Protective Effect Of Vitamin D₃ On Intestinal Failure Associated Liver Disease:Clinical Observation And Experimental Study

With the successful implementation of parenteral nutrition (PN) on canine in 1968, PN has been widely used in clinical practice. Numerous intestinal failure (IF) or intestinal dysfunction patients could be long-term survivals with the support of PN. It has become an important treatment to save lives. However, only 3 years later, there were some reports about intestinal failure associated liver disease (IFALD). Studies have confirmed that IFALD develops in 40～60% of infants who require long term total parenteral nutrition (TPN) for IF and 15～40% of adults on home parenteral nutrition (HPN). The three key types of the hepatobiliary disorders associated with IFALD were steatosis, cholestasis, and formation of gallbladder stones. Even in the 21st century, the incidence of IFALD was still maintained between 70～80% in long-term PN support patients and the mortality for three years of IFALD patients was about 20%. Therefore, the high incidence and mortality of IFALD was always the difficult problem in PN field. In addition, it hindered the development of nutrition support and nutrition therapy.Vitamin D belongs to a group of steroids with a broken ring known as secosteroids. The most important forms of vitamin D are vitamin D3 (cholecalciferol), produced in the human skin; and vitamin D2 (ergocalciferol), derived from plants. The most active metabolite of vitamin D3. 1α,25-dihydroxyvitamin D3(1.25(OH)2D3. calcitriol), is synthesised in a highly regulated multi-step process. When human skin is exposed to sunlight, UVB irradiation causes the photolysis of provitamin D3 (7-dehydrocholesterol) to pre-vitamin D3, a thermodynamically unstable isomer that is rapidly converted into vitamin D3. This passes into the bloodstream and is transported to the liver bound to the vitamin D-binding protein (DBP) and, to a lesser extent, to albumin. Alternatively, vitamin D3 can be absorbed in the intestine from dietary sources. Vitamin D3 is first hydroxylated in the liver by several mitochondrial and microsomal vitamin D3-25-hydroxylases. The resultant 25-hydroxyvitamin D3 (25-OHD3, calcidiol) is the main circulating form of vitamin D3, and it is further hydroxylated in the kidney by mitochondrial 25-OHD3-1a-hydroxylase, yielding the hormonally active form 1,25(OH)2D3.The most widely and classically accepted physiological role for vitamin D, mediated mostly by 1,25(OH)2D3, is the regulation of calcium and phosphate homeostasis and bone mineralisation. In the past decades, evidence has emerged that the importance of 1,25(OH)2D3 for health extends far beyond bone. The discovery in 1981 that 1,25(OH)2D3 induces the differentiation of myeloid leukaemia cells and inhibits the growth of melanoma cells triggered the interest of cancer researchers. Since then, numerous observations have indicated a much broader range of autocrine and/or paracrine activities for 1,25(OH)2D3, including the regulation of cell differentiation, proliferation, maturation, apoptosis, invasion and angiogenesis. What’s more, studies have confirmed that 1,25(OH)2D3 could induce the activation of farnesoid X receptor (FXR), which is the most important bile acid sensor.In this study, we explored the prevalence of suboptimal vitamin D status in patients with short bowel syndrome, and investigate the protective effect of vitamin D3 on IFALD patients. Then, we stablished stable IFALD rat models and studied the protective effect and mechanism of vitamin D3 on bile acid metabolism in rat model with IFALD.Part ⅠPrevalence of suboptimal vitamin D status in patients with short bowel syndromeObjective:The purpose of this study was to determine vitamin D status in patients with short bowel syndrome and investigate its correlation with bone mineral density.Methods:We performed a 2-year retrospective review of patients with short bowel syndrome who were admitted to our hospital between January 2014 and January 2016. Serum 25-OHD and bone mineral density were determined in all subjects. Patients were then categorized as deficient (< 20 ng/ml), insufficient (20-30 ng/ml), or normal (≧30 ng/ml) based on their lowest vitamin D level. Meanwhile, patients were categorized as normal bone mineral density (-1< T level≦+1), osteopenia (-2.5< T level≦-1), and osteoporosis (T level≦-2.5) based on their bone mineral density test.Results:Suboptimal vitamin D status were identified in all individuals, including 3 (5.0%) with vitamin D insufficiency and 57 (95.0%) with vitamin D deficiency. Residual small bowel length (B,0.072,P=0.001) and duration of short bowel syndrome (B,-0.066,P=0.020) were both significantly correlated with suboptimal vitamin D levels. Overall, only two (3.4%) patients presented a normal bone mineral density; osteopenia and osteoporosis was noted in 41 (68.3%) and 17 (28.3%) individuals, respectively. Low 25-OHD concentration was associated with a decreased bone mineral density (B,0.065, P=0,029). There were no other demographic characteristics or clinical examinations associated with suboptimal vitamin D levels and bone loss.Conclusions:Suboptimal VtD status and bone loss are common in patients with short bowel syndrome. This emphasizes the critical importance of routine surveillance of serum vitamin D levels and consideration of enteral or parenteral supplementation when indicated.Part IIProtective effect of vitamin D3 on intestinal failure associated liver disease:clinical observationObjective:The aim of this study was to investigate the protective effect of vitamin D3 on intestinal failure associated liver disease.Methods:From July 2014 to July 2015, we recruited 11 patients with short bowel syndrome who were treated in our center and met the inclusion criteria. We assigned them into two groups randomly:control group and experimental group. In the experimental group, patients were given an oral vitamin D3 supplementation. The dose and duration of vitamin D3 supplementation was 800 IU/time three times daily for one month. Blood samples were collected each week for analysis of liver function.Results:Serum bilirubin and liver enzyme were highly increased in control group than that in experimental group (P<0.05). After four weeks, there were significant differences as to serum bilirubin and liver enzyme between control group and experimental group, the intestinal failure associated liver disease relevant indicators of experimental group were significantly better than that of control group.Conclusions:Supplementation of vitamin D3 was effective in the protection of intestinal failure associated liver disease in patients with short bowel syndrome.Part IIIProtective effect and mechanism of vitamin D3 on bile acid metabolism in rat model with intestinal failure associated liver diseaseStudy 3.1 Protective effect of vitamin D3 on bile acid metabolism in rat model with intestinal failure associated liver diseaseObjective:The purpose of this study was to explore the effect of vitamin D3 to intestinal failure associated liver disease in a short bowel syndrome rat model.Methods:Twelve adult male rats with a central catheter placed in the right jugular vein were assigned into two groups:control group (N=6) and experimental group (N=6). All rats underwent 75% intestinal resection and accepted total parenteral nutrition support for 14 days. The experimental group received intraperitoneal injection of vitamin D3 at a dose of 4μg/kg body weight once a day for 14 days. Blood samples were drawn at preoperative day,7 day and 14 day postoperatively for analysis of serum alanine transaminase (ALT), aspartate transaminase (AST), direct bilirubin (DBIL), and total bilirubin (TBIL). After blood collection at 14 day postoperatively, we detected bile flow and obtained the liver tissue specimens for pathological examination.Results:The serum values of liver function were increased as time went on. The serum ALT (55.23±6.41 U/L vs.64.01±7.23 U/L, P< 0.05), AST (177.22±26.42 U/L vs.209.00±31.24 U/L, P< 0.05), TBIL (3.23±0.21μmol/L vs.3.65±0.23μmol/L, P< 0.05), DBIL (0.99±0.03 μmol/L vs.1.15±0.06μmol/L, P< 0.05) of experimental group were significantly lower than those of control group at day 14 postoperatively. In addition, the bile flow of experimental group (8.23±1.26 μml/min) was significantly higher than that of control group (5.85±1.41μml/min)(P<0.05). The pathological score of experimental group (1.23±0.26) were significantly lower than those of control group (1.85±0.41) (P< 0.05).Conclusions:Vitamin D3 could lead to amelioration in serum ALT, AST, TBIL and DBIL in short bowel syndrome patients depending on parenteral nutrition. In addition, vitamin D3 could reduce liver damage of intestinal failure associated liver disease. This might be considered as an adjunctive therapy to protect liver function for short bowel syndrome patients depending on parenteral nutrition. However, more work is needed to explore the specific mechanism.Study 3.2 Mechanism of vitamin D3 regulating bile acid metabolism in rat model with intestinal failure associated liver diseaseObjective:The purpose of this study was to explore the mechanism of vitamin D3 regulating bile acid metabolism in rat model with intestinal failure associated liver disease.Methods:Twelve adult male rats with a central catheter placed in the right jugular vein were assigned into two groups:experimental group (N=6) and control group (N=6). All rats underwent 75% intestinal resection and accepted total parenteral nutrition support for 14 days. The experimental group received intraperitoneal injection of vitamin D3 at a dose of 4μg/kg body weight once a day for 14 days. At 14 day postoperatively, we obtained the liver tissue specimens for analysis by western-blot and immunohistochemistry.Results:Compared with control group, results of western-blot showed that the protein expressions of liver VDR, FXR, BSEP, OSTa, OSTP in experimental group were significantly higher (P< 0.05), which was also confirmed by immunohistochemistry analysis. The results of western-blot also showed that the protein expressions of liver CYP7A1 and NTCP in experimental group were significantly lower than that of control group (P<0.05). In addition, the protein expressions of intestine OSTα and OSTβ in experimental group were significantly higher than that of control group (P< 0.05), while the protein expression of intestine ASBT was significantly lower than that of control group (P< 0.05).Conclusions:Vitamin D3 could release the suppression of protein expressions of BSEP, OSTα, OSTβ in liver and OSTα, OSTβ in small intestine, it could also inhibit the protein expressions of CYP7A1 and NTCP in liver and ASBT in small intestine. Therefore, vitamin D3 could regulate the metabolism of bile acid and protect the liver function of intestinal failure associated liver disease.