ObjectiveTo investigate the etiology and clinical features of intrahepatic cholestasis and the diagnostic value of whole exome sequencing （WES） through a retrospective analysis of the medical history， pathological results， and gene sequencing data of 62 patients with unexplained intrahepatic cholestasis. MethodsA retrospective analysis was performed for the clinical data of 480 patients who underwent WES due to unexplained liver function abnormalities in Nanjing Second Hospital from January 2017 to December 2023， among whom 62 patients with unexplained intrahepatic cholestasis were selected based on laboratory data， and a confirmed diagnosis was made based on imaging data， pathological findings， and gene sequencing data. The patients with unexplained intrahepatic cholestasis were analyzed in terms of demographic features， clinical manifestation， etiology spectrum， and genetic profile. ResultsA total of 62 patients with unexplained intrahepatic cholestasis were included， among whom there were 35 male patients and 27 female patients， with a median age of 42 （7 — ‍77） years. WES was used to make a definite diagnosis in 21 patients （33.87%）， among whom the patients with familial intrahepatic cholestasis accounted for the highest proportion of 52.38% （11/21）； genetic metabolic disorders were excluded by WES in 34 patients， with drug-induced liver injury and sepsis-associated liver injury accounting for the highest proportion of 55.88% （19/34）， followed by primary biliary cholangitis and primary sclerosing cholangitis accounting for 20.59% （7/34） and intrahepatic bile duct stones accounting for 17.65% （6/34）， while the patients with a lack of confirmed diagnosis accounted for 11.29% （7/62）. A total of 21 novel mutation sites which were not reported in previous articles were identified in this study. ConclusionGenetic metabolic disorders constitute a significant proportion of unexplained intrahepatic cholestasis， and WES plays a crucial role in the diagnosis of unexplained intrahepatic cholestasis.

Objective:To analyze the distribution characteristics of UGT1A1 mutant genes (including enhancers, promoters, and exons 1-5) and further explore the correlation between UGT1A1 genotype and clinical phenotypes in patients with inherited hyperunconjugated bilirubinemia.Methods:Patients diagnosed with hereditary hyperunconjugated bilirubinemia at Nanjing Second Hospital from June 2015 to December 2022 were retrospectively analyzed. The UGT1A1 gene was examined using Sanger sequencing in all patients. Complete blood count, liver function, and abdominal imaging examinations were performed. Comparison of categorical variable data using χ2 testor Fisher percision tests. Comparison of continaous veriable data with normal distribution using t-test. Results:112 cases (male:female ratio 81:31, aged 9-70 years) had inherited hyperunconjugated bilirubinemia, with a total of 14 mutation sites identified, of which seven were confirmed mutations, and the frequency ranged from high to low: (TA)n accounted for 50%, c.211G>A (p.G71R) accounted for 49.10%, 1456T>G (p.Y486D) accounted for 16.96%, c.686C>A (p.R229W) accounted for 12.5%, 1091C>T (p.P364L) accounted for 8.04%, and c- 3279T>G accounted for 0.982%. Simultaneously, all patients had one to four mutations, of which only one mutation was the most common (55.36%), followed by two mutations (37.5%), and rare three and four mutations (5.36% and 1.78%). There was no statistical significance in total bilirubin (TBil) levels among the four groups ( F=0.652, P=0.583). One mutation was most common in (TA)n and c.211G>A (p.G71R), among which TA6/TA7 ( n=10) and TA7/TA7 ( n=14) mutations were statistically significant in TBil ( t=2.143, P=0.043). The c.211G>A (p.G71R) heterozygous ( n=9) and isolated ( n=15) mutation had no statistical significance in TBil ( t=0.382, P=0.706). The GS group accounted for 75%, the intermediate group accounted for 16.9%, and the CNS-Ⅱ group accounted for 8%. TBil was statistically significant among the three groups ( F=270.992, P<0.001). There was no statistically significant difference ( χ2=3.317, P=0.19) between mutation 1 (44 cases, 14 cases, and 4 cases, respectively) and mutations ≥ 2 (40 cases, 5 cases, and 5 cases, respectively) in the GS group, intermediate group, and CNS-II group. Conclusion:The number of UGT1A1 gene mutation sites may have no synergistic effect on TBil levels in patients with inherited hyperunconjugated bilirubinemia. TA7/TA7 mutations are not uncommon, and TBil levels are relatively high.

Objective:To explore the independent risk factors for bleeding in patients following percutaneous liver biopsy examination.Methods:The clinicopathological data of patients who underwent percutaneous liver biopsy examination at Nanjing Second Hospital from January 2012 to December 2021 were retrospectively collected. Univariate and multivariate logistic regression analysis were used to investigate the effect of age, gender, lesion type (diffuse liver parenchymal lesions, focal liver lesions), number of biopsies, tissue length, presence or absence of cirrhosis, presence or absence of portosystemic shunt, erythrocytes, white blood cells, hemoglobin, platelets, prothrombin time, fibrinogen, international normalized ratio, and liver biochemical indicators on bleeding following liver biopsy, as well as to screen independent risk factors.Results:A total of 3 331 patients were examined by percutaneous liver biopsy, and 3 060 cases were actually included by excluding 271 cases who took consultation from other hospitals. The overall postoperative hemorrhagic rate was 1.6% (49/3 060). Of which, forty-four cases (1.4%) had overt bleeding (hemodynamic changes or hemoglobin decreased by more than 20 g/L), five cases (0.2%) had minor bleeding, three cases had subcapsular hepatic hemaotma, and two cases had local bleeding from liver biopsy. Among the overt bleeding cases, two cases were in the off-label group (platelet<50×10 9/L or international normalized ratio>1.5), and the rest were in the non-off-label group. The results of univariate analysis showed that factors such as focal liver lesions, portosystemic shunt, prolonged prothrombin time, increased international normalized ratio, bilirubin, and alkaline phosphatase were associated with bleeding after liver biopsy in the non-off-label group. The multivariate collinearity diagnosis revealed statistically significant differences for the indicators. Multivariate logistic regression analysis finally included factors such as lesion type, portosystemic shunt, international normalized ratio, total bilirubin, and alkaline phosphatase. The results showed that patients with focal liver lesions were more prone to bleed after surgery than patients with diffuse liver parenchymal lesions ( OR=3.396, P=0.002, 95% CI: 1.596-7.228). Patients with portosystemic shunt were more prone to bleed than those without portosystemic shunt ( OR=3.301, P=0.018, 95% CI: 1.232-8.845). Patients were more likely to experience bleeding following liver biopsy when their total bilirubin levels were elevated ( OR=1.006, P<0.001, 95% CI:1.003-1.008). Conclusion:Focal liver lesions, portosystemic shunts, and elevated total bilirubin are independent risk factors for bleeding after percutaneous liver biopsy.

Biochemical liver function tests are important methods to determine liver function in clinical practice， but abnormal liver biochemical parameters are not completely equivalent to liver damage. Some genetic and immune factors can also cause abnormal liver biochemical parameters， but with good prognosis in most cases. This article summarizes the causes of some benign abnormal liver biochemical parameters， so as to help clinicians to broaden their thinking of diagnosis and treatment， take into account genetic and immune factors， and avoid misdiagnosis and mistreatment.

Objective To investigate the role of PPARγ/GluT4 axis in insulin resistance(IR),cell proliferation and apoptosis of granulosa cells.Methods A total of 45 married women with PCOS who received routine IVF-ET assisted pregnancy treatment in Center of Reproductive Medicine,Qinghai Provincial People's Hospital were enrolled in this study from August 2018 to August 2020.All the patients were divided into IR group(PCOS-IR group,HOMA-IR≥2.57,n=23)and non-IR group(PCOS-NIR group,HOMA-IR<2.57,n=22)according to HOMA-IR.Meanwhile,21 married patients with infertility due to male or fallopian tube factors were enrolled as control group(Con).miR-27a mimics,miR-27a inhibitors(miR-27a inhibitor)and corresponding controls(mimics NC and inhibitor NC)transfected PCOS-IR granulosa cells,which were then divided into miR-27a mimics group,miR-27a inhibitor group,mimics-NC group and inhibitor-NC group.Double luciferase report test confirmed that miR-27a binded to PPARγ.Cell proliferation and apoptosis were detected by CCK-8 method and Annexinv-FITC/PI method.The expression of miR-27a,GluT4,PPARγ,Bax related to B lymphomas-2,Cleaved caspase-3 and B lymphomas-2(Bcl-2)were detected by RT-qPCR and Western blot respectively.Results Compared with Con group,the expression of miR-27a increased(P<0.01),while the expression of PPARγ mRNA and protein decreased in PCOS-NIR and PCOS-IR groups(P<0.01).Compared with PCOS-NIR group,the expression of miR-27a increased(P<0.05),while the expression of PPARγ mRNA and protein decreased in PCOS-IR group(P<0.01).The double luciferase report showed that there was a targeted binding site between PPARγ and miR-27a.Compared with inhibitor-NC group,the cell activity increased at 24 h,48 h,72 h and 96 h in miR-27a inhibitor group(P<0.05 or P<0.01),while the apoptosis rate decreased inmiR-27a inhibitor and mimics-NC group(P<0.05 or P<0.01).Compared with miR-27a inhibitor group,the apoptosis rate increased,and the cell activity decreased at 24,48,72 and 96 h in mimics-NC and miR-27a mimics groups(P<0.05 or P<0.01).Compared with the inhibitor-NC group,the expression of miR-27a,Bax and Cleaved caspase-3 increased(P<0.05 or P<0.01),while the expression of GluT4,PPARγ and Bcl-2 decreased in miR-27a mimics group(P<0.01).In miR-27a inhibitor group,the protein expressions of GluT4,PPARγ and Bcl-2 increased(P<0.05 or P<0.01),while miR-27a,Bax and Cleaved caspase-3 decreased(P<0.01).Compared with miR-27a inhibitor group,the expressions of miR-27a,Bax and Cleaved caspase-3 increased(P<0.01),while the expressions of GluT4,PPARγ and Bcl-2 decreased in mimics-NC and miR-27a mimics groups(P<0.05 or P<0.01).Conclusion The expression level of miRNA-27a is related to IR,cell proliferation,and apoptosis of granulosa cells,which may be related to PPARγ signal path.

Hepatic granuloma is a kind of disease caused by both infection or non-infection etiologies, and it is found in approximately 2% to 15% of liver biopsies. Some of which could be identified by the pathological morphology. This article reviews the common etiology, pathological manifestations, diagnosis, and differential diagnosis of hepatic granuloma, which is hopeful to improve clinicians' and pathologists' awareness.

Objective To investigate the clinical and pathological features of progressive familial intrahepatic cholestasis type 3 (PFIC3). Methods A retrospective analysis was performed for 1326 patients with unexplained liver disease who attended Nanjing Second Hospital from January 2017 to December 2019, among whom 8 patients were diagnosed with PFIC3 based on clinical/pathological manifestation and gene sequencing results (1 patient did not undergo liver biopsy due to contraindication). Clinical, laboratory, imaging, and pathological findings were analyzed and a literature review was performed for the pathology of ABCB4-related diseases to summarize the clinical and pathological features of PFIC-3. Results Among the 8 patients with PFIC3, there were 5 male patients and 3 female patients, with a median age of 29.5 years. Of all 8 patients, 4 (50%) manifested as chronic cholestasis and 4 (50%) manifested as biliary cirrhosis, among whom 3 (75%) had the manifestation of portal hypertension. As for biochemical examination, 75% (6/8) had an increase in alkaline phosphatase, and 100% (8/8) had an increase in gamma-glutamyl transpeptidase. As for imaging examination, 50% (4/8) had cholecystitis, 25% (2/8) had gallstones, 25% (2/8) had bile duct dilatation, 75% (6/8) had splenomegaly, and 25% (2/8) had liver cirrhosis. As for liver biopsy, all 7 patients manifested as bile duct injury and/or reduction, and 57.1% (5/7) had absence of the bile duct. Multidrug resistance P-glycoprotein 3 (MDR3) immunohistochemical staining showed normal expression in 42.9% (3/7) of the patients and reduced expression in 57.1% (4/7) of the patients. Literature review obtained 17 articles with a description of the bile duct or MDR3 immunohistochemistry. Among the 7 patients with low phospholipid-associated cholelithiasis, 71.4% (5/7) had normal bile duct, 14.3% (1/7) had bile duct reduction, and 14.3% (1/7) had absence of the bile duct; among the 6 patients with intrahepatic cholestasis of pregnancy, 16.7% (1/6) had normal bile duct, 50% (3/6) had bile duct reduction, and 33.3% (2/6) had absence of the bile duct; among the 8 patients with PFIC3, 25% (2/8) had bile duct reduction and 75% (6/8) had absence of bile duct; among the 21 patients with PFIC3, 9.5% (2/21) had normal expression of MDR3, 23.8% (5/21) had a reduction in the expression of MDR3, and 66.7% (14/21) had absence of the expression of MDR3. Conclusion PFIC3 mainly manifests as cholestasis, cholelithiasis, and hepatic fibrosis. Pathological manifestation includes bile duct injury and bile duct reduction or absence of the bile duct in severe cases, and the degree of injury is associated with disease severity. MDR3 immunohistochemistry may show normal expression, reduced expression, or absence of expression, and diagnosis cannot be excluded in patients with normal expression. Genetic testing can be performed for diagnosis when necessary.

OBJECTIVE To establish the fingerprint of Tibetan medicine Adhatoda vasica ，and determine the contents of vasicine and vasicinone ，so as to comprehensively evaluate its quality combined with chemical pattern recognition. METHODS Using vasicine as control ，HPLC fingerprints of 11 batches of A. vasica were established with Similarity Evaluation System for Chromatographic Fingerprints of TCM （2012 edition）. The common peaks were identified and their similarities were evaluated. Cluster analysis （CA），principal component analysis （PCA）and orthogonal partial least squares-discriminant analysis （OPLS-DA） were performed by using SPSS 25 software and SIMCA 14.1 software. The variable importance in the projection （VIP）value＞1.0 was used as the standard to screen the differential components affecting the quality of A. vasica ；the contents of vasicine and vasicinone were determined by HPLC simultaneously. RESULTS A total of 23 common peaks were found ，and peak 2 was identified as vasicine ，and peak 4 was identified as vasicinone. Their similarities ranged 0.920-0.994. The results of CA showed that 11 batches of samples were clustered into 3 categories（distance was 14）：S1-S8 as one category （origin：Yunnan，Tibet），S9 as one category （origin：Yunnan），S10-S11 as one category （origin：Sichuan）；the results of P CA and OPLS-DA showed that S 9 and S10-S11 were divided into one category respectively ，and S1-S8 were further divided into 2 categories：S1，S4 as one category，S2-S3，S5-S8 as one category ；the common peaks with VIP value ＞1.0 included peak 2，peak 16，peak 21，peak 17，peak 1 and peak 13. Among 11 batches of samples ， contents of vasicine and vasicinone were 4.12-10.22 and 0.60-3.26 mg/g， respectively. CONCLUSIONS Established edu.cn HPLC fi ngerprint and content determination method are simple and accurate ，and can be used for the quality evaluation of Tibetan medicine A. vasica ，by combining with chemical pattern recognition. Vasicine and other components may be the differential components that affect the quality of the drug.

Objective: To understand the etiology of hepatopathy of unknown etiology in patients undergoing liver biopsy. Methods: Demographic data and pathological examination reports of patients with hepatopathy of unknown etiology who underwent liver biopsy examination at outpatient and inpatient of the Second Hospital of Nanjing between January 2017 and June 2018 were retrospectively collected. All liver histopathological sections combined with clinical and pathological features based on liver biopsy examinations were diagnosed by a reputed clinician and a pathologist. Results: A total of 470 cases with hepatopathy of unknown etiology who underwent liver biopsy were enrolled. Of these, 425 cases (90.4%) had a definite diagnosed disease after comprehensive analysis of pathological and clinical data. The diagnosis of hepatopathy of unknown etiology included 11 diseases: 90 cases with autoimmune hepatitis had autoimmune liver disease (19.1%), 38 cases had primary biliary cholangitis (8.1%), 43 cases with overlap syndrome of autoimmune hepatitis had primary biliary cholangitis (9.1%), 118 cases had drug-induced liver injury (25.1%), 75 cases had nonalcoholic fatty liver disease (NAFLD) (16.0%), 12 cases had alcoholic liver disease (2.6 cases) %), 15 cases (3.2%) had vascular liver disease, 7 cases (1.5%) had hereditary metabolic liver disease, 5 cases (1.1%) had other systemic diseases, 16 cases (3.4%) had more than two kinds of liver diseases, and 6 cases (1.3%) had others rare liver diseases. Conclusion Over 90% cause of the hepatopathy of unknown etiology in the long run can be determined, and the main causes are autoimmune liver disease, drug-induced liver injury, and nonalcoholic fatty liver disease, which needs multidisciplinary cooperation to diagnose, and clinicians need to master the basic and clinical knowledge of liver diseases as well as liver pathology, hepatobiliary imaging, and genetics.