Astaxanthin Attenuates Nonalcoholic Steatohepatitis with Downregulation of Osteoprotegerin in Ovariectomized Mice Fed Choline-Deficient High-Fat Diet.

BACKGROUND: Postmenopausal estrogen decline increases the risk of developing nonalcoholic steatohepatitis (NASH), and it might accelerate progression to cirrhosis and hepatocellular carcinoma. AIMS: This study aimed to investigate a novel therapy for postmenopausal women who are diagnosed with NASH. METHODS: Seven-week-old female C57BL/6 J mice were divided into three experimental groups as follows: (1) sham operation (SHAM group), (2) ovariectomy (OVX group), and (3) ovariectomy + 0.02% astaxanthin (OVX + ASTX group). These three groups of mice were fed a choline-deficient high-fat (CDHF) diet for 8 weeks. Blood serum and liver tissues were collected to examine liver injury, histological changes, and hepatic genes associated with NASH. An in vitro study was performed with the hepatic stellate cell line LX-2. RESULTS: The administration of ASTX significantly improved pathological NASH with suppressed steatosis, inflammation, and fibrosis, in comparison with those in the OVX-induced estrogen deficiency group. As a result, liver injury was also attenuated with reduced levels of alanine aminotransferase and aspartate transaminase. In addition, our study found that ASTX supplementation decreased hepatic osteoprotegerin (OPG) in vivo, a possible factor that contributes to NASH development. In vitro, this study further confirmed that ASTX has an inhibitory effect on the secretion of OPG in LX-2 human hepatic stellate cells. CONCLUSIONS: Our findings suggest that ASTX alleviates CDHF-OVX-induced pathohistological NASH with downregulated OPG, possibly via suppression of the transforming growth factor beta pathway. ASTX could has promise for use in postmenopausal women diagnosed with NASH.

Resveratrol Modulates Bone Mineral Density and Bone Mineral Content in A Rat Model of Male Hypogonadism.

OBJECTIVE: To determine whether resveratrol (Res) can correct osteoporosis induced in a rat model of male hypogonadism. METHODS: Thirty-two rats were randomly divided into 4 groups, 8 in each group; 1) a control sham group: underwent a similar surgical procedure for induction of orchiectomy (ORCD) without ligation of any arteries or veins or removal of the testis and epididymis; 2) a control + Res-treated group (Con+Res): underwent sham surgery similar to the control, but was then treated with Res, as described below; 3) an ORCD-induced group: bilateral ORCD surgery as described above, and 4) a ORCD+Res-treated group: bilateral ORCD surgery followed by Res treatment. Res treatment began 4 weeks after ORCD and continued for 12 weeks. After 12 weeks, bone mineral density (BMD) and bone mineral content (BMC) were measured in the tibia and femur of each rat's right hind leg. Blood levels of bone turnover indicators such as deoxypyridinoline (Dpd), N-telopeptide of type I collagen (NTX I), alkaline phosphatase (ALP), and osteocalcin (OC), as well as receptor activator of nuclear factor kappa B (RANK) and osteoprotegerin (OPG) were assessed. RESULTS: ORCD significantly decreased BMD (P<0.01) and significantly increased bone resorption, manifested by increased RANK. In addition, it inhibited serum levels of OPG and OC. Res treatment after ORCD effectively increased serum levels of bone formation markers such as OPG and OC, compared with testisectomized rats (P<0.05). CONCLUSION: Res could ameliorate bone loss induced by male hypogonadism, possible via restoration of the normal balance between RANK and OPG.

Isoorientin ameliorates osteoporosis and oxidative stress in postmenopausal rats.

CONTEXT: Isoorientin has many biological activities, including antioxidant, anti-inflammatory, antitumor. However, the effect of isoorientin on postmenopausal osteoporosis remains unclear. OBJECTIVE: To evaluate the effect of isoorientin on postmenopausal osteoporosis. MATERIALS AND METHODS: Sprague-Dawley rats were divided into five groups (n = 5): sham, model, 17-ß-oestradiol (E2, 10 µg/kg/day), low-dose isoorientin (L-Iso, 50 mg/kg), and high-dose isoorientin (H-Iso, 100 mg/kg). The rats were ovariectomized, treated by gavage daily for 12 weeks, and serum and femur samples were collected. Bone mineral density, bone metabolism, and oxidative stress were assessed. H&E staining, immunohistochemistry, and western blotting were employed. RESULTS: Isoorientin improved the bone mineral density of the lumbar vertebrae (2.01 ± 0.05 g/cm3 in H-Iso group vs. 1.74 ± 0.07 g/cm3 in model group) and femur (1.46 ± 0.06 g/cm3 vs. 1.19 ± 0.03 g/cm3), increased the trabecular bone number (1.97 ± 0.03 vs. 1.18 ± 0.13) and thickness (0.27 ± 0.02 vs. 0.16 ± 0.03 mm). Isoorientin decreased the separation degree of trabecular bone, ameliorated bone histomorphology changes, and significantly improved the mechanical properties. Isoorientin diminished MDA (by 60%) and increased SOD (by 49.2%), and GSH-Px (by 159%) activity. Furthermore, osteoprotegerin (OPG), nuclear factor erythroid 2-like 2 (Nrf2), haem oxygenase (HO-1), NAD(P)H quinone dehydrogenase 1(NQO1), and oestrogen receptor 1(ESR1) protein expression increased, while receptor activator of nuclear factor-κB ligand (RANKL) protein expression decreased after treatment. CONCLUSIONS: Isoorientin ameliorates osteoporosis via upregulating OPG and Nrf2/ARE signalling, suggesting isoorientin maybe a potential therapeutic drug for PMOP.

Effects of osteoprotegerin/TNFRSF11B in two models of abdominal aortic aneurysms.

Osteoprotegerin (OPG), additionally termed tumor necrosis factor receptor superfamily member 11B, is produced by vascular smooth muscle cells (VSMCs) and endothelial cells in the vasculature, and its release may be modulated by pro­inflammatory cytokines, including interleukin­1ß and tumor necrosis factor­α. The present study investigated the effects of treatment with low­dose human recombinant OPG on abdominal aortic aneurysm (AAA) development in mice. Mice were treated with 1 µg human recombinant OPG four times (or vehicle) for 2 weeks prior to inducing AAA. A total of two different models for inducing AAA were used to investigate the hypothesis as to whether OPG is involved in key events of AAA development, using osmotic mini­pumps with angiotensin II in apolipoprotein­E (ApoE­/­) mice for 28 days or using periaortic application of CaCl2 on the aorta in C57Bl/6J mice for 14 days. OPG was continuously administered during the experimental period. Histological staining using Masson's trichrome, Verhoeff's van­Gieson and picro­sirius red, in addition to reverse transcription­quantitative polymerase chain reaction analysis of various markers, were used to analyze phenotypic alterations. Treatment with OPG had no inhibitory effect on AAA development in the angiotensin II model in ApoE­/­ mice, which developed suprarenal aneurysms, although it increased vessel wall thickness of the aorta and total collagen in C57Bl/6J mice using the CaCl2 model that induced infrarenal dilation of the aorta. Treatment with OPG did not inhibit aneurysm development and key events, including inflammation, extracellular matrix or VSMC remodeling, in aortas from OPG­treated mice with periaortic treatment with CaCl2. The results indicated that mice treated with low levels of human recombinant OPG may have a more stable aneurysmal phenotype due to compensatory production of collagen and increased vessel wall thickness of the aorta, potentially protecting the aneurysm from rupture. Further studies investigating rupture models of AAA in addition to using higher levels of OPG are require to verify this speculation. Furthermore, treatment with low levels of OPG in patients with AAA may represent a novel therapeutic strategy for the treatment of AAA as well as attenuate the adverse effects associated with the administration of normal and high dosages of OPG.

RANK-Independent Osteoclast Formation and Bone Erosion in Inflammatory Arthritis.

OBJECTIVE: Proinflammatory molecules promote osteoclast-mediated bone erosion by up-regulating local RANKL production. However, recent evidence suggests that combinations of cytokines, such as tumor necrosis factor (TNF) plus interleukin-6 (IL-6), induce RANKL-independent osteoclastogenesis. The purpose of this study was to better understand TNF/IL-6-induced osteoclast formation and to determine whether RANK is absolutely required for osteoclastogenesis and bone erosion in murine inflammatory arthritis. METHODS: Myeloid precursors from wild-type (WT) mice or mice with either germline or conditional deletion of Rank, Nfatc1, Dap12, or Fcrg were treated with either RANKL or TNF plus IL-6. Osteoprotegerin, anti-IL-6 receptor (anti-IL-6R), and hydroxyurea were used to block RANKL, the IL-6R, and cell proliferation, respectively. Clinical scoring, histologic assessment, micro-computed tomography, and quantitative polymerase chain reaction (qPCR) were used to evaluate K/BxN serum-transfer arthritis in WT and RANK-deleted mice. Loss of Rank was verified by qPCR and by osteoclast cultures. RESULTS: TNF/IL-6 generated osteoclasts in vitro that resorbed mineralized tissue through a pathway dependent on IL-6R, NFATc1, DNAX-activation protein 12, and cell proliferation, but independent of RANKL or RANK. Bone erosion and osteoclast formation were reduced, but not absent, in arthritic mice with inducible deficiency of RANK. TNF/IL-6, but not RANKL, induced osteoclast formation in bone marrow and synovial cultures from animals deficient in Rank. Multiple IL-6 family members (IL-6, leukemia inhibitory factor, oncostatin M) were up-regulated in the synovium of arthritic mice. CONCLUSION: The persistence of bone erosion and synovial osteoclasts in Rank-deficient mice, and the ability of TNF/IL-6 to induce osteoclastogenesis, suggest that more than one cytokine pathway exists to generate these bone-resorbing cells in inflamed joints.

Recombinant osteoprotegerin effects during orthodontic movement in a rat model.

BACKGROUND AND OBJECTIVES: Anchorage is one of the most challenging sides in orthodontics. The use of biological modulators that inhibit osteoclasts could be a solution to address these problems and provide new adjunctive approaches. The aim of this study was to assess the effectiveness of recombinant osteoprotegerin fusion protein (OPG-Fc) in orthodontic anchorage. MATERIALS AND METHODS: Two groups of male Sprague-Dawley rats were utilized. The animals in the experimental group received twice-weekly injections with high dose of OPG-Fc (5.0mg/kg) in mesial and distal mucosa of the first molars, and those in the control group received no drugs. Right first maxillary molars were mesialized using a calibrated nickel-titanium spring connected to an anterior mini-screw. Tooth movement was measured by two blinded observers using scanned and magnified stone casts. Receptor activator of nuclear factor κB (RANK), run-related transcription factor 2 (Runx2), type I collagen, vimentin, matrix metalloproteinases 2 and 9, S100 protein and the putative mechanoproteins acid-sensing ion channel (ASIC2) and transient receptor potential vainilloid 4 (TRPV4) were evaluated using immunohistochemistry. RESULTS: OPG-Fc group showed an important decreased in mesial molar movement with only 52%, 31%, and 22% of the total mesial molar movement compared with control group at Days 7, 14, and 21, respectively (P < 0.001). RANK ligand and Runx2 positive cells were severely reduced after OPG-Fc treatment. Periodontal ligament architecture, cell arrangement, and immunohistochemical patter for vimentin, type I collagen and the mechanoproteins TRPV4 and ASIC2 were altered by tooth movement and all these parameters altered by the applied treatment. CONCLUSIONS: OPG-Fc effectively inhibits osteoclastogenesis resulting in improved bone quantity and orthodontic anchorage. Based on present results, OPG-Fc could have clinical utility in preventing undesired tooth movements.

The inhibitory effects of a RANKL-binding peptide on articular and periarticular bone loss in a murine model of collagen-induced arthritis: a bone histomorphometric study.

INTRODUCTION: We designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis. METHODS: Twenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed. RESULTS: The OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites. CONCLUSION: The peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.

Muscadine grape skin extract can antagonize Snail-cathepsin L-mediated invasion, migration and osteoclastogenesis in prostate and breast cancer cells.

To develop new and effective chemopreventive agents against bone metastasis, we assessed the effects of muscadine grape skin extract (MSKE), whose main bioactive component is anthocyanin, on bone turnover, using prostate and breast cancer cell models overexpressing Snail transcription factor. MSKE has been shown previously to promote apoptosis in prostate cancer cells without affecting normal prostate epithelial cells. Snail is overexpressed in prostate and breast cancer, and is associated with increased invasion, migration and bone turnover/osteoclastogenesis. Cathepsin L (CatL) is a cysteine cathepsin protease that is overexpressed in cancer and involved in bone turnover. Snail overexpression in prostate (LNCaP, ARCaP-E) and breast (MCF-7) cancer cells led to increased CatL expression/activity and phosphorylated STAT-3 (pSTAT-3), compared to Neo vector controls, while the reverse was observed in C4-2 (the aggressive subline of LNCaP) cells with Snail knockdown. Moreover, CatL expression was higher in prostate and breast tumor tissue compared to normal tissue. MSKE decreased Snail and pSTAT3 expression, and abrogated Snail-mediated CatL activity, migration and invasion. Additionally, Snail overexpression promoted osteoclastogenesis, which was significantly inhibited by the MSKE as effectively as Z-FY-CHO, a CatL-specific inhibitor, or osteoprotegerin, a receptor activator of nuclear factor kappa B ligand (RANKL) antagonist. Overall, these novel findings suggest that Snail regulation of CatL may occur via STAT-3 signaling and can be antagonized by MSKE, leading to decreased cell invasion, migration and bone turnover. Therefore, inhibition using a natural product such as MSKE could potentially be a promising bioactive compound for bone metastatic cancer.

Osteoprotegerin enhances osteogenesis of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and hold promise for applications of bone regeneration such as bone tissue engineering. However, current approaches for in vitro osteogenesis cannot effectively induce osteogenesis and need to be modified to produce quality bone for clinical applications. Previous studies have shown that the conditioned medium (CM) from osteoblast culture enhances osteogenesis of MSCs, and soluble osteogenic factors in the CM may be involved in the regulation. However, these factors are not fully identified. In this study, we profiled soluble factors secreted from MG-63 cells using a comparative protein array and found that osteoprotegerin (OPG), known as a potent anti-osteoclastogenic protein, was at the highest relative level among 507 soluble molecules detected by the array. Furthermore, treating hMSCs with OPG before osteogenic induction significantly increased the expression of osteocalcin mRNA transcript and the production of calcium deposits compared to the untreated control cells, suggesting that OPG is capable of priming undifferentiated hMSCs for the enhancement of subsequent osteogenesis. Furthermore, we showed that the nuclear factor-kappaB (NF-κB) was activated by OPG in undifferentiated hMSCs and that blocking NF-κB activation before osteogenic induction decreased osteogenesis of OPG-pretreated cells upon receiving osteogenic stimuli. Taken together, our results suggest that OPG is a pro-osteogenic factor that can be used as an osteogenic supplement in a growth medium to prime the osteogenic capacity of undifferentiated hMSCs to enhance osteogenesis for bone tissue engineering.

OPG-Fc treatment in growing pigs leads to rapid reductions in bone resorption markers, serum calcium, and bone formation markers.

Inhibition of the receptor activator of NF-κB ligand (RANKL) is a novel therapeutic option in the treatment of osteoporosis and related diseases. The aim of this study was to evaluate bone metabolism and structure in pigs after RANKL inhibition. 12 growing pigs were assigned to 2 groups with 6 animals each. The OPG group received recombinant human OPG-Fc (5 mg/kg IV) at day 0, the control group was given 0.9% NaCl solution. Serum levels of OPG-Fc, calcium (Ca), phosphorus (P), and bone turnover markers were evaluated every 5 days, and pigs were euthanized on day 20. Serum OPG-Fc concentration peaked at day 5 and coincided with significantly decreased Ca, P, and bone turnover markers. By day 15, measureable OPG-Fc serum levels could only be detected in 2/6 animals. With OPG-Fc clearance starting at day 10, serum Ca and P concentrations were not different between the 2 groups. TRACP5b, P1CP, and BAP levels significantly decreased by 40-70% relative to vehicle controls in the OPG-Fc group between days 5 and 10, indicating that pharmacologic concentration of OPG-Fc led to systemic concomitant inhibition of bone formation and resorption in young growing pigs. Dual X-ray absorptiometry data derived from the proximal femur did not differ between the 2 groups. µCT analysis of selected bone sites demonstrated an OPG-Fc-induced improvement of specific bone architectural indices and bone mineralization.

Effects of RANKL, osteoprotegerin, calcium and phosphorus on survival and activation of Muscovy duck osteoclasts in vitro.

The aim of this study was to determine whether receptor activator of nuclear factor NF-kappaB ligand (RANKL), osteoprotegerin (OPG) and a calcium:phosphorus (Ca:P) ratio of 2:1 could affect survival and activation of Muscovy duck osteoclasts (OCs). Bone marrow cells were obtained from 5-day-old Muscovy ducks and cultured with (Group A) No added factors, (B) 30ng/mL soluble RANKL (sRANKL), (C) 30ng/mL sRANKL and 10ng/mL OPG, (D) 10ng/mL OPG, (E) 50ng/mL OPG, (F) 100ng/mL OPG and (G) 30ng/mL sRANKL, 6mmol/L Ca and 3mmol/L P. sRANKL promoted the survival of OCs on day 2, whereas the number of OCs decreased with addition of OPG in a dose-dependent manner. OPG and Ca:P (2:1) both inhibited OC survival induced by RANKL. RANKL stimulated bone resorption by OCs, whereas OPG, but not Ca:P (2:1), inhibited the activity of OCs induced by RANKL. RANKL promotes the survival and activation of OCs from Muscovy ducks, whereas OPG and, to a lesser extent, Ca:P (2:1) reduce the life span and inhibited the activation of OCs induced by RANKL.

Bone resorption increases tumour growth in a mouse model of osteosclerotic breast cancer metastasis.

Osteosclerotic metastases account for 20% of breast cancer metastases with the remainder osteolytic or mixed. In mouse models, osteolytic metastases are dependent on bone resorption for their growth. However, whether the growth of osteosclerotic bone metastases depends on osteoclast or osteoblast actions is uncertain. In this study, we investigate the effects of high and low bone resorption on tumour growth in a mouse model of osteosclerotic metastasis. We implanted human breast cancer, MCF-7, cells into the tibiae of mice. Low and high levels of bone resorption were induced by osteoprotegerin (OPG) treatment or calcium deficient diet respectively. We demonstrate that OPG treatment significantly reduces tumour area compared to vehicle (0.42 +/- 0.06 vs. 1.27 +/- 0.16 mm2, P < 0.01) in association with complete inhibition of osteoclast differentiation. In contrast, low calcium diet increases tumour area compared to normal diet (0.90 +/- 0.30 vs. 0.58 +/- 0.20 mm2, P < 0.05) in association with increased osteoclast numbers (84.44 +/- 5.18 vs. 71.11 +/- 3.56 per mm2 bone lesion area, P < 0.05). Osteoblast surfaces and new woven bone formation were similarly increased within the tumour boundaries in all treatment groups. Tumour growth in this model of osteosclerotic metastasis is dependent on ongoing bone resorption, as has been observed in osteolytic models. Bone resorption, rather than bone formation, apparently mediates this effect as osteoblast surfaces in the tumour mass were unchanged by treatments. Treatment of breast cancer patients through correction of calcium deficiency and/or with anti-resorptive agents such as OPG, may improve patient outcomes in the adjuvant as well as palliative settings.

Accelerated bone resorption, due to dietary calcium deficiency, promotes breast cancer tumor growth in bone.

The skeleton is a major site of breast cancer metastases. High bone turnover increases risk of disease progression and death. However, there is no direct evidence that high bone turnover is causally associated with the establishment and progression of metastases. In this study, we investigate the effects of high bone turnover in a model of breast cancer growth in bone. Female nude mice commenced a diet containing normal (0.6%; 'Normal-Ca') or low (0.1%; 'Low-Ca') calcium content. Mice were concurrently treated with vehicle or osteoprotegerin (1 mg/kg/d s.c; n = 16 per group). Three days later (day 0), 50,000 Tx-SA cells (variant of MDA-MB-231 cells) were implanted by intratibial injection. On day 0, mice receiving Low-Ca had increased serum parathyroid hormone (PTH) and tartrate-resistant acid phosphatase 5b levels, indicating secondary hyperparathyroidism and high bone turnover, which was maintained until day 17. Osteoprotegerin increased serum PTH but profoundly reduced bone resorption. On day 17, in mice receiving Low-Ca alone, lytic lesion area, tumor area, and cancer cell proliferation increased by 43%, 24%, and 24%, respectively, compared with mice receiving Normal Ca (P < 0.01). Osteoprotegerin treatment completely inhibited lytic lesions, reduced tumor area, decreased cancer cell proliferation, and increased cancer cell apoptosis. Increased bone turnover, due to dietary calcium deficiency, promotes tumor growth in bone, independent of the action of PTH. Breast cancer patients frequently have low dietary calcium intake and high bone turnover. Treatment to correct calcium insufficiency and/or treatment with antiresorptive agents, such as osteoprotegerin, may be of benefit in the adjuvant as well as palliative setting.

Osteoclasts play a part in pain due to the inflammation adjacent to bone.

Bone disorders with increased osteoclastic bone resorption are frequently associated with bone pain and inhibitors of osteoclasts reduce bone pain. Osteoclasts degrade bone minerals by secreting protons through the vacuolar H+-ATPase, creating acidic microenvironments. Because acidosis is a well-known cause of pain, we reasoned that osteoclasts cause pain through proton secretion. We explored this using an animal model in which a single subcutaneous injection of the complete Freund's adjuvant (CFA) in the hind-paw caused inflammatory hyperalgesia (hyper-responsiveness to noxious stimuli). Osteoclastic bone resorption was increased in the metatarsal bones in the CFA-injected hind-paws. CFA-induced hyperalgesia was significantly suppressed by the bisphosphonates, zoledronic acid (ZOL) and alendronate and osteoprotegerin. c-src-deficient mice in which osteoclasts are inherently dysfunctional exhibited reduced CFA-induced hyperalgesia. Repeated subcutaneous injections of parathyroid hormone-related protein into the hind-paw also induced hyperalgesia with increased osteoclastic bone resorption. The hyperalgesia was associated with increased mRNA expression of acid-sensing ion channel (ASIC) 1a, 1b and 3 in the ipsi-lateral dorsal root ganglions (DRGs) by RT-PCR and c-Fos in the ipsi-lateral spinal dorsal horn by immunohistochemistry. Of note, ZOL decreased the ASIC1a mRNA expression and c-Fos. Treatment of the DRG cell line F-11 with acid (pH5.5) increased ASIC1a, 1b and 3 mRNA expression and nuclear c-Fos expression. The ASIC blocker amiloride inhibited acid-induced c-Fos expression in F-11 cells. Moreover, F-11 cells transfected with the transient receptor potential channel vanilloid subfamily member 1 (TRPV1) showed increased acid-induced nuclear c-Fos expression compared with parental F-11 cells. Finally, bafilomycin A1, an inhibitor of the vacuolar H+-ATPase, reversed the hyperalgesia and down-regulated ASIC1a mRNA expression in the DRGs. These results led us to propose that osteoclasts play a part in CFA-induced inflammatory pain through an activation of the acid-sensing receptors including ASICs and TRPV1 by creating acidosis.