+ (0.5, 1, 2, 4, 8 mmol/L) levels treatment on the proliferation rate of osteoblast cells, intracellular free Ca2+ ([Ca2+]i) and endoplasmic reticulum (ER) stress apoptosis pathway related gene levels of rabbit OB cells. Our results demonstrated that F exposure had a pronounced negative effect on osteoblast survival, further different Ca2+ levels treatment suggested that low concentration of Ca2+ (0.5-1 mmol/L) relieved the damaged effect, on the contrary, high concentration of Ca2+ (2-8 mmol/L) enhanced the effect. In addition, F significantly increased [Ca2+]i levels and the expression of ER stress-induced cell apoptosis pathway related genes. Treatment with 0.5-1 mmol/L Ca2+ markedly reversed the F-induced harmful effects, but high dose Ca2+ (2-8 mmol/L) enhanced these effects. In summary, 0.5-1 mmol/L Ca2+ can alleviate F-induced OB cells injure through ER stress apoptosis pathway, which provided a dose basis for the future study on the treatment of skeletal fluorosis with Ca2+.