化痰祛湿活血方对代谢相关脂肪性肝炎大鼠解整合素金属蛋白酶17/髓系细胞触发受体2介导的巨噬细胞胞葬的影响
DOI: 10.12449/JCH260214
Effect of Huatan Qushi Huoxue prescription on macrophage efferocytosis mediated by a disintegrin and metalloproteinase 17 and triggering receptor expressed on myeloid cells 2 in rats with metabolic dysfunction-associated steatohepatitis
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摘要:
目的 观察化痰祛湿活血方对代谢相关脂肪性肝炎(MASH)大鼠的治疗效果及作用机制。 方法 选取60只SPF级SD大鼠随机分为空白对照组、模型A组(单纯性脂肪肝模型)、模型B组(MASH模型)、西药组(多烯磷脂酰胆碱,143.64 mg/kg)、中药高剂量组(化痰祛湿活血方,20.16 g/kg)和中药中剂量组(化痰祛湿活血方,10.08 g/kg),每组10只。除空白对照组外,其余各组均给予高脂饲料。模型A组于第8周取材,其余各组于第12周开始每日给药1次,连续8周,模型B组灌胃等体积的生理盐水,于第20周取材。检测大鼠体重、肝湿重、肝指数;采用微板法检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)和游离脂肪酸(FFA);酶联免疫吸附法(ELISA)检测血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、IL-6、可溶性髓系细胞触发受体2(sTREM2);苏木精-伊红染色、油红O染色观察肝组织病理变化;免疫荧光法检测肝组织CD68+TREM2+细胞并计算巨噬细胞胞葬率;实时荧光定量聚合酶链反应检测肝组织中鞘氨醇-1-磷酸(S1P)、鞘氨醇1磷酸酯受体1(S1PR1)、解整合素金属蛋白酶17(ADAM17)和髓系细胞触发受体2(TREM2)的mRNA表达水平;免疫组织化学法检测肝组织中S1P、S1PR1、ADAM17和TREM2蛋白表达。符合正态分布的计量资料,方差齐者采用单因素方差分析进行组间比较,进一步两两比较采用LSD-t检验;方差不齐者采用Welch’s检验进行组间比较,进一步两两比较则采用Tamhane’s检验。不符合正态分布的计量资料组间比较采用Kruskal-Wallis H检验,进一步两两比较采用Dunn’s检验。 结果 与空白对照组比较,模型A组和模型B组的大鼠体重、肝湿重显著升高;模型B组的肝指数显著升高(P值均<0.05)。HE染色结果显示,模型A组大鼠肝组织呈现弥漫性大泡性脂肪变性,模型B组大鼠肝组织显示大量气球样变性肝细胞,小叶内及汇管区可见混合性炎症细胞浸润、轻度窦周纤维化。与空白对照组比较,模型A组、模型B组大鼠NAS、油红O阳性面积显著升高(P值均<0.05);且模型B组大鼠NAS、油红O阳性面积较模型A组显著升高(P值均<0.05)。与空白对照组比较,模型A组、模型B组的血清TC、TG、LDL-C、FFA、IL-1β、IL-6、sTREM2水平均显著升高,血清HDL-C水平显著降低,模型B组的血清ALT、AST和TNF-α水平显著升高(P值均<0.05);且模型B组的血清ALT、AST、TC、TG、FFA、TNF-α、IL-1β、IL-6和sTREM2水平较模型A组显著升高,血清HDL-C水平较模型A组显著降低(P值均<0.05)。免疫荧光法结果显示,与空白对照组比较,模型A组巨噬细胞胞葬率显著升高(P<0.05);模型B组巨噬细胞胞葬率显著低于模型A组(P<0.05)。实时荧光定量聚合酶链反应结果显示,与空白对照组比较,模型A组、模型B组TREM2的mRNA水平显著升高(P值均<0.05),模型B组S1P、S1PR1的mRNA水平显著升高(P值均<0.05);且模型B组S1PR1、TREM2的mRNA水平较模型A组升高(P值均<0.05)。免疫组织化学法结果显示,与空白对照组比较,模型A组和模型B组S1P、S1PR1、ADAM17蛋白表达水平显著升高,模型A组TREM2蛋白表达水平显著升高(P值均<0.05);且模型B组S1P、S1PR1和ADAM17蛋白表达水平较模型A组升高,TREM2蛋白表达水平较模型A组显著降低(P值均<0.05)。与模型B组比较,各用药组大鼠体重、肝湿重和肝指数显著降低(P值均<0.05);各用药组肝组织脂肪变及炎症损伤改善明显,NAS及油红O阳性面积显著降低(P值均<0.05);血清ALT、AST、TC、TG、FFA、IL-1β、IL-6水平显著降低(P值均<0.05),血清HDL-C水平显著升高(P<0.05),中药高剂量组血清TNF-α水平显著降低(P<0.05);各用药组巨噬细胞胞葬率显著升高(P值均<0.05);中药高剂量组、中药中剂量组ADAM17蛋白表达水平显著降低,中药高剂量组TREM2蛋白表达水平显著升高(P值均<0.05)。 结论 化痰祛湿活血方改善MASH大鼠肝脂质代谢及炎症水平可能与调控肝脏巨噬细胞胞葬有关。 Abstract:Objective To investigate the therapeutic effect and mechanism of Huatan Qushi Huoxue prescription on rats with metabolic dysfunction-associated steatohepatitis (MASH). Methods A total of 60 specific pathogen-free Sprague-Dawley rats were randomly divided into blank control group, model A group, model B group, Western medicine group (polyene phosphatidylcholine, 143.64 mg/kg), high-dose Chinese medicine group (Huatan Qushi Huoxue prescription, 20.16 g/kg), and middle-dose Chinese medicine group (Huatan Qushi Huoxue prescription, 10.08 g/kg). All rats except those in the blank control group were given high-fat diet. Samples were collected from the model A group at week 8, and since week 12, the other groups were given the corresponding drug once a day for 8 consecutive weeks, with samples collected at week 20. Body weight, liver wet weight, and liver index were measured for all rats; the microplate method was used to measure the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and free fatty acids (FFA); ELISA was used to measure the serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and soluble triggering receptor expressed on myeloid cells 2 (sTREM2); HE staining and oil red O staining were performed to observe liver histopathological changes; immunofluorescence assay was used to measure CD68+TREM2+ cells in liver tissue and calculate the phagocytosis rate of macrophages; quantitative real-time PCR was used to measure the mRNA expression levels of sphingosine 1-phosphate (S1P), sphingosine 1-phosphate receptor 1 (S1PR1), a disintegrin and metalloproteinase 17 (ADAM17), and triggering receptor expressed on myeloid cells 2 (TREM2) in liver tissue, and immunohistochemistry was used to measure the protein expression levels of S1P, S1PR1, ADAM17, and TREM2 in liver tissue. A one-way analysis of variance was used for comparison of normally distributed continuous data with homogeneity of variance between groups, and the least significant difference t-test was used for further comparison between two groups; the Welch’s test was used for comparison of normally distributed continuous data with heterogeneity of variance between groups, and the Tamhane’s test was used for further comparison between two groups. The Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between groups, and the Dunn’s test was used for further comparison between two groups. Results Compared with the blank control group, the model A group and the model B group had significant increases in body weight and liver wet weight, and the model B group had a significant increase in liver index (all P<0.05). HE staining showed diffuse macrovesicular steatosis of liver tissue in the model A group and a large number of hepatocytes with ballooning degeneration in liver tissue in the model group B, with the presence of mixed inflammatory cell infiltration and mild perisinusoidal fibrosis in the lobules and the portal area. Compared with the blank control group, the model A group and the model B group had significant increases in NAS score and oil red O-positive area (all P<0.05), and the model B group had significant increases in these two indicators than the model A group (both P<0.05). Compared with the blank control group, the model A group and the model B group had significant increases in the serum levels of TC, TG, LDL-C, FFA, IL-1β, IL-6, and sTREM2 and a significant reduction in the serum level of HDL-C, and the model B group had significant increases in the serum levels of ALT, AST, and TNF-α (all P<0.05); compared with the model A group, the model B group had significant increases in the serum levels of ALT, AST, TC, TG, FFA, TNF-α, IL-1β, IL-6, and sTREM2 and a significant reduction in the serum level of HDL-C (all P<0.05). Immunofluorescence assay showed that compared with the blank control group, the model A group had a significant increase in the phagocytosis rate of macrophages (P<0.05), while the model B group had a significantly lower phagocytosis rate of macrophages than the model A group (P<0.05). Quantitative real-time PCR showed that compared with the blank control group, the model A group and the model B group had a significant increase in the mRNA expression level of TREM2, and the model B group had significant increases in the mRNA expression levels of S1P and S1PR1 (both P<0.05); moreover, compared with the model A group, the model B group had significant increases in the mRNA expression levels of S1PR1 and TREM2 (both P<0.05). Immunohistochemistry showed that compared with the blank control group, the model A group and the model B group had significant increases in the protein expression levels of S1P, S1PR1, and ADAM17, and the model A group had a significant increase in the protein expression level of TREM2 (all P<0.05); compared with the model A group, the model B group had significant increases in the protein expression levels of S1P, S1PR1, and ADAM17 and a significant reduction in the protein expression level of TREM2 (all P<0.05). Compared with the model B group, each medication group had significant reductions in body weight, liver wet weight, and liver index (all P<0.05); each medication group had significant improvements in hepatic steatosis and inflammatory damage, with significant reductions in NAS score and oil red O-positive area (all P<0.05); each medication group had significant reductions in the serum levels of ALT, AST, TC, TG, FFA, IL-1β, and IL-6 (all P<0.05) and a significant increase in the serum level of HDL-C (P<0.05), and the high-dose Chinese medicine group had a significant reduction in the serum level of TNF-α (P<0.05); each medication group had a significant increase in the phagocytosis rate of macrophages (all P<0.05); the high- and middle-dose Chinese medicine groups had a significant reduction in the protein expression level of ADAM17, and the high-dose Chinese medicine group had a significant increase in the protein expression level of TREM2 (all P<0.05). Conclusion Huatan Qushi Huoxue prescription improves lipid metabolism and inflammation in the liver of MASH rats by regulating hepatic macrophage phagocytosis. -
表 1 引物序列
Table 1. Primer information
基因 引物序列(5'-3') 碱基数 GAPDH 上游
下游CTGGAGAAACCTGCCAAGTATG
GGTGGAAGAATGGGAGTTGCT22 21 S1P 上游
下游TCATCACGTCCCCTGAAAAGAG
CAAAAACAGCAACCCTGACATTAG22 24 S1PR1 上游
下游GCTGAACATCGGAGTGGAGAAG
GAGCCACAAACATACTTCCTTCC22 23 TREM2 上游
下游CCAAGCCCTCAACACCACA
ACCGTGCTCCCATTCTGCTT19 20 ADAM17 上游
下游AAGGGATCTACAGTCTGCGACA
CCTAGAGTCAGGCTCACCAACC22 22 注:GAPDH,甘油醛-3-磷酸脱氢酶;S1P,鞘氨醇-1-磷酸;S1PR1,鞘氨醇1磷酸酯受体1;TREM2,髓系细胞触发受体2;ADAM17,解整合素金属蛋白酶17。
表 2 各组大鼠体重、肝湿重、肝指数
Table 2. Rat weight, liver wet weight, and liver index of each group of rats
组别 动物数(只) 体重(g) 肝湿重(g) 肝指数(%) 空白对照组 10 542.80±27.48 16.23±1.27 2.99±0.22 模型A组 10 593.50±10.461)2) 20.43±2.551)2) 3.45±0.462) 模型B组 10 742.70±79.011) 39.80±10.461) 5.30±0.951) 西药组 10 622.50±62.622) 22.23±2.522)3) 3.57±0.222) 中药高剂量组 10 583.60±21.022) 18.68±1.902) 3.20±0.282) 中药中剂量组 10 602.80±51.712) 22.09±3.462) 3.67±0.512) F值 12.954 20.209 15.394 P值 <0.01 <0.01 <0.01 注:与空白对照组比较,1)P<0.05;与模型B组比较,2)P<0.05;与中药高剂量组比较,3)P<0.05。
表 3 各组大鼠肝组织染色结果
Table 3. Staining results of liver tissue in each group of rats
组别 动物数
(只)肝细胞脂肪变 小叶内炎症 (20倍计数坏死灶) 肝细胞气球样变 NAS(分) 油红O阳性面积
(%)空白对照组 5 1.00(1.00~1.50) 0.00(0.00~0.00) 0.00(0.00~0.50) 1.40±0.55 3.70±0.40 模型A组 5 2.00(2.00~3.00) 0.00(0.00~0.00) 2)0.00(0.00~1.00) 2)2.80±0.841)2) 17.44±3.081)2) 模型B组 5 3.00(3.00~3.00) 1)1.00(1.00~2.00) 1)2.00(1.50~3.00) 1)6.60±1.141) 61.91±2.281) 西药组 5 2.00(2.00~3.00) 1.00(1.00~1.50) 1.00(1.00~2.00) 5.00±0.712)3) 27.36±1.852)3) 中药高剂量组 5 2.00(1.50~2.00) 0.00(0.00~1.00) 1.00(1.50~1.00) 3.00±1.002) 18.66±2.182) 中药中剂量组 5 2.00(1.50~2.50) 1.00(1.00~1.50) 1.00(1.00~2.00) 4.20±1.642) 29.51±1.632)3) 统计值 H=17.690 H=118.964 H=118.495 F=1 15.538 F=1450.275 P值 <0.01 <0.01 <0.01 < 0.01 <0.01 注:与空白对照组比较,1)P<0.05;与模型B组比较,2)P<0.05;与中药高剂量组比较,3)P<0.05。
表 4 各组大鼠血清肝脏酶学、血脂水平
Table 4. Serum liver enzyme and blood lipid levels of rats in each group
组别 动物数
(只)ALT
(U/L)AST
(U/L)TC
(mmol/L)TG
(mmol/L)HDL-C
(mmol/L)LDL-C
(mmol/L)FFA
(mmol/L)空白对照组 10 20.24±8.82 22.85±7.83 2.03±0.22 0.77±0.21 2.40±0.70 0.65±0.21 0.94±0.55 模型A组 10 26.25±10.072) 24.63±8.362) 3.68±0.411)2) 1.36±0.131)2) 1.18±0.331)2) 1.85±0.441) 2.38±0.591)2) 模型B组 10 162.36±37.601) 46.11±8.891) 6.45±1.451) 2.12±0.571) 0.58±0.181) 1.88±0.731) 5.73±2.101) 西药组 10 58.07±16.702)3) 34.26±14.572)3) 3.34±0.542) 1.10±0.372) 1.28±0.342)3) 1.56±0.81 2.36±0.652)3) 中药高剂量组 10 33.70±11.712) 21.31±6.382) 2.73±0.482) 0.87±0.272) 2.13±0.592) 1.04±0.29 1.14±0.722) 中药中剂量组 10 59.97±13.122) 33.41±10.872) 3.57±0.642) 1.27±0.512) 0.95±0.132)3) 1.08±0.38 2.52±0.632)3) F值 37.111 9.158 44.310 17.883 25.613 15.462 17.479 P值 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 注:与空白对照组比较,1)P<0.05;与模型B组比较,2)P<0.05;与中药高剂量组比较,3)P<0.05。ALT,丙氨酸氨基转移酶;AST,天冬氨酸氨基转移酶;TC,总胆固醇;TG,甘油三酯;HDL-C,高密度脂蛋白胆固醇;LDL-C,低密度脂蛋白胆固醇;FFA,游离脂肪酸。
表 5 各组大鼠血清TNF-α、IL-1β、IL-6、sTREM2水平
Table 5. Serum TNF-α, IL-1β, IL-6 and sTREM2 levels in each group of rats
组别 动物数
(只)TNF-α
(pg/mL)IL-1β
(pg/mL)IL-6
(pg/mL)sTREM2
(ng/mL)空白对照组 10 229.18±104.00 97.03±21.08 26.42±6.20 7.21±0.71 模型A组 10 316.47±45.692) 203.71±31.491)2) 57.24±11.691)2) 8.66±0.601)2) 模型B组 10 808.13±164.831) 355.85±57.041) 120.96±40.591) 21.13±1.961) 西药组 10 601.78±112.253) 267.74±48.802)3) 61.83±19.492) 18.52±3.303) 中药高剂量组 10 371.56±64.322) 197.96±37.522) 37.41±11.592) 11.20±1.442) 中药中剂量组 10 601.12±231.133) 249.31±56.642)3) 63.11±36.342)3) 13.71±1.542)3) F值 29.504 38.013 24.509 115.212 P值 <0.01 <0.01 <0.01 <0.01 注:与空白对照组比较,1)P<0.05;与模型B组比较,2)P<0.05;与中药高剂量组比较,3)P<0.05。TNF-α,肿瘤坏死因子-α;IL-1β,白细胞介素-1β;IL-6,白细胞介素-6;sTREM2,可溶性髓系细胞触发受体2。
表 6 各组大鼠巨噬细胞胞葬率
Table 6. The phagocytic rate of macrophages in each group of rats
组别 动物数(只) 巨噬细胞胞葬率(%) 空白对照组 5 11.55±2.30 模型A组 5 51.42±1.901)2) 模型B组 5 15.14±1.69 西药组 5 19.21±2.052)3) 中药高剂量组 5 49.65±5.822) 中药中剂量组 5 27.07±4.592)3) F值 129.133 P值 <0.01 注:与空白对照组比较,1)P<0.05;与模型B组比较,2)P<0.05;与中药高剂量组比较,3)P<0.05。
表 7 各组大鼠肝组织S1P、S1PR1、ADAM17、TREM2 mRNA表达水平
Table 7. The mRNA levels of S1P, S1PR1, ADAM17, and TREM2 in liver tissues of rats in each group
组别 动物数(只) S1P S1PR1 ADAM17 TREM2 空白对照组 6 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 模型A组 6 2.91±1.13 2.60±1.082) 2.63±1.34 2.84±0.601)2) 模型B组 6 6.73±2.331) 7.11±2.981) 7.93±3.68 7.16±0.931) 西药组 6 5.90±0.70 6.34±2.47 5.61±1.90 6.76±1.37 中药高剂量组 6 5.94±2.14 8.20±7.41 3.03±1.04 8.50±1.87 中药中剂量组 6 5.90±1.86 6.70±3.39 4.45±1.49 6.58±1.42 F值 12.058 3.536 9.710 34.864 P值 < 0.01 < 0.05 < 0.01 < 0.01 注:与空白对照组比较,1)P<0.05;与模型B组比较,2)P<0.05。S1P,鞘氨醇-1-磷酸;S1PR1,鞘氨醇1磷酸酯受体1;ADAM17,解整合素金属蛋白酶17;TREM2,髓系细胞触发受体2。
表 8 各组大鼠肝组织S1P、S1PR1、ADAM17、TREM2蛋白表达水平
Table 8. Expression levels of S1P, S1PR1, ADAM17, and TREM2 proteins in liver tissues of rats in each group
组别 动物数(只) S1P S1PR1 ADAM17 TREM2 空白对照组 5 6.24±0.87 3.43±0.90 5.00±1.01 5.24±0.30 模型A组 5 11.87±1.771)2) 8.21±0.781)2) 13.26±1.721)2) 34.15±9.151)2) 模型B组 5 22.47±2.961) 35.42±7.861) 26.31±1.801) 8.52±2.27 西药组 5 20.72±1.46 30.95±3.53 22.54±2.393) 9.38±2.20 中药高剂量组 5 20.99±1.37 33.77±5.14 15.37±1.322) 22.74±5.662) 中药中剂量组 5 22.42±2.57 31.25±3.93 20.57±1.422)3) 15.02±2.91 F值 59.345 50.987 129.890 27.657 P值 <0.01 <0.01 <0.01 <0.01 注:与空白对照组比较,1)P<0.05;与模型B组比较,2)P<0.05;与中药高剂量组比较,3)P<0.05。S1P,鞘氨醇-1-磷酸;S1PR1,鞘氨醇1磷酸酯受体1;ADAM17,解整合素金属蛋白酶17;TREM2,髓系细胞触发受体2。
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