Mechanism of hyperproteinemia-induced blood cell homeostasis imbalance in an animal model
摘要: 高蛋白血症是一种以血浆蛋白浓度（PPC）显著升高为指标的严重代谢紊乱疾病，临床常并发于多种恶性疾病或重度感染。然而，由于临床无法剥离原发性疾病的影响，目前对高PPC的分子机理研究甚少。该文在无脊椎模式动物家蚕中构建了一个原发性高蛋白血症动物模型，调查了高PPC对循环血细胞的影响。结果发现，高PPC严重影响血细胞稳态，导致活性氧水平升高，诱导了依赖内质网-钙离子信号途径的血细胞程序性死亡发生。另一方面，高PPC通过激活血细胞的JAK/STAT信号通路，诱导以颗粒细胞为主的血细胞增殖。补充内分泌激素活性物质20E治疗后，能够显著减轻高PPC对循环血细胞稳态的影响。因此，该文发现了一条高PPC影响血细胞稳态不同于高血糖、高血脂和高胆固醇的信号通路。此外发现，造血因子Gcm的基因表达下调可作为潜在的高蛋白血症早期检测指标。Abstract: Hyperproteinemia is a metabolic disorder associated with increased plasma protein concentration (PPC) and is often clinically complicated by malignant diseases or severe infections. At present, however, research on the molecular mechanism underlying high PPC (HPPC) is scant. Here, an animal model of primary hyperproteinemia was constructed in an invertebrate (Bombyx mori) to investigate the effects of HPPC on circulating blood cells. Results showed that HPPC affected blood cell homeostasis, leading to increased reactive oxygen species levels, and induced programmed cell death dependent on the endoplasmic reticulum-calcium ion signaling pathway. HPPC induced the proliferation of blood cells, mainly granulocytes, by activating the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. Supplementation with the endocrine hormone active substance 20E significantly reduced the impact of HPPC on blood cell homeostasis. Thus, we identified a novel signaling pathway by which HPPC affects blood cell homeostasis, which differs from hyperglycemia, hyperlipidemia, and hypercholesterolemia. In addition, we showed that down-regulation of gene expression of the hematopoietic factor Gcm could be used as a potential early detection indicator for hyperproteinemia.
- Animal model /
- Blood cell homeostasis /
- High plasma protein concentration (HPPC) /
- JAK/STAT pathway /
- Programmed cell death
Figure 1. HPPC affected circulating hemocyte (blood cell) composition
A: Results of blood cell classification after AO-PI staining. Pro, Prohemocytes; Pla, Plasmatocytes; Gra, Granulocytes; Sph, Spherulocytes. These hemocytes showed green fluorescence after AO staining and could be distinguished based on intensity of fluorescence and morphology of hemocytes. Oen, Oenocytoids. These hemocytes showed red fluorescence after PI staining; blood cell nuclei with necrosis or membrane rupture were also stained red by PI and could be distinguished from other hemocytes in morphology. B: Blood cell density (n=10). CK, control group; AM, model group. C: Percentage composition of circulating hemocytes (n=3). AH, could not distinguish types and often showed deformities. D, E: Densities of different blood cell types (n=10). D: Pla; E: Gra. Ordinate represents number of hemocytes per mm3 of hemolymph. F: Gene transcription level of hematopoietic-related factor Gcm (n=3). Internal reference gene was Bombyx mori Rp49. G, H: Interference with Gcm gene at individual level affected blood cell composition. Gcm gene transcriptional level in hemocytes (G) and percentage composition of blood cells (H) were investigated 48 h after intervention treatment (n=3). CK, control group. CK+RNAi, individual intervention in CK group. mAM, mild model group. mAM+RNAi, individual intervention in mild model group. Each silkworm in the siRNA group was injected with 10 μg of Gcm-siRNA, control (NC in Figure 1G and CK or mAM in Figure 1H) was injected with the same amount of negative control siRNA (si-NC), with each group effectively containing 18 injected individuals (six individuals×three repetitions). Data are mean±standard error of the mean (SEM). ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001, Student’s t-test.
Figure 2. HPPC induced autophagy of circulating hemocytes (blood cells) in early and mid-stage period after modeling
MDC and Lyso-Tracker Red staining marked autophagic hemocytes in hemolymph at 48, 96, and 192 h after modeling. Positive rate (%)=(fluorescent cell number/total cell number)×100. Scale bars: 100 μm. A: Fluorescence image of MDC-stained hemocytes 96 h after modeling (n=3). B: MDC staining positive rate in hemocytes. Significant differences between groups are indicated with different letters (P≤0.05, n=3). C: MDC staining positive rate in different types of hemocytes. D: Lyso-Tracker Red staining fluorescence image of hemocytes 48 h after modeling. E: Lyso-Tracker Red staining positive rate of hemocytes (n=3). F, G: Transcription levels of autophagy genes (F) Atg6 and (G) Atg8 in hemocytes analyzed by qPCR (n=3). Internal reference gene was Bombyx mori Rp49. Data are mean±SEM. ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001, Student’s t-test.
Figure 3. HPPC induced apoptosis of circulating hemocytes (blood cells) via endoplasmic reticulum-calcium ion release signaling pathway
TUNEL and Ca-FL staining show hemocytes with apoptosis and elevated cytoplasmic calcium level, respectively, in hemolymph at 48, 96, and 192 h after modeling, and DAPI shows hemocyte nuclei. Positive rate (%)=(fluorescent cell number/total cell number)×100. Scale bars: 100 μm. A: TUNEL-stained fluorescence image at 192 h after modeling. B: TUNEL staining positive rate of hemocytes. C: TUNEL staining positive rate of different types of hemocytes. D: Transcription level of apoptosis-initiating gene Dronc analyzed by qPCR, with internal reference gene Rp49 (n=3). E: Ca-FL-stained fluorescence image of hemocytes at 96 h after modeling. F: Positive rate of hemocyte Ca-FL staining. In Figure 3B, F, significant differences between groups are indicated with different letters. Data are mean±SEM, P≤0.05, n=3. In Figure 3D, data are mean±SEM. *: P<0.05; **: P<0.01; ***: P<0.001, Student’s t-test.
Figure 4. HPPC increased oxidative stress in hemocytes
DCFH-DA staining of ROS in hemocytes at 48, 96, and 192 h after modeling. Scale bars: 100 μm. A: DCFH-DA-stained fluorescence image at 96 h after modeling. B: Relative level of ROS in circulating hemocytes. Fluorescence intensity was calculated using Image Pro software, relative ROS intensity (×105)=fluorescence intensity/total hemocytes count. C–E: Three main components of ROS in plasma. C: H2O2 content; D: Superoxide anion (OFR) content; E: Hydroxyl radical (∙OH) inhibition ability. F–H: SOD and CAT activity and GSH content in plasma. A–H, n=3. Data are mean±SEM. ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001, Student’s t-test.
Figure 5. Hyperproteinemia delayed proliferation of hemocytes in later stages of modeling
A–C: DNA staining of proliferating hemocytes. EdU and DAPI staining marked proliferating cell nuclei and all cell nuclei, respectively, in hemolymph at 48, 96, 144, 192, 216, and 240 h after modeling. Positive rate (%)=(EdU-positive cell number/DAPI-positive cell number)×100. Scale bars: 100 μm. A: EdU staining fluorescence at 192 h after modeling. B: EdU-positivity rate in hemocytes (CK 48–240 h, n=3, 3, 3, 4, 4, 4; AM 48–240 h, n=3, 4, 4, 3, 3, 4. P≤0.05). Significant differences between groups are indicated with different letters. C: Composition of EdU-positive blood cells. Positive rate (%)=(number of EdU-positive blood cells of same type/total number of EdU-positive cells)×100. D: PI-FCM detected cell cycle phase of circulating hemocytes (n=3). Cell cycle was divided into G0/G1, S, and G2/M phases. E: Quantitative diagram of cell cycle phases. Ordinate indicates proportion of cells in G0/G1, S, and G2/M phases to total number of cells. F: Proliferation index (PI) (n=3), indicating proportion of proliferating hemocytes to total number of circulating hemocytes. PI (%)=(S+G2/M)/(G0/G1+S+G2/M)×100. Data are mean±SEM. ns: P>0.05; ***: P<0.001, Student’s t-test. G, H: Culture HPO-wing disc complex in vitro. After 24 h of modeling, HPO-wing disc complex was removed, and level of hematopoiesis was investigated after 48 h of culture in vitro. PR, Prohemocytes. PL, Plasmatocytes. G: HPO-Wing morphology. Scale bars: 0.5 mm; H: Number of hemocytes released by single culture of HPO-wing disc complex (n=5). Data are mean±SEM. Significant P-values were calculated by Student’s t-test.
Figure 6. HPPC activated JAK/STAT signaling pathway
STAT immunofluorescence and DAPI staining indicate cells expressing STAT protein and all cell nuclei, respectively, in hemolymph at 48, 96, and 192 h after modeling. A: STAT immunofluorescence image of hemocytes at 192 h after modeling. Scale bars: 100 μm. B: STAT-positivity rate in hemocytes (n=3). STAT-positivity rate (%)=(STAT-positive cell number/total cell number)×100. C: Composition of STAT-positive blood cells. Positive rate (%)=(number of STAT-positive blood cells of same type/total number of STAT-positive cells)×100. D: Levels of STAT protein and phosphorylated-STAT protein in blood cells were analyzed by western blotting and referenced by β-Tubulin at 48, 96, and 192 h after modeling (n=3). Only one STAT protein was identified in silkworm, i.e., BmSTAT. E, F: Relative expression level of phosphorylated-STAT (E) and total STAT protein (F) in blood cells. G–J: qPCR analysis of transcription level of JAK/STAT pathway genes. G: STAT; H: HOP; I: SOCS2; J: SOCS6. Reference gene was Rp49 (n=3). Data are mean±SEM. ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001, Student’s t-test. K–M: Culture hematopoietic organs in vitro (n=7). K: Scheme design. At stage 5L2d (day 2 of fifth instar Bombyx mori larvae), anterior pair of HPO-wing disc complexes were dissected and cultured in a 10 μL hanging drop culture system supplemented with JAK inhibitor AG490 (100 nmol/L) or vehicle DMSO for 72 h, with released blood cells then collected. L: Hemocytes were produced in vitro. Scale bars: 200 μm. M: Number of hemocytes. Number of hemocytes in DMSO group from same individual was normalized. Data are mean±SEM. Significant P-values were calculated by Student’s t-test.
Figure 7. Endocrine hormone treatment of hyperproteinemia affected hemocyte proliferation and differentiation
A–C: EdU staining was used to investigate hemocyte proliferation after injection of JAK inhibitor A490 (n=3). AM+AG490, AM group injected with AG490 (50 μmol/10 μL) at 144 h after modeling, with hemolymph samples then collected at 192 h. A: Level of phosphorylated-STAT protein was analyzed by western blotting. B: EdU-stained fluorescence image. C: EdU-positivity rate in blood cells. Scale bars: 100 μm. D–F: Investigation of JAK/STAT pathway and Gcm gene transcription in peripheral blood of patients with HPPC (n=4). NPPC, patients with normal total protein levels in clinical cases; HPPC, patients with total protein levels outside normal range in clinical cases. D: Total protein level, reference value 65–85 g/L. Transcription levels of JAK/STAT pathway (E) and Gcm-related genes in hemocytes (F) were analyzed by qPCR. Internal reference gene was Homo sapiens β-Actin. G–I: Percentage of hemocytes in mAM group after 20E rescue. CK, control; mAM, mild model; mAM+20E, 20E was injected at 24 h after mAM modeling, and hemolymph samples were collected at 144 h after injection (corresponding to 192 h after AM group modeling). G: PPC level (n=5). H: Circulating hemocyte image. PR, Prohemocytes; PL, Plasmatocytes; GR, Granulocytes. Scale bars: 100 μm. I: Percentage composition of circulating hemocytes. J–L: Transcription levels of related genes in mAM group after 20E rescue (n=3) were analyzed by qPCR. Internal reference gene was Bombyx mori Rp49. mAM+20E, mAM group was injected with 20E at 0 h after modeling. J: Gcm; K: HOP; L: STAT. Data are mean±SEM. ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001, Student’s t-test.
Figure 8. Summary of effects of HPPC on blood cell homeostasis
HPPC increases ROS in hemocytes, and then induces the PCD via the endoplasmic reticulum-calcium ion release signal pathway. HPPC also activates JAK/STAT signaling pathway and induces proliferation of hemocytes, especially granulocytes; significant down-regulation of Gcm gene further aggravates this process, thereby affecting circulating hemocyte homeostasis, resulting in an increase in the percentage of granulocytes and oenocytoids and decrease in the percentage of prohemocytes, plasmatocytes, and spherulocytes. Black solid line arrow indicates positive correlation induction effect, and black dotted line arrow indicates correlation promotion effect; red and blue lines indicate inhibitory (reverse regulation) effects based on our results and results from cited literature (Bazzi et al., 2018; Cui et al., 2018; Hu et al., 2015), respectively. White arrow indicates change in number of hemocytes in hemolymph induced by HPPC, and length of arrow indicates changed maximum ratio of density between model and control (Gra, Pro, Pla, Oen, and Sph was 2.38, 3.99, 9.82, 2.71 and 18.77 times, respectively). Width of arrow indicates absolute value of maximum difference between model and control in percentage of various types of hemocytes (maximum AM-CK) (Gra, Pro, Pla, Oen, and Sph was 27.16%, 10.99%, 21.28%, 1.90%, and 0.79%, respectively). Upward arrow indicates that AM is higher than CK, and downward arrow indicates that AM is lower than CK.
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ZR-2021-397 Supplementary Materials.pdf