A Mouse Model of Damp-Heat Syndrome in Traditional Chinese Medicine and Its Impact on Pancreatic Tumor Growth

Abstract

Background: Damp-heat syndrome is one of the most important syndrome types in the traditional Chinese medicine (TCM) syndrome differentiation and treatment system, as well as the core pathogenesis of pancreatic cancer (PC) which remains a challenge to medical researchers due to its insidious onset and poor prognosis. Great attention has been given to the impact of damp-heat syndrome on tumorigenesis and progression, but less attention has been given to damp-heat modeling per se. Studying PC in a proper damp-heat syndrome animal model can recapitulate the actual pathological process and contribute to treatment strategy improvement.

Methods: Here, an optimized damp-heat syndrome mouse model was established based on our prior experience. The Fibonacci method was applied to determine the maximum tolerated dosage of alcohol for mice. Damp-heat syndrome modeling with the old and new methods was performed in parallel of comparative study about general appearance, food intake, water consumption and survival. Major organs, including the liver, kidneys, lungs, pancreas, spleen, intestines and testes, were collected for histological evaluation. Complete blood counts and biochemical tests were conducted to characterize changes in blood circulation. PC cells were subcutaneously inoculated into mice with damp-heat syndrome to explore the impact of damp-heat syndrome on PC growth. Hematoxylin-eosin staining, Masson staining and immunohistochemistry were performed for pathological evaluation. A chemokine microarray was applied to screen the cytokines mediating the proliferation-promoting effects of damp-heat syndrome, and quantitative polymerase chain reaction and Western blotting were conducted for results validation.

Results: The new modeling method has the advantages of mouse-friendly features, easily accessible materials, simple operation, and good stability. More importantly, a set of systematic indicators was proposed for model evaluation. The new modeling method verified the pancreatic tumor-promoting role of damp-heat syndrome. Damp-heat syndrome induced the proliferation of cancer-associated fibroblasts and promoted desmoplasia. In addition, circulating and tumor-located chemokine levels were altered by damp-heat syndrome, characterized by tumor promotion and immune suppression.

Conclusions: This study established a stable and reproducible murine model of damp-heat syndrome in TCM with systematic evaluation methods. Cancer associated fibroblast-mediated desmoplasia and chemokine production contribute to the tumor-promoting effect of damp-heat syndrome on PC.

Keywords: cancer-associated fibroblasts; chemokines; damp-heat syndrome; evaluation indicators; mouse model; pancreatic tumor.

Graphical Abstract

Figure 1

Determination of the alcohol MTD for mice. (A) Dose incremental scheme of alcohol. (B) Exploration of the alcohol MTD for mice through the Fibonacci method. (C) Survival of mice at the critical points and above the critical point of alcohol dosage (n = 10 in each group). (D) Body weight of mice receiving different dosages of alcohol (n = 10 in each group). (E) Rectal temperature of mice receiving different dosages of alcohol (n = 6 in each group). (F) Urine volume of mice receiving different dosages of alcohol (n = 6 in each group). *p < 0.05, **p < 0.01.

Figure 2

A damp-heat syndrome mouse model was established with the new and old methods. (A) Diagram for establishing a mouse model of damp-heat syndrome. (B) Activity status and anatomical view of mice in the three groups. (C) Body weight of mice over time. (D) Food intake of mice in the three groups over time. (E) Water consumption of mice in the three groups over time. (F) Survival of mice in the three groups over time.

Figure 3

HE liver staining results of mice in the three groups at different time points after damp-heat induction and pathological observation of the intestine at the endpoint.

Figure 4

Complete blood count and biochemical analyses. (A) Differential complete blood count indicators between the control group and the damp-heat syndrome group. (B) Differential biochemical indicators between the control group and the damp-heat group. Control: healthy mice (n = 23), Model: mice with damp-heat syndrome (n = 54). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Impact of damp-heat syndrome on the pancreatic tumor. (A) Appearance of mice in the four groups. (B) Tumors of mice with and without damp-heat syndrome. (C) Tumor weight of mice at the end of the experiment. (D) Tumor volume changes over time. (E) Body weight of mice in the four groups. (F) Histopathological observation of tumor tissue. C: control group, T: pancreatic tumor group, D: damp-heat syndrome group, TD: pancreatic tumor with damp-heat syndrome group. n = 6 in each group. *p < 0.05, **p < 0.01 compared with the T group.

Figure 6

Chemokine differences between tumor-bearing mice with and without damp-heat syndrome. (A) Heatmap indicating serum chemokine distribution between the two groups (n = 10 in the T group, n = 9 in the TD group). (B) Heatmap indicating tumor tissue chemokine distribution between the two groups (n = 3 in each group). (C) Relative mRNA expression of tissue chemokines tested by qPCR (n = 10 in the T group, n = 9 in the TD group). (D) Relative protein expression of tissue chemokines tested by western blotting (n = 8 in each group). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the T group.