# Cmpk2 Gene and Protein Expression in Saliva or Salivary Glands of Dyslipidemic Mice
## 4 Discussion  
To date, limited information exists regarding the expression of _Cmpk2_ mRNA in saliva. In this investigation, we demonstrate that _Cmpk2_ mRNA secretion into saliva is elevated in the presence of lipid abnormalities. Immunofluorescence analysis of salivary glands confirmed the localization of _CMPK2_ protein in serous cells rather than mucous cells within the submandibular gland. The relationship between increased body weight and abnormal lipid levels has been demonstrated [49; 50]; however, it is not observed universally [51; 52]. In this study, two dyslipidemia animal models were employed, an HFHC diet group and a genetic model (ApoE-deficient mice), to elucidate the effects of varying body weights and fat accumulation while exhibiting similar states. In the HFHC diet group, there was an increase in body weight and accumulation of fat (Figure 3). In ApoE-deficient mice, an increase in body weight with increasing weekly age was noted, yet without significant lipid accumulation  
Figure 7: Immunofluorescence staining of the submandibular gland and its quantitative data. (**A**) The submandibular gland was stained with pan-cytokeratin (pan-CK), DAPI (DAPI-Fluoromount-G), and _CMPK2_. Pan-CK binds to salivary gland epithelial cells, while DAPI binds to cell nuclei. (**B**) The quantification of _CMPK2_ fluorescence intensity was quantified using ImageJ software (National Institutes of Health). Data are mean \(\pm\) SD (n = 4). * \(p\) \(<\) 0.05, *** \(p\) \(<\) 0.001, *** \(p\) \(<\) 0.0001: Student’s _t_-test for 9 w mice, one-way ANOVA with [PERSON]’s post-hoc test for 46 w mice. (**C**) Focus on serous demilune in H-E and immunofluorescence staining images of the submandibular gland (merge image of pan-CK, _CMPK2_, and DAPI); _CMPK2_ is localized in the serous acini. SA: serous acini, MA: mucous acini. SeD: serous demilune (white dotted line and arrow). **C:** C57 BL/6 mice, **A:** ApoE-deficient mice, **C**\(\rightarrow\): C57 BL/6 mice fed a normal diet, **C**+: C57 BL/6 mice fed a HFHC diet, **A**\(\rightarrow\): ApoE-deficient mice fed a normal diet. (Figure 3). Conversely, serum LDL-C and T-CHO significantly increased in both groups at 46 w (Figure 4). These findings are consistent with previous studies [53; 54], indicating that the two animal models can be reliably established in this experiment. mRNA has been extensively investigated in human saliva [32]. Particularly after the onset of the COVID-19 pandemic, the relationship between saliva studies and the mRNA transcripts of COVID-19 has increased significantly [55]. The salivary glands are located in close proximity to blood vessels, allowing the exchange of metabolites between the oral cavity and the circulatory system [56]. As a result, certain proteins in saliva are similar to those found in blood. Research has shown that a large number of biomarkers found in saliva correlate with their levels in the blood [57]. In addition, studies have shown that salivary biomarkers have excellent accuracy, sensitivity, and specificity in distinguishing the early stages of oral squamous cell carcinoma [58]. This suggests that saliva is a potential alternative to blood-based tests. Although blood tests are essential for diagnosing dyslipidemia, the non-invasive nature of saliva offers significant advantages. It is our contention that early prevention of dyslipidemia could be achieved if screening could be conducted by saliva testing during physical examinations. However, salivary mRNA studies in rodents remain extremely limited. In this experiment, we initially utilized two commercially available total RNA collection methods. However, no distinct waveform was observed at A260 using the NanoDrop (Figure S2). Conversely, the waveform was confirmed by the TRIzolol-based method, leading to the implementation of RNA extraction using the TRIzolol method in the subsequent experiment. Even using the TRIzolol methods, the existence of phenol seems to be identified at the peak at A270 [59].