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Gene proteins, growth factors/their receptors in the wall of chronic calculous cholecystitis-affected gallbladder children

BMC Pediatrics volume 25, Article number: 288 (2025) Cite this article

Abstract

Background

Chronic calculous cholecystitis is the main cause of cholecystectomies in children, and 50.5% of patients with gallstones are asymptomatic at the time of diagnosis. However, the morphopathogenesis of chronic cholecystitis with cholelithiasis is unclear and may involve various genes, gene proteins, and growth factors.

Methods

Tissues were obtained from four males (aged 6–18 years) and two females (aged 15 and 14 years) during planned cholecystectomies. Five healthy gallbladder tissues were obtained from the archival postmortem tissue of children. SHH, IHH, HGF, IGF1, IGF1R, and HOXB3 were detected by immunohistochemistry and evaluated semiquantitatively. Statistical analysis was used to identify statistically significant differences and correlations between the factors.

Results

Decreased numbers of SHH-, IHH-, and IGF1R-positive cells, along with an increased number of HOXB3-positive cells, were observed in patients. SHH-positive epitheliocytes and connective tissue cells; IHH-positive cells in all locations; IGF1R-positive epitheliocytes, endotheliocytes, and smooth muscle cells; and HOXB3-positive smooth muscle cells were significantly different among the groups. However, the strongest negative correlation was found between HOXB3-positive smooth myocytes and SHH- and IHH-positive connective tissues, and the strongest positive correlation was detected among epithelial IHH, SHH, and IGF1R, as well as between IGF1R in the epithelium and endothelium of the blood vessels.

Conclusions

The reduced number of cells positive for the primary endodermal proteins SHH/IHH and the decreased number of IGFR1-positive cells suggest their potential roles in the development of chronic calculous cholecystitis. Additionally, the increased number of HOXB3-positive cells under these conditions likely implies stimulated growth properties, whereas HGF and IGF1 appear to have a reduced contribution to the pathogenesis of chronic calculous cholecystitis.

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Introduction

Chronic calculous cholecystitis involves prolonged inflammation of the gallbladder, which is accompanied by cholelithiasis. Gallstone formation is observed in most cases of chronic cholecystitis, causing mechanical dysfunction of gallbladder emptying [1]. In an urban population of children, the gallstone prevalence was 1% [2], whereas it was 0.13% according to a 1989 ultrasonographic survey [3]. The sex distribution of gallstone incidence worldwide has shown a female predominance of 1.5:1 [4].

Approximately 50.5% of gallstone disease cases are asymptomatic at the time of diagnosis [5]. Therefore, cholelithiasis as a main indication for cholecystectomy is found in only 15.6% of cases [6]. A 2017 study revealed that most cases of cholecystectomies are caused by chronic cholecystitis combined with cholelithiasis [7]. Therefore, the prevalence of calculous cholecystitis in children is 1.9–4% [8]. Nonetheless, the incidence of cholelithiasis and cholecystectomies is increasing because it is associated with obesity [9].

Changes in the levels of proteins such as Sonic hedgehog (SHH), Indian hedgehog (IHH), homeobox B3 (HOXB3), growth factors/their receptors hepatocyte growth factor (HGF) and insulin growth factor 1 (IGF1) may play a role in the development of chronic calculous cholecystitis; however, the precise impact of these factors in children is not fully understood.

SHH is a signalling molecule found in vertebrates [10] that is expressed by the endoderm of the gut and its derivatives [11–12]. In adult mammalian tissues, SHH remains silent and is activated only during cell differentiation and proliferation to induce repair and regeneration. However, increased expression of SHH occurs in inflammatory, dysfunctional, and cancerous cells, including those in the gallbladder [13–14]. To promote repair, SHH is expressed by epithelial cells of the bile duct and fibroblasts in response to injuries [15]. However, increased SHH expression is found in chronic cholecystitis and gallbladder carcinoma [16].

IHH is a secretory signalling protein that is expressed throughout the length of the intestinal epithelium during development and adulthood [17]. This molecule coordinates cell differentiation patterns and the vascular system through the growth and sprouting of blood vessels, organizes anatomical structures of the digestive tract during embryonic development and the progression of the epithelial cell to its mature state, and negatively regulates apoptotic processes [18]. The highest expression of IHH in epithelial cells of the bile duct is in the gallbladder, and the expression decreases from the common bile duct, reaching its lowest level in the peripheral ducts [19].

HOXB3 is a human homeobox gene (Hox) protein that controls cell differentiation and developmental patterns in vertebrates by DNA-specific binding, activating or repressing transcription [20]. Homeobox genes encode anatomic segment identities and regulate stem cell differentiation [21]. Research has shown that HOXB3 is expressed in various tissues, mostly in the sigmoid colon but also minimally expressed in the pancreas and liver. HOXB3 levels in the liver and gallbladder are intermediate [22].

HGF, or scatter factor, is a pleiotropic protein that influences cell growth, increasing DNA synthesis in various epithelial cells. This molecule is also a multifactorial factor involved during embryogenesis in wound healing, organ or tissue regeneration, and organogenesis [23,24,25]. HGF is synthesized and secreted from fibroblasts [26] and endothelial and fat-storing cells [27]. Importantly, HGF therapy suppresses inflammation in obstructed nephropathy [28] and reverses cholestatic damage to cholangiocellular cilium morphology [29]. HGF production is stimulated by IGF1 and through insulin growth factor 1 receptor (IGF1R) [30].

IGF1 is a widely locally synthesized peptide hormone that acts as a growth factor with endocrine, paracrine, and autocrine functions [31] and is produced mainly in the liver. Therefore, IGF1 activates proliferating pathways in epithelial cells of the bile duct [32]. This molecule is pleiotropic and stimulates the synthesis of DNA, proteoglycans, glycosaminoglycans, and proteins. Moreover, IGF1 induces glucose uptake in cells. IGF1 binds to a specific IGF1R.

IGF1R expression is tissue- and development stage-specific. In a variety of cell types, IGF1R is present and activates cell proliferation and differentiation as well as the inhibition of apoptosis [31]. IGF1R is expressed in the biliary epithelium, and IGF1, which binds to the receptor, protects epithelial cells of the bile duct against cholestatic injury in vitro by increasing cholangiocyte and smooth muscle proliferation. On the one hand, supranormal levels of IGF1 induce extensive proliferation of the bile duct epithelium [33]; on the other hand, low serum levels of IGF1 are associated with hepatic lobular inflammation [34].

Despite all the abovementioned factors involved in gallbladder formation, little is known about their appearance and distribution, especially in chronic calculous cholecystitis. Thus, the present study aimed to explore the appearance, distribution, and interactions of different proteins and growth factors in chronic calculous cholecystitis.

Materials and methods

Study participants

A total of six cholecystectomies were performed on two females and four males aged 6 to 18 years. For the controls, five samples were used; the samples were retrieved postmortem from similar-aged subjects after car accidents. All the tissue samples were referred for investigation to the Institute of Anatomy and Anthropology of Riga Stradins University by the Department of Paediatric Surgery of the Children’s University Hospital from 2007 to 2010. A gallbladder was included in the study if it contained chronic inflammation with concrements with or without adhesions of the wall; gallbladder dyskinesia caused by chronic calculous cholecystitis was observed; no medical treatment was used in the preoperative period; the patient was under the age of 18 years; a diagnosis of chronic calculous cholecystitis was present for 1.5-2 years; and one exacerbation occurred before surgery. The diagnosis was based on ultrasound sonography and clinical presentation (abdominal pain, vomiting, elevated body temperature, changes in blood biochemistry). The exclusion criteria included the absence of gallstones, acute cholecystitis, and the absence of functional disorders. The patients’ clinical information is summarized in Table 1.

The research on tissue samples was carried out in 2024 and approved by the ethical committee at Riga Stradins University (the permit was issued on the 10th of May 2007) and was performed in accordance with the Helsinki Declaration. A consent form was signed by the participants and parents.

Table 1 Characteristics of study participants
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Immunohistochemical (IHC) analysis

The samples were fixed for 24 h in 2% formaldehyde, 0.2% picric acid, and 0.1 M phosphate buffer with a pH of 7.2. The tissues were processed in Tyrode buffer, which contained 10% saccharose, for 12 h. Furthermore, the material was embedded in paraffin and cut into 4–6 μm thick sections. Haematoxylin and eosin staining was performed to assess the routine morphology of the samples. With the standard biotin-streptavidin method, the tissues were prepared and further studied to detect SHH, IHH, HGF, IGF1, IGF1R, and HOXB3 with the appropriate antibodies (Table 2).

Table 2 Description of the antibodies used in the immunohistochemical analysis
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The examined tissues contained the whole gallbladder wall with an area of 5mm2. Light microscopy and semiquantitative scoring analysis were performed to evaluate the quantity and distribution of the immunoreactive cells in the gallbladder epithelium, connective tissue, blood vessels, and smooth myocytes. Two independent morphologists individually analysed all the tissue samples. The number of positively stained cells of the gallbladder in the light microscopy visual field was rated using the following scale: +w– occasional positive cells, +– few positive cells, ++w– weakly stained moderate number of positive cells, ++– moderate number of positive cells, +++w– weakly stained numerous positive cells, +++– numerous positive cells, ++++w– weakly stained abundant positive cells, ++++– abundant positive cells [35]. Microphotographs were taken with a Leica DC 300 F digital camera (Leica Microsystems Digital Imaging, Cambridge, UK) at a magnification of 200×.

Statistical analysis

The IHC results were transformed into a numerical form: 0 represents 0, +w corresponds to 0.5, + corresponds to 1, ++w corresponds to 1.5, ++ corresponds to 2, +++w corresponds to 2.5, +++ corresponds to 3, ++++w corresponds to 3.5, and ++++ corresponds to 4. Statistically significant differences were determined via the Mann‒Whitney U test. If the p value was < 0.05, the difference was statistically significant. Spearman’s rank correlation coefficient (R) was also calculated and analysed to find correlations between the chosen proteins or growth factors. R < 0.199 indicates a very weak correlation, 0.20 < R < 0.399 is a weak correlation, 0.40 < R < 0.599 is a moderate correlation, 0.60 < R < 0.799 is a strong correlation, and 0.80 < R < 1.000 is a very strong correlation. The data were processed using IBM SPSS software version 29.0 (IBM Company, North Castle, Armonk, NY, USA).

Results

Routine staining

In almost all gallbladder tissue samples, many inflammatory cells were observed in the subepithelium (Fig. 1a). The infiltration reached all gallbladder wall levels, and the cells were intermixed with chaotically organized smooth muscle and collagen fibre bundles at the adhesion site of the gallbladder (Fig. 1b). Controls were evaluated according to the common standard tissue picture of the gallbladder (Fig. 1c).

Fig. 1
figure 1

Microphotographs of routinely stained gallbladder walls. (a) Note inflammatory cell infiltration in the subepithelium in the patient sample (arrow). Haematoxylin and eosin, ×200. (b) The adhesion site shows inflammatory cells intermixed with collagen fibre bundles (arrow). Haematoxylin and eosin, ×200. (c) Control image of the practically unchanged gallbladder wall; haematoxylin and eosin, ×400

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SHH

In the patient group, the median number of SHH-positive cells in the epithelium was numerous (+++), fluctuating from weakly stained abundant (++++w) to numerous (+++) in median quantity. In contrast, a few to weakly stained moderate (+/++w) positive cells were found in connective tissue, fluctuating from few (+) to moderate (++) quantity. However, in the blood vessels and smooth myocytes, the median number of SHH-positive cells was moderate to weakly stained numerous (++/+++w), fluctuating from moderate (++) to numerous (+++) (Table 3; Fig. 2a).

Table 3 Semiquantitative evaluation of the proteins and growth factors/their receptors in patient gallbladder structures
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In the control group, the median number of SHH-positive cells in the epithelium was high (++++), and that in the connective tissue was moderate (++); however, the number of SHH-positive cells did not fluctuate. In blood vessels and smooth myocytes, the median number of SHH-positive cells was also moderate (++), fluctuating from a moderate (++) number of positive cells to numerous (+++) cells in blood vessels and fluctuating from few (+) to moderate (++) in smooth myocytes (Table 4; Fig. 2b).

Table 4 Semiquantitative evaluation of the gene proteins and growth factors/their receptors in control gallbladder structures
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Fig. 2
figure 2

Microphotographs of SHH-positive gallbladder structures in patient and control samples. (a) Patient sample with numerous (+++) SHH protein-positive cells in the epithelium, few (+) in connective tissue, moderate (++) number of positive cells in blood vessels, and weakly stained numerous (+++w) positive cells in smooth myocytes. SHH IMH, ×200. (b) Control sample with abundant (++++) SHH-positive cells in a small fragment of the epithelium and weakly stained moderate (++w) number of positive cells in connective tissue, blood vessels, and smooth myocytes. SHH IMH, ×200

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IHH

In the patient group, the median quantity of IHH-positive cells in the epithelium and blood vessels was moderate (++), fluctuating from occasional (+w) to moderate (++) numbers of positive cells in the epithelium and fluctuating from few (+) to moderate (++) numbers of positive cells in blood vessels. In contrast, a few to weakly stained moderate (+/++w) number of positive cells were found in connective tissue, fluctuating from occasional (+w) to weakly stained moderate (++w) quantity. However, in myocytes, a moderate (++w) number of positive cells were weakly stained, fluctuating from a few (+) to a moderate (++) number of protein-positive cells (Table 3; Fig. 3a).

In the control group, the median quantity of IHH-positive cells in the epithelium and blood vessels was abundant (++++), with no fluctuations in the epithelium and fluctuating from weakly stained abundant (++++w) positive cells to abundant (++++) cells in blood vessels. However, in connective tissue, the median number of IHH-positive cells was high (+++), fluctuating from moderate (++) to abundant (++++). However, numerous (+++w) weakly stained positive cells were found in smooth myocytes, fluctuating from weakly stained numerous (+++w) to numerous (+++) positive cells (Table 4; Fig. 3b).

Fig. 3
figure 3

Microphotographs of the IHH-positive gallbladder structures in the patient and control materials. (a) Patient sample with a moderate (++) number of IHH-positive cells in the epithelium and blood vessels, few (+) in connective tissue, and occasional (+w) positive cells in smooth myocytes. IHH IMH, ×200. (b) Control sample with abundant (++++) IHH-positive cells in the epithelium and blood vessels, numerous (+++) cells in connective tissue, and weakly stained numerous (+++w) cells in smooth myocytes. IHH IMH, ×200

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HGF

In the patient group, the median quantity of HGF-positive cells in the epithelium and smooth myocytes was weakly stained abundant (++++w), fluctuating from occasional (+w) to abundant (++++) in the epithelium and fluctuating from a moderate (++) number of positive cells to abundant (++++) in smooth myocytes. However, an abundant (++++) number of positive cells were found in connective tissue and blood vessels, fluctuating from numerous (+++) to abundant (++++) in connective tissue and with no fluctuations in blood vessels (Table 3; Fig. 4a).

In the control group, the median quantity of HGF-positive cells in the epithelium, connective tissue, and blood vessels was abundant (++++) with no fluctuations. In contrast, the median quantity of HGF-positive cells in smooth myocytes was weakly stained abundant (++++w), with no fluctuations (Table 4; Fig. 4b).

Fig. 4
figure 4

Microphotographs of the HGF protein-positive gallbladder structures in the patient and control materials. (a) Patient sample with abundant (++++) HGF-positive cells in the epithelium, blood vessels and smooth myocytes but numerous (+++) HGF-positive cells in connective tissue. HGF IMH, ×200. (b) Control sample with abundant (++++) HGF-positive cells in the epithelium, connective tissue, and blood vessels but abundant (++++w) weakly stained smooth myocytes. HGF IMH, ×200

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IGF1

In the patient group, the median number of IGF1-positive cells in the epithelium was high (+++), fluctuating from weakly stained moderate (++w) numbers of positive cells to abundant (++++) cells. However, an abundant (++++) number of positive cells were found in connective tissue, fluctuating from numerous (+++) to abundant (++++). In contrast, the median quantity of factor-positive cells in blood vessels was weakly stained abundant (++++w), fluctuating from numerous (+++) to abundant (++++). Similarly, numerous to weakly stained abundant (+++/++++w) cells were found in smooth myocytes, fluctuating from weakly stained numerous (+++w) to weakly stained abundant (++++w) (Table 3; Fig. 5a).

In the control group, the median quantity of IGF1-positive cells in the epithelium and blood vessels was abundant (++++) with no fluctuations. In contrast, in connective tissue, the number of IGF1-positive cells was high (+++), and in weakly stained abundant (++++w) in smooth myocytes; both values fluctuated from numerous (+++) to abundant (++++) (Table 4; Fig. 5b).

Fig. 5
figure 5

Microphotographs of the IGF1-positive gallbladder structures in the patient and control materials. (a) Patient sample with numerous (+++) IGF1-positive cells in the epithelium and abundant (++++) cells in connective tissue and blood vessels but weakly stained abundant (++++w) cells in smooth myocytes. IGF1 IMH, ×200. (b) Control sample with abundant (++++) IGF1-positive cells in the epithelium and blood vessels and numerous (+++) cells in the connective tissue but weakly stained abundant (++++w) smooth myocytes. IGF1 IMH, ×200

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IGF1R

In the patient group, the median number of IGF1R-positive cells in the epithelium and connective tissue was weakly stained numerous (+++w), fluctuating from a few (+) to numerous (+++). However, moderate to weakly stained numerous (++/+++w) positive cells were found in the blood vessels, fluctuating from weakly stained moderate (++w) to numerous (+++). In contrast, in smooth myocytes, the median quantity of positive cells was moderate (++), fluctuating from a few (+) to numerous (+++) (Table 3; Fig. 6a).

In the control group, the median quantity of IGF1R-positive cells in the epithelium, connective tissue, and blood vessels was abundant (++++), with no fluctuations in the epithelium or blood vessels and fluctuating from weakly stained numerous (+++w) to abundant (++++) in the connective tissue. However, in smooth myocytes, IGF1R-positive cells were weakly stained abundant (++++w), fluctuating from numerous (+++) to abundant (++++) (Table 4; Fig. 6b).

Fig. 6
figure 6

Microphotographs of the IGF1R-positive gallbladder structures in patient and control samples. (a) Patient sample with numerous (+++) IGF1R-positive cells in the epithelium, moderate (++) cells in connective tissue and smooth myocytes, but weakly stained numerous (+++w) cells in blood vessels. IGF1R IMH, ×200. (b) Control sample with abundant (++++) IGF1R-positive cells in the epithelium, connective tissue, blood vessels, and smooth myocytes. IGF1R IMH, ×200

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HOXB3

In the patient group, the median number of HOXB3-positive cells in the epithelium was weakly stained abundant to abundant (++++w/++++), fluctuating from weakly stained to moderate (++w) positive cells to abundant (++++). However, an abundant (++++) number of positive cells were found in connective tissue, blood vessels, and smooth myocytes, with no fluctuations in connective tissue or blood vessels, but the number of positive cells fluctuated from weakly stained abundant (++++w) to abundant (++++) in smooth myocytes (Table 3; Fig. 7a).

In the control group, the median quantity of HOXB3-positive cells in the epithelium, connective tissue, and blood vessels was abundant (++++), with no fluctuations in the epithelium or blood vessels but fluctuating from numerous (+++) to abundant (++++) in the connective tissue. In contrast, in smooth myocytes, the number of HOXB3-positive cells was numerous (+++), fluctuating from a moderate (++) number of positive cells to numerous (+++) (Table 4; Fig. 7b).

Fig. 7
figure 7

Microphotographs of HOXB3-positive gallbladder structures in patient and control samples. (a) Patient sample with intensively stained abundant (++++) HOXB3-positive cells in the epithelium, connective tissue, blood vessels, and smooth myocytes. HOXB3 IMH, ×200. (b) Control sample with abundant (++++) HOXB3-positive cells in the epithelium, connective tissue, and blood vessels but numerous (+++) smooth myocytes. HOXB3 IMH, ×200

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Correlation analysis among the epithelium, connective tissue, blood vessels, and smooth myocytes

A statistically significant difference (p = 0.004 (U = 30.0)) was found between patient and control samples in SHH-positive epithelium, all IHH-positive structures, IGF1R-positive epithelium and blood vessels, and HOXB3-positive smooth myocytes. Additionally, a statistically significant difference (p = 0.017) was found in SHH-positive connective tissue and IGF1R-positive smooth myocytes between the control group and patient group (Table 5).

Table 5 Mann‒Whitney U test values and p values for proteins and growth factors/their receptors for gallbladder structures between patient and control samples
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Correlation analysis using Spearman’s rank coefficient (R) revealed 41 correlations, which consisted of SHH-positive cells in the epithelium and connective tissue; IHH-positive cells in all structures; HOXB3-positive cells in smooth myocytes; and growth factor receptor IGF1R-positive cells in the epithelium, blood vessels and smooth myocytes of the gallbladder. Of these, 80.5% were positively correlated, with 21 very strong correlations with R > 0.8 and 12 strong correlations with R coefficients between 0.6 and 0.8. In contrast, negative correlations accounted for 19.5% of all correlations, indicating three pairs of very strong correlations with R< (-0.8) and five pairs with strong correlations where the R coefficient was between − 0.8 and − 0.6 (Table 6; Fig. 8).

A very strong positive correlation was observed between SHH-positive epithelium and IHH-positive cells in the epithelium (R = 0.933), blood vessels, smooth myocytes and connective tissue. Furthermore, the SHH-positive epithelium correlated with the IGF1R-positive epithelium and blood vessels. A very strong positive correlation was detected between SHH-positive connective tissue and IHH-positive connective tissue. Additionally, IHH-positive structures include the epithelium and blood vessels, the epithelium and connective tissue, the epithelium and smooth myocytes, and smooth myocytes and blood vessels. In addition, IHH-positive structures (epithelium, smooth myocytes, and blood vessels) were strongly correlated with IGF1R-positive cells in the epithelium and blood vessels. Moreover, IHH-positive cells in blood vessels and IGF1R in smooth myocytes were compared. Furthermore, strong correlations were detected between the following IGF1R-positive structures: the epithelium and blood vessels, the epithelium and smooth myocytes, and smooth myocytes and blood vessels.

A strong positive correlation was found between IHH-positive structures, e.g., between connective tissue and smooth myocytes and between connective tissue and blood vessels. Additionally, IHH-positive structures (epithelium, smooth myocytes, connective tissue) correlated with IGF1R-positive cells in smooth myocytes and IHH-positive cells in connective tissue with IGF1R-positive structures (epithelium, blood vessels). Furthermore, IGF1R-positive cells in smooth myocytes correlated with SHH-positive cells in the epithelium, and IGF1R in blood vessels correlated with SHH in connective tissue. Additionally, a strong correlation was detected between SHH-positive cells in the epithelium and connective tissue. SHH-positive cells in connective tissue correlated with IHH-positive structures in the epithelium and smooth myocytes.

A very strong negative correlation was observed between HOXB3-positive cells in smooth myocytes and SHH- and IHH-positive cells in connective tissue, with R values of-0.879 and − 0.882, respectively. Furthermore, a very strong negative correlation was observed between HOXB3-positive cells in smooth myocytes and SHH-positive cells in the epithelium of the gallbladder. Additionally, a strong negative correlation was observed between HOXB3 in smooth myocytes and IHH-positive structures (epithelium, smooth myocytes, and blood vessels). Furthermore, IGF1R expression in the epithelium and blood vessels correlated with HOXB3 in smooth myocytes.

Table 6 Notable Spearman’s rank correlations between proteins and growth factors/their receptors in patients’ tissues
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Fig. 8
figure 8

Spearman’s rank (p < 0.05) correlation coefficients between proteins and growth factors/their receptors in the patient gallbladder structures. The correlation legend is shown on top, and the cells are filled on the basis of correlation values. Red indicates a negative correlation, whereas green indicates a positive correlation. Abbreviations: SHH−Sonic hedgehog; IHH−Indian hedgehog; HGF−hepatocyte growth factor; IGF1−insulin-like growth factor 1; IGF1R−insulin-like growth factor 1 receptor; HOXB3−homeobox B3; E−epithelium; CT−connective tissue; BV−blood vessels; SM−smooth myocytes

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Discussion

Our research revealed that the proteins SHH and IHH and the growth factors/receptors HGF, IGF1, and IGF1R were downregulated, as the number of positive cells was lower than that in the control group. In contrast, HOXB3 was upregulated in smooth myocytes. Although HGF and IGF1 had the highest number of positive cells in both groups, the results were not statistically significant. Although it is difficult to determine whether this result is a primary or a secondary change, the functions of these factors may also be affected.

SHH induces expression of bone morphogenic protein 4 (Bmp4) in the gut, which controls gut-specific smooth muscle development. The thinning of the smooth muscle layer is due to the ability of SHH to negatively regulate growth [11]. For this reason, a substantial reduction in SHH leads to aberrantly thick smooth muscle formation in the gallbladder, and increased thickness of the gallbladder was also detected in chronic cholecystitis patients by Limaiem et al. [36]. The fact that SHH was downregulated in the epithelium and fibroblasts of our patients compared with those in the control group provides some evidence that SHH is connected to chronic calculous cholecystitis. In contrast, Xie et al. reported the absence of SHH expression in normal gallbladders and increased expression of SHH in chronic cholecystitis. The authors proposed that, during repeated damage caused by chronic inflammation, SHH expression is increased because of the overexpression of cytokines to induce regeneration [16]. The difference could occur because Xie et al. studied the gallbladders of adults, ranging from 28 to 62, whereas our study focused on pediatric patients aged from 6 to 18 years. Additionally, the previous study was not focused on chronic calculous cholecystitis, as not all the participants were presented with stones, in contrast to our study. We suppose that the downregulation of SHH was not the primary because then it should be connected with some genetic disorders; therefore, more likely, it changed as a secondary response to the mechanical influence of gallstones and inflammation, where HH signaling inhibition was also detected by Zacharias et al. [37]. Gallstones continuously irritate the gallbladder wall, sustaining persistent inflammation and, therefore, resulting in muscle layer changes.

A reduced number of IHH-positive tissue cells were found in all tissue types of the gallbladders affected by chronic calculous cholecystitis. The processing of the IHH and signal production are identical to those of SHH since they are structurally similar and use the same receptor through the same intracellular signalling pathway [17, 38]. In the study carried out by Hatanaka et al. in the normal gallbladder, the IHH protein was missing [39]. In contrast, our study revealed abundant IHH-positive cells in normal gallbladders, suggesting that a decreased IHH level probably contributes to chronic calculous cholecystitis. This finding is also indirectly demonstrated by the fact that Hedgehog (HH) family ligands act as morphogens for the development of the smooth muscle layer [38, 40]. Hence, decreased HH signalling promotes epithelial cell proliferation and crypt expansion [41]. We suggest that decreased IHH results in a thickened gallbladder wall and further chronic calculous cholecystitis, as the results were statistically significant in all gallbladder structures, especially in the epithelium. Additionally, a strong correlation between SHH and IHH indicates the involvement of both proteins in the function of healthy and inflamed gallbladders with gallstones.

The synergy of SHH and IGF1 is found in somite myogenesis because IGF1R levels depend on Smoothened protein (Smo) activation. Smo is involved in the SHH pathway; therefore, loss of Smo decreases the amount of IGF1R [42]. The present study also revealed a positive correlation among SHH, IHH, and IGF1R in the epithelium and smooth myocytes; however, only epithelial expression was statistically significant. We propose that the downregulation of IHH is also connected to decreased IGF1R levels because it acts through the same Smo pathway as SHH. This result was also established by other authors, for example, in a study on antler chondrocytes conducted in 2017, which also suggested that IGF1 can stimulate the expression of IHH [43].

Our study revealed a decreased number of IGF1- and IGF1R-positive structures. This receptor is present in multiple cells, as it promotes cell proliferation and differentiation, inhibits apoptosis, and synthesizes the extracellular matrix in fibroblasts and endotheliocytes [31]. IGF1R is expressed in the biliary epithelium, protecting it from cholestatic injury and promoting smooth muscle proliferation [33], whereas IGF1 activates proliferating pathways by stimulating the synthesis of DNA [31–32]. Research carried out in 2023 revealed that IGF1R inhibition leads to decreased cell proliferation in hepatocellular carcinoma cells [44]. Interestingly, IGF1R inactivation induces cholestasis in mouse models and a lack of cholangiocyte growth [45]. Therefore, the biliary epithelium expresses IGF1R and protects epithelial cells in the bile duct against cholestatic injury [33]. We suggest that with the deficiency of IGF1R, the protective response of epithelial cells in the bile duct decreases, as there are not enough receptors to bind with growth factors, resulting in gallbladder inflammation and chronic calculous cholecystitis in our patients. The involvement of the IGF1 pathway in inflammatory inhibition was also proven by Li et al. [46]. Therefore, a decreased number of IGF1Rs reduces IGF1 pathway activation and leads to inflammation.

The only upregulated growth factor in our study was HOXB3, which showed statistically significant results in smooth myocytes of the patient material. HOXB3 controls cell differentiation by acting as a positive or negative regulator of transcription by DNA-specific binding [20, 21]. This molecule also regulates the differentiation of smooth muscle cells via an epigenetic mechanism [21]. We suggest that the upregulation of HOXB3 is a compensatory mechanism for the downregulation of HH ligands. The association of HOXB3 with gallbladder pathologies is a novel finding, as, to the best of our knowledge, no prior studies have reported such a link. Therefore, this is the first time we can link HOXB3 with calculous cholecystitis. Given that the upregulation of HOXB3 was detected in the patient samples and not in the control, we suggest that it could be connected with the pathogenesis of chronic calculous cholecystitis. Moreover, elevated levels of other HOX family genes were previously connected with gallbladder cancer [47,48,49]. We further believe that HOXB3 upregulation may serve as compensation for long-lasting unresolved inflammation in chronic calculous cholecystitis. This is supported by findings that Interleukin-1β, similarly, can upregulate HOXC10 expression [50], and an increased amount of HOXB4 levels have been shown to dramatically decrease pro-inflammatory factors in endothelial cell injury [51]; therefore, leading to a secondary increase of HOXB3 in calculous cholecystitis to resolve the inflammation.

We suggest that HGF is not the most important growth factor found in chronic calculous cholecystitis-affected gallbladders, as in both control and patient samples, there was an abundant or weakly abundant number of HGF-positive cells. However, HGF is an important factor in gallbladder formation during foetal development as a bile duct-inducing or stabilizing factor from periportal connective tissue [52]. Furthermore, in a study conducted in the 2000s, Mori et al. reported that HGF promotes DNA synthesis and cell migration [53]. Additionally, inhibition of HGF results in the resolution of neutrophil-induced inflammation [54]. Furthermore, research carried out in 2008 revealed that HGF gene therapy suppressed the infiltration of inflammatory T cells and macrophages in obstructed nephropathy, indicating anti-inflammatory effects [28]. In cholestatic-induced gallbladder damage, HGF decreases gallbladder swelling and dark colouring and reverses the changes in cholangiocellular cilium morphology [29].

This study has potential limitations, such as the limited number of gallbladders affected by chronic calculous cholecystitis, as it was conducted on a comparatively small patient group. Additionally, Western blot analysis, ELISA (enzyme-linked immunosorbent assays), or qPCR (quantitative polymerase chain reaction) are needed to better understand the protein and growth factor/receptor concentration patterns in specific chronic calculous cholecystitis-affected gallbladder walls. Finally, specific gene expression might also be important for revealing those genes that are actually responsible for functional and structural aberrant gallbladder development.

Conclusions

The decrease in SHH-, IHH-, and IGF1R-positive structures in chronic calculous cholecystitis-affected gallbladders suggests the involvement of these proteins and growth factor receptors in their correct formation and function.

An increased number of HOXB3-positive smooth myocytes and a negative correlation with SHH, IHH, and IGF1R could indicate a compensatory reaction to reverse improper cell proliferation and promote the correct formation and function of the gallbladder.

However, HGF and IGF1 are unlikely to be the key factors in the development of chronic calculous cholecystitis, as the results did not reveal a statistically significant difference between the number of positive cells in the control and patient samples.

Data availability

All data supporting reported results can be found in the result section of this manuscript.

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Acknowledgements

The resource of the Department of Morphology in Riga Stradiņš University is kindly acknowledged. Provision of material by Professor Arnis Enģelis is greatly appreciated. Assistance of the lab. technician Natalia Moroza is greatly acknowledged. Our gratitude is extended to Eva Nazarova for the language review of this manuscript.

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  1. Institute of Anatomy and Anthropology, Riga Stradins University, Kronvalda Boulevard 9, Riga, LV-1010, Latvia

    Darja Derbeneva & Mara Pilmane

  2. Department of Paediatric Surgery, Riga Stradins University, Dzirciema street 16, Riga, LV-1007, Latvia

    Aigars Petersons

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Conceptualization, methodology, validation, data curation, investigation, project administration M.P, D.D., A.P; statistical analysis, visualization, writing-original draft preparation, D.D.; writing-review and editing, M.P. and A.P.; supervision M.P.; resources M.P. and A.P. All authors have read and agreed to the published version of the manuscript.

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Derbeneva, D., Pilmane, M. & Petersons, A. Gene proteins, growth factors/their receptors in the wall of chronic calculous cholecystitis-affected gallbladder children. BMC Pediatr 25, 288 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12887-025-05650-4

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